I *‘- H...- ‘ W i s LIBRARY ,. Michigan State is) University (J .. ..._..* This is to certify that the thesis entitled THE DENATURATION OF DNA REPAIR PROTEINS AS A POSSIBLE RATE LIMITING STEP IN THE THERMAL DEATH OF SINGLE CELLS presented by Robert Shenkar has been accepted towards fulfillment of the requirements for Ph-Tl- fiegreeinjjnphxsica C Q EKG/l: {A ”wk—u Q/Ltt Major professor Date June 7, 1979 0-7639 [1 “V“‘ L V “151.11%." ‘\ ‘Y‘\ 2935!” . 1 '.t.\~ y,” ovznoug mes: 25¢ per on por tu- museum ugmv mrtngnls: I. Place in book return to move’ charge from circulation records ITHE DENATURATION OF DNA REPAIR PROTEINS AS A POSSIBLE RATE LIMITING STEP IN THE THERMAL DEATH OF SINGLE CELLS By Robert Shenkar A DISSERTATION submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biophysics 1980 ABSTRACT THE DENATURATION OF DNA REPAIR PROTEINS AS A POSSIBLE RATE LIMITING STEP IN THE THERMAL DEATH OF SINGLE CELLS By Robert Shenkar The experiments to be described support the hypothesis that the denaturation of DNA repair proteins is a rate limiting step in the thermal death of cells grown in tissue culture. Mammalian fibroblasts were subjected to small changes in temperature before ultraviolet (UV) irradiation in order to detect changes in repair of UV induced damage. Confluent fibroblasts were incubated for three days in a medium deficient in arginine and isoleucine. After this period the cells were incubated for one hour with hydroxyurea and then treated with 25h nm UV radiation at various doses. During various spans of time immediately before UV treatment, the cells were incubated at different temperatures. Repair of DNA was mea- sured by several methods after UV treatment. In some experiments repair was measured by incorporating 3H- Thymidine at 37°C for different lengths of time after UV irradi— ation. Human skin and hamster (V-79) fibroblasts incubated at either hO°C or hl°C for 3 days immediately prior to UV treatment showed significantly less 3H-Thymidine incorporation into their DNA than cells that were constantly incubated at 37°C prior to Robert Shenkar UV treatment. This result was observed regardless of the length of the period of 3H-Thymidine incorporation. Cells incubated at 33°C prior to UV treatment did not differ significantly from cells that were always incubated at 37°C in their incorporation of 3H- Thymidine. Autoradiography showed that V-79 cells incubated at hl°C prior to UV treatment had a fairly wide distribution of grains per nu- cleus, but with average number of grains significantly less than the average number of grains in cells always incubated at 37°C. This suggests that prior heat treatment impairs the repair in all of the cells rather than causes the thermal death of a large por- tion of the cells. Finally, an endonuclease specific assay was conducted to de- termine whether the above effects were due to the thermal denatur- ation of the endonuclease repair enzyme or rather to some heat sensitive process involved in the transport and phosphorylation of exogenous 3H-Thymidine to 3H-dTTP into the DNA inside the cells. By using a dimer specific endonuclease from M, luteus, the number of dimers produced and repaired was measured directly. For both hamster and human fibroblasts, significantly less repair was ob- served in cells incubated at hl°C prior to UV treatment than in cells incubated at 37°C before UV treatment. This suggests that the endonuclease repair enzymes are denatured at the higher tem- perature. The evidence shows that the thermal denaturation of the endonuclease is the rate limiting step for one of the process- es leading to the death of V-79 cells. © Copyright by ROBERT SHENKAR 1980 To my parents George and Stella Shenkar iii ACKNOWLEDGEMENTS Appreciation is due to the many individuals who have assisted in the research presented in this dissertation. Professors Gabor Kemeny, Barnett Rosenberg, Estelle McGroarty, and James E. Trosko deserve special recognition for many valuable suggestions throughout the course of the research and for helpful comments for the text of this manuscript. The advice for the overall conduct of the research from Professor Gabor Kemeny, the chairman of my dissertation committee, was invaluable. I am very grateful to Drs. Barnett Rosenberg, James E. Trosko, and Richard B. Setlow for the Opportunity of working in their laboratories and for the materials that were necessary for the experiments. I appreciate the technical assistance from Dr. Pamela McAllister, Mrs. Mary Banderski, Mr. Roger Schultz, and Mrs. Eleanor Grist. The computer assistance from Mr. Keith Thompson and Mr. Donald Brunder is greatly appreciated. Finally, I am grateful for the support received from the College of Osteopathic Medicine at Michigan State University. iv TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . LIST OF FIGURES . . Chapter I INTRODUCTION . PROTEIN DENATURATION AS THE CAUSE OF THERMAL DEATH THE CORRELATION BETWEEN THE MAXIMUM LIFE SPAN OF MAMMALS AND REPAIR OF DNA DAMAGED BY ULTRAVIOLET RADIATION THE HYPOTHESIS THAT DENATURATION OF DNA REPAIR PROTEINS IS THE RATE LIMITING STEP IN THE THERMAL DEATH OF SINGLE CELLS EXPERIMENTS THAT WERE CONDUCTED IN ORDER TO SUPPORT THE HYPOTHESIS THAT DENATURATION OF DNA REPAIR PROTEINS IS A RATE LIMITING STEP IN THE THERMAL DEATH OF SINGLE CELLS II THE EFFECT OF TEMPERATURE ON THE REPAIR OF ULTRAVIOLET INDUCED PYRIMIDINE DIMERS IN THE DNA OF MAMMALIAN CELLS AS MEASURED BY UNSCHEDULED DNA SYNTHESIS INTRODUCTION . . . . MATERIALS AND METHODS . Cells . . . . . xi 13 1h 17 17 18 18 Media . . . . . . . . . . . . . . . . . . . Experimental Culture Procedure . . . . . . Ultraviolet Irradiation and Repair . . . . Analysis of "Unscheduled DNA Synthesis" . . RESULTS 1. The effect of 2 hours of different pre- UV incubation temperatures on "unsche- _ duled DNA synthesis" in V-79 cells . . The effect of 8 and 21 hours of hl°C pre-UV incubation on "unscheduled DNA synthesis" in V-79 cells. . . . . . . . The effect of 3 days of hl°C pre-UV in- cubation on "unscheduled DNA synthesis" in V-79 cells . . . . . . . . . . . . . The effect of cycloheximide and 3 days of hl°C pre-UV incubation on "unsche- duled DNA synthesis" in V-79 cells . . The effect of incubation at hl°C for various spans of time before ultra- violet irradiation on "unscheduled DNA synthesis" in V-79 cells . . . . . The effect of incubation at 33°C for various spans of time before ultraviolet irradiation on 3H-Thymidine incorporation in V—79 cells . . . . . . vi 19 19 21 22 23 23 26 32 39 A3 A6 7. The effect of incubation at 33°C for various spans of time before ultra- violet irradiation on various periods of 3H-Thymidine incorporation in V-79 cells . . . . . . . . . . . . . . . . 5h 8. The effect of incubation at 33°C for 5 days before ultraviolet irradiation on various periods of 3H—T’hymidine incorporation in V-79 cells . . . . . . . 57 9. The effect of incubation at 33°C for 5 days before ultraviolet irradiation on "unscheduled DNA synthesis" conducted for various durations in V-79 cells with added hydroxyurea . . . . . . . . . . 6O 10. The effect of incubation at hO°C for 3 days before ultraviolet irradiation on various periods of "unscheduled DNA synthesis" in V-79 cells . . . . . . . . . 72 11. The effect of incubation at h1°C for 3 days before ultraviolet irradiation on 2 and 2h hours of 3H-Thymidine in- corporation in human skin fibroblasts . . 76 12. The effect of incubation at 37°C and hl°C for l, 8, and 2h hours in deficient medium on subsequent "unscheduled DNA synthesis" in human fibroblasts . . . . . 80 DISCUSSION . . . . . . . . . . . . . . . . . . . . 81 vii SUMMARY . III THE EFFECT OF TEMPERATURE ON THE REPAIR OF ULTRAVIOLET INDUCED PYRIMIDINE DIMERS IN THE DNA OF MAMMALIAN CELLS AS MEASURED BY AUTORADIOGRAPHY INTRODUCTION . . . . . . . . . MATERIALS AND METHODS . Cells . . . . . . . . . . Media . . . Experimental Culture Procedure . . . . . . Ultraviolet Irradiation and Repair . . . . . Autoradiography . . . . . . . . Staining . . . . . . . . . . . RESULTS . . . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . . . . SUMMARY . IV THE EFFECT OF TEMPERATURE ON THE REPAIR OF ULTRAVIOLET INDUCED PYRIMIDINE DIMERS IN THE DNA OF MAMMALIAN CELLS AS MEASURED BY ENDONUCLEASE SITE SPECIFICITY INTRODUCTION . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . Cells . . . . . . . . . . . . . . . . Media Experimental Culture Procedure . . . . . Ultraviolet Irradiation and Repair . . . . Cell Lysis and Phenol Extraction of the DNA The Endonuclease Assay . . . . . . . . . . Calculations of Endonuclease-Sensitive Sites . viii 91 93 93 9b 9b 9h 9h 9h 95 96 96 103 106 107 107 108 108 108 108 109 110 111 112 RESULTS Experiments Involving Chinese Hamster Lung Fibroblasts Experiments Involving Human Skin Fibroblasts DISCUSSION . . . . . . . . . . . . . . . SUMMARY V DISCUSSION . . . . . . . . . THE CALCULATION OF THE ACTIVATION ENTHALPY FOR THERMAL IMPAIRMENT OF EXCISION REPAIR IN V-79 CELLS AND HUMAN SKIN FIBROBLASTS . EVIDENCE THAT THE DENATURATION OF THE DNA REPAIR PROTEINS IS A RATE LIMITING STEP IN THE THERMAL DEATH OF SINGLE CELLS . THE ACTIVATION ENTHALPIES OF THE DNA REPAIR PROTEINS DETERMINING THE MAXIMUM LIFE SPAN OF A MULTICELLULAR ORGANISM VI CONCLUSIONS VII RECOMMENDATIONS . . . . . . . . . . . . . . . . APPENDIX A APPENDIX B . . . . . . . . . . . . . . . . . . . . . LIST OF REFERENCES . . . . . . . . . . . . . . . . . . ix 113 113 121 1A8 150 151 151 156 159 161 16A 167 168 172 Table LIST OF TABLES The effect of 2 hours of various temperature treatments before ultraviolet irradiation on 3H-Thymidine incorporation into the DNA of Chinese hamster lung fibroblasts . . . . . . . . . . . . . 2h Endonuclease sensitive sites removed in human 1A2 skin fibroblasts . . . . . . . . . . . . . . . . . . The thermal inhibition of repair . . . . . . . . . . . . . 15h A program to determine average molecular weights . . . . . 169 Figure LIST OF FIGURES Experimental Culture Procedure . . . . . . . . . . . . . . . 20 The amount of 3H-Thymidine (5 uCi/ml medium) incorporated into the DNA of V-79 cells for 2 hours at 37°C as a func- tion of the previous duration of incubation at hl°C. 3H—Thymidine incorporation followed the h1°C incubation period, hydroxyurea (5 mM) treatment for 1 hour, and 25h nm ultraviolet irradiation at 15 J/m2 (hollow circles) or non-irradiation (solid circles) . . . . . . . . . . . . . . 28 The amount of 3H-Thymidine (5 uCi/ml medium) incorporated into the DNA of V-79 cells for 2 hours at 37°C as a func- tion of the previous duration of incubation at A1°C. 3H-Thymidine incorporation followed the hl°C incubation period, hydroxyurea (5 mM) treatment for 1 hour, and 25h nm ultraviolet irradiation at 15 J/m2 (hollow circles) or non-irradiation (solid circles) . . . . . . . . . . . . . . 31 The amount of 3H—Thymidine (5 uCi/ml medium) incorporated into the DNA of V-79 cells for 2 hours at 37°C as a func- tion of the previous duration of incubation at Al°C. 3H-Thymidine incorporation followed the hl°C incubation period, hydroxyurea (5 mM) treatment for 1 hour, and 25h nm ultraviolet irradiation at 15 J/m2 (hollow circles) or non-irradiation (solid circles) . . . . . . . . . . . . . . 35 The amount of 3H-Thymidine (5 uCi/ml medium) incorporated into the DNA of v-79 cells for 2 hours at 37°C following 25h nm ultraviolet irradiation at 15 J/m2 (hollow circles) or non-irradiation (solid circles) as a function of the temperatures of (a) the 72 hour incubation in deficient medium before hydroxyurea was added ("pre-HU"), (b) the 1 hour incubation with hydroxyurea (5 mM) before 3H-Thymidine incorporation ("HU"), and (c) the 2 hour 3H-Thymidine incorporation ("repair") . . . . . . . . . . . 37 xi 10. 11. The amount of 3H-Thymidine into the DNA of V-79 cells tion of previous durations 3H-Thymidine incorporation (5 uCi/ml medium) incorporated for 2 hours at 37°C as a func- of incubation at hl°C. followed the hl°c incubation period, treatment for 1 hour with hydroxyurea (5 mM) and with (hollow symbols) or without (solid symbols) cyclo- heximide (5 ug/ml), and 25h nm ultraviolet irradiation at 15 J/m2 (squares) or non-irradiation (circles) . . . . The amount of DNA isolated (top) and 3H—Thymidine (5 uCi/ml medium) incorporated into the DNA (bottom) of V-79 cells for 2 hours at 37°C as a function of the previous duration of incubation at hl°C. incorporation followed the hydroxyurea (5 mM) treatment for 1 hour, and 25h nm ultra- 3H-Thymidine hl°C incubation period, violet irradiation at 15 J/m2 (hollow circles) or non- irradiation (solid circles) O O O O O O I O I O O O The net amount of 3H-Thymidine (5 uCi/ml medium) incor— porated into the DNA of V-79 cells for 2 hours at 37°C, which was used for the excision repair of ultraviolet radiation-induced dimers, as a function of previous dura— tions of incubation at hl°C. Each point is derived from the difference of the averaged values of points in Figure 7 between those representing ultraviolet irradiated cells and non-irradiated cells . . . . . . . . . . . . The amount of 3H-Thymidine into the DNA of V-79 cells tion of previous durations 3H—Thymidine incorporation period, hydroxyurea (5 mM) nm ultraviolet irradiation The amount of 3H-Thymidine into the DNA of V-79 cells tion of previous durations 3H—Thymidine incorporation period, hydroxyurea (5 mM) nm ultraviolet irradiation The amount of 3H-Thymidine into the DNA of V-79 cells (5 uCi/ml medium) incorporated for 2 hours at 37°C as a func- of incubation at 33°C. followed the 33°C incubation treatment for 1 hour, and 25h at 15 J/m2 . . . . . . (5 uCi/ml medium) incorporated for 2 hours at 37°C as a func- of incubation at 33°C. followed the 33°C incubation treatment for 1 hour, and 25h at 15 J/m2 . . . . . . . . . (5 uCi/ml medium) incorporated for various periods at 37°C as a function of previous durations of incubation at 33°C. 3H—Thymidine incorporation 10 hours (hollow circles), and 20 hours (squares) followed the 33°C incubation period, for 5 hours (solid circles), hydroxyurea (5 mM) treatment for 1 hour, and 25h nm ultraviolet irradiation at 15 J/m2 xii Al AS h8 51 53 12. 13. 1A. 15. 16. The amount of DNA isolated (top) and 3H—Thymidine (5 uCi/ml medium) incorporated into the DNA (bottom) of V-79 cells at 37°C as a function of the duration of 3H-Thymidine incorporation, following a 120 hour incubation period at either 37°C (solid circles) or 33°C (hollow circles), hydroxyurea (5 mM) treatment for 1 hour, and 25A nm ultraviolet irradiation at 15 J/m2 . . . . . . . . . . . . . . . . . . . . . . . . . . 59 The amount of DNA isolated (top) and 3H-Thymidine (5 uCi/ml medium) incorporated into the DNA (bottom) of V-79 cells at 37°C as a function of the duration of 3H-Thymidine incorporation, following a 120 hour incubation period at either 37°C (solid symbols) or 33°C (hollow symbols) and 25A nm ultraviolet irradiation at 15 J/m2 (squares) or non-irradiation (circles). Hydroxyurea ("HU") was added at 17, A3, and 68 hours after ultraviolet irradiation, as well as 1 hour before irradiation, in increasing increments of 5 mM_each. . . . . 62 The net amount of 3H-Thymidine (5 uCi/ml medium) incor— porated into the DNA of V-79 cells at 37°C, which was used for the excision repair of ultraviolet radiation-induced dimers, as a function of the duration of 3H—Thymidine incorporation. Each point is derived from the difference of the averaged values of points in Figure 13 between those representing ultraviolet irradiated cells and non- irradiated cells, which were previously incubated for 120 hours at either 37°C (solid circles) or 33°C (hollow circles). . . . . . . . . . . . . . . . . . . . . . . . . . 6h The amount of 3H-Thymidine (5 uCi/ml medium) incorporated into the DNA of V-79 cells at 37°C as a function of the duration of 3H—Thymidine incorporation, following a 120 hour incubation period at either 37°C (circles) or 33°C (squares) and 25A nm ultraviolet irradiation at 15 J/m2 (hollow symbols) or non-irradiation (solid symbols). Hydroxyurea ("HU") was added at 21, A5, 68, and 96 hours after ultraviolet irradiation, as well as 1 hour before irradiation, in increasing increments of 5 mM_each . . . . 69 The net amount of 3H-Thymidine (5 uCi/ml medium) incor- porated into the DNA of V-79 cells at 37°C, which was used for the excision repair of ultraviolet radiation— induced dimers, as a function of the duration of 3H—Thymidine incorporation. Each point is derived from the difference of the averaged values of points in Figure 15 and Figure 17 between those representing ultraviolet irradiated cells and non-irradiated cells, which were previously incubated for 120 hours (Figure 15) at either 37°C (solid circles) or 33°C (squares) or were previously incubated for 72 hours (Figure 17) at either 37°C (hollow circles) or at A0°C (triangles) . . . . . . . . . . . . . . 71 xiii 17. 18. 19. 20. 21. 22. The amount of DNA isolated (top) and 3H-Thymidine (5 uCi/ml medium) incorporated into the DNA (bottom) of V-79 cells at 37°C as a function of the duration of 3H—Thymidine incorporation, following a 72 hour incuba- tion period at either 37°C (circles) or AO°C (triangles) and 25A nm ultraviolet irradiation at 15 J/m2 (hollow symbols) or non—irradiation (solid symbols). Hydroxy— urea was added at 18, A2, and 66 hours after ultraviolet irradiation, as well as 1 hour before irradiation, in increasing increments of 5 mM each . . . . . . . . . . . . 75 The amount of 3H—Thymidine (5 uCi/ml medium) incorporated into the DNA of human skin (736-NF) fibroblasts at 37°C as a function of the duration of 3H-Thymidine incor— poration, following a 72 hour incubation period at either 37°C (circles) or Al°C (triangles), hydroxyurea (5 mM) treatment for 1 hour, and 25A nm ultraviolet irradiation at 15 J/m2 (hollow symbols) or non-irradiation (solid symbols). . . . . . . . . . . . . . . . . . . . . . . . 79 The amount of 3H-Thymidine (5 uCi/ml medium) incorporated into the DNA of human skin (736-NF) fibroblasts at 37°C as a function of previous durations of incubation at either 37°C (circles) or Al°C (triangles), after an initial 72 hour incubation period at 37°C. Just prior to 3H-Thymidine incorporation, the cells were treated with hydroxyurea (5 mM) for 1 hour and either exposed (hollow symbols) or not exposed (solid symbols) to 25A nm ultraviolet irradia— tion at 15 J/m2 . . . . . . . . . . . . . . . . . . . . . 83 Autoradiograph of V-79 nuclei after 72 hour incubation at 37°C, treatment for 1 hour with hydroxyurea (5 mM), ultra- violet irradiation (25A nm) at 15 J/m2, and exposure to 3H—Thymidine (5 uCi/ml medium) for 2 hours at 37°C . . . . 98 Autoradiograph of V-79 nuclei after 72 hour incubation at Al°C, treatment for 1 hour with hydroxyurea (5 mM), ultra— violet irradiation (25A nm) at 15 J/m2 , and exposure to 3H-Thymidine (5 UCi/ml medium) for 2 hours at 37°C . . . . loo Distribution curves representing the number of cells with indicated grains per nucleus. V-79 cells were incubated at either 37°C (solid circles) or Al°C (hollow circles) for 72 hours in a medium deficient in arginine and isoleucine, treated for 1 hour with hydroxyurea (5 mM), ultraviolet irradiated (25A nm) at 15 J/m2 , and exposed to 3H-Thymidine (5 uCi/ml medium) for 2 hours. Seven hundred cells were counted in each treatment group. The absence of a data point means there were no cells with that grain number . . 102 xiv 23. 2A. 25. 26. 27. 28. Distribution curves representing the numbers of cells with indicated grains per nucleus. The solid circles represent the experimental data from Figure 22 for V-79 cells which were incubated at Al°C for 72 hours before ultraviolet irradiation. The hollow circles represent the expected values if the cells incor- porated 33% as much 3H-Thymidine as cells that were incubated at 37°C for 72 hours before ultraviolet irradiation. The triangles represent the expected values if 67% of the cells died and incorporated no 3H-Thymidine, while the remaining 33% of the cells incorporated as much 3H-Thymidine as cells that were incubated at 37°C for 72 hours before ultraviolet irradiation . . . . . . . . . . . . . . . . . . 105 Sedimentation profiles of extracted DNA from V-79 cells after treatment with M. luteus endonuclease. Values of Mw were 6A.A3 x 166 (Fractions 1 to 27) for non-irradiated cells (solid circles) and 23.A9 x 106 (Fractions 5 to 28) for cells ultraviolet irradiated at 2.5 J/m2 (hollow circles) . . . . . . . . . . 115 Sedimentation profiles of extracted DNA from V—79 cells after treatment with M, luteus endonuclease. Cells were exposed to 2.5 J m2 ultraviolet radiation. Values of MV were 20.77 x 10 (Fractions 2 to 26) for DNA extracted immegiately after irradiation (solid circles) and 26.50 x 10 (Fractions 2 to 26) for DNA extracted 6 hours after irradiation (hollow circles). . . . . . . . . 118 Sedimentation profiles of extracted DNA from V-79 cells after treatment with M, luteus endonuclease. Cells were exposed to 2.5 J/m2 ultraviolet radiation and the DNA was extracted 6 hours after irradiation. Values of MV were 25.37 x 106 (Fractions 1 to 25) for cells that were incubated at 37°C for 72 hours before irradiation (solid circles) and 23.18 x 106 (Fractions 1 to 25) for cells that were incubated at Al°C for 72 hours before irradia— tion (hollow circles) . . . . . . . . . . . . . . . . . . . 120 Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M. luteus endonuclease. Values of M were 51.75 x 106 (Fractions 1 to 29) for non—irradiateg cells (solid circles) and 7.06 x 106 (Fractions 1A to 29) for cells ultraviolet irradiated at 20 J/m2 (hollow circles) . . . . . . . . . . 123 Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M. luteus endonuclease. Cells were exposed to 20 J/m2 ultraviolet irradiation. Values of Mw were 5.77 x 106 (Fractions 8 to 25) for DNA extracted immediately after irradiation (solid circles) and 7.7A x 106 (Fractions 8 to 25) for DNA extracted 2A hours after irradiation (hollow circles) . 126 XV 29. 30. 31. 32. 33. Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M. luteus endonuclease. Cells were exposed to 20 J/m2 ultraviolet radiation and the DNA was extracted 2 hours after irra- diation. Values of Mw were 8.06 x 10 (Fractions 5 to 25) for cells that were incubated at 37°C for 72 hours before irradiation (solid circles) and 7.66 x 106 (Fractions 5 to 25) for cells that were incubated at Al°C for 72 hours before irradiation (hollow circles) . . . . . . . . . . . . 128 Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M, luteus endonuclease. Confluent cells were incubated at 37°C for 2A hours in medium deficient in arginine and isoleucine before exposure to 20 J/m2 ultraviolet radiation. Values of MV were 3.78 x 106 (Fractions 7 to 32) for DNA extracted immediately after irradiation (solid circles) and 7.96 x . 106 (Fractions 1 to 32) for DNA extracted 2A hours after irradiation (hollow circles) . . . . . . . . . . . . . . . 131 Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M, luteus endonuclease. Confluent cells were incubated at 37°C for 72 hours in medium deficisnt in arginine and isoleucine before exposure to 20 J/m ultraviolet radiation. Values of Mw were A.05 x 106 (Fractions 7 to 31) for DNA extracted imgediately after irradiation (solid circles) and 7.88 x 10 (Fractions 1 to 30) for DNA extracted 2A hours after irradiation (hollow circles) . . . . . . . . . . . . . . . 133 Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M, luteus endonuclease. Confluent cells were incubated at 37°C for 1AA hours in medium deficient in arginine and isoleucine before exposure to 20 J/m2 ultraviolet radiation. Values of Mw were A.0A x 106 (Fractions 8 to 31) for DNA extracted imgediately after irradiation (solid circles) and 7.98 x 10 (Fractions 1 to 32) for DNA extracted 2A hours after irradiation (hollow circles) . . . . . . . . . . . . . . . 135 Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M, luteus endonuclease. Confluent cells were incubated at Al°C for 2A hours in medium deficient in arginine and isoleucine before exposure to 20 J/m2 ultraviolet radiation. Values of MV were A.l7 x 106 (Fractions 9 to 32) for DNA extracted imgediately after irradiation (solid circles) and 7.31 x 10 (Fractions 1 to 29) for DNA extracted 2A hours after irradiation (hollow circles) . . . . . . . . . . . . . . . 137 xvi 3A. 35. 36. 37. 38. Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M, luteus endonuclease. Confluent cells were incu- bated at Al°C for 72 hours in medium deficient in arginine and isoleucine before exposure to 20 J/m2 ultraviolet radiation. Values of MV were A.01 x 10 (Fractions 1 to 31) for DNA extracted immediateéy after irradiation (solid circles) and 6.8A x 10 (Fractions 1 to 31) for DNA extracted 2A hours after irradiation (hollow circles) . . . . . . . . . . . . . . . 139 Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M, luteus endonuclease. Confluent cells were incubated at Al°C for 1AA hours in medium deficient in arginine and isoleucine before exposure to 20 J/g2 ultraviolet radiation. Values of Mw were A.0A x 10 (Fractions 5 to 31) for DNA extracted immegiately after irradiation (solid circles) and 5.20 x 10 (Fractions 2 to 31) for DNA extracted 2A hours after irradiation (hollow circles). 1A1 Sedimentation profiles of extracted DNA from human skin fibroblasts (735—NF) after treatment with M, luteus endonuclease. Confluent cells were incubated at Al°C for 2A hours in medium deficient in arginine and isoleucine before exposure to 20 J/m2 ultraviolet radiation. Values of Mw were A.0A x 106 (Fractions 9 to 25) for DNA extract- ed immediately after irradiation (solid circles) and 7.12 x 106 (Fractions 1 to 25) for DNA extracted 2A hours after irradiation (hollow circles) . . . . . . . . . . . . 1A5 Sedimentation profiles of extracted DNA from human skin fibroblasts (736-NF) after treatment with M, luteus endonuclease. Confluent cells were incubated at Al°C for 72 hours in medium deficient in arginine and isoleucine before exposure to 28 J/m2 ultraviolet radiation. Values of Mw were 5.55 x 10 (Fractions 7 to 25) for DNA extract- ed immedigtely after irradiation (solid circles) and 7.91 x 10 (Fractions 1 to 25) for DNA extracted 2A hours after irradiation (hollow circles) . . . . . . . . . . . . 1A7 Plots of Equations (2A) from page 155 for temperatures (T) at 37°C, AO°C, and Al°C. The dotted lines represent the rate constant, kD (T), as a function of the activation enthalpy, AHI. The solid curves represent the rate constant differences, kD (T) - kD (37°c), for T at Ao°c and at Al°C. The points represent data obtained from Table 3. . . . . . 153 xvii CHAPTER I INTRODUCTION The experiments that are to be described were conducted to test the hypothesis that the denaturation of DNA repair proteins is the rate limiting step in the thermal death of single cells. This hypothesis arose from the evidence presented by Rosenberg and coworkers1 that protein denaturation is the cause of thermal death in living systems; and the correlation between maximum life span of mammals and extent of repair of DNA damaged by ultraviolet radiation as described by Hart and Setlow2. PROTEIN DENATURATION AS THE CAUSE OF THERMAL DEATH It is well known that temperature affects biological systems in a very dramatic manner. For example, at temperatures higher than 315°K, most proteins denature with pseudo-first order kinet- ics since the renaturation rate is negligible3. The rate is des- cribed by dn/dt = -an (l), where kD is the first order denaturation constant and is expressed by the absolute rate theory equationh as k T _ .;§_ k K h + + D ehs /R e-AH /RT (2). The transmission coefficient, K, is taken to be equal to unity; and k h, and R are the Boltzmann, Planck, and gas constants, B, 1 2 respectively. The activation enthalpy, AH+, and the activation entropy, AS+, for thermal denaturation of proteins follow a com- 5-9 pensation law , 1.. AS = AH+ + b (3), .1. T c where Tc and b are constants. Furthermore, the thermal death of viruseslo, bacteriall-l3, yeastsl", and mammalian cells in tissue culturelS-lg obey the same compensation law with the same constants: TC = 325°K and b = -6A.5 cal/mol °K. These observations led to the hypothesis that protein denaturation is the rate limiting step in the thermal death of single cells. The life span of multicellular poikilotherms also has a strong temperature dependence. Most of these organisms show senescense, an increased probability of dying with age. The first mathematical expression of survivorship curves of multicellular organisms was reported by Gompertz (1825)2O: at (A). 1: II I Z|l—' Ala ll :11 (D The rate of mortality, u, is expressed by a time dependent function containing two constants R0 and a. The power law has recently been applied to the survivorship of multicellular organisms1 N(t) = - fidgét) = Atn‘l (5). 01‘ N(t) _ -Atn/n NO - e (6), where A and n are constants. 3 The power law has several advantages over the Gompertz law and its modificationsl. Survivorship curves for many multicellu- lar organisms are better fitted by the power law than by the Gom- pertz function. Furthermore, the power law contains only two con- stants. Although the Gompertz function also contains two constants, its modifications by Makeham21 and the addition of Perk's function22, which provide a better fit to the data, contain additional constants. Only one temperature dependent constant is contained in the power law, while both constants are temperature dependent in the Gompertz function. The logarithm of A, the temperature dependent constant of the power law, plotted against the reciprocal temperature yields an Arrhenius plot. The activation enthalpy for death can be obtained from the slope of this Arrhenius plot. The activation enthalpy cannot be obtained from the Gompertz function. Finally, the number of rate limiting steps is n + l, where n is the temperature independent constant of the power law. The number of rate limiting steps cannot be obtained from the Gompertz function. The power law can be used in a thermodynamic analysis of death because of these advantages over the Gompertz function. For example, it can be applied to Drosophila melanogaster. From the survivorship plots of ln ln (NO/N) against 1n t, the constants A and n can be obtained as a function of temperaturel. Over a small temperature range (25°C — 33°C), n is found to vary from 5.2 to 5.8, or n = 5.5 i 6.6% for Drosophilal. Thus n is fairly temperature independent. The number of rate limiting steps, n + 1, for the death of Drosophila is then probably 6 or 7. The values of A vary by four orders of magnitude over the range 25°C - 33°Cl. Finally, the plot of log A versus the reciprocal absolute h temperature yields a good straight linel. This is an Arrhenius plot with the equation, 1.. A = Ace-AH (RT (7). The activation enthalpy for the mortality rate is obtained from the slope of the Arrhenius plot. The activation enthalpy for death in Drosophila melanogaster is 190 kcal/moll. The sum of the activation enthalpies of the rate limiting steps for the mortality rate is equal to the activation enthalpy for death of a multicellular organism. This is a consequence of the sequential chain model for aging from which the power law can be derived23. The pertinent details are shown below. The sequential model for aging consists of an abstract chain containing m identical but independent links23. A link is intact when in state 1,2,...n. A direct transition from state 3 is only permitted to state 3 + l (J = 1,2,...n). The transition probabil- ities, p, are assumed to be equal and independent of time. Let pj(t) be the probability that a given link is in state 3 at time t. The deterioration of the link can be described by the equations 3% p302) = pipHm - pj(t)] (8). where J = 1,2,...n, p0(t) = o for all t (9), pJ(O) = l; J = l = 0; J # l (10). If fJ(s) is the Laplace transform of pj(t), 1 f1(s) = E15 J-l = _E_. = _£L____ fJ(S) s+p fJ-l(s) (8+p)i (12)’ for j = 1,2,...n. Taking the inverse transform of fj(s), p3 = e'pt3‘1/z (19>. Thus, n-l 1n n = n ln p + 1n [mt /(n—l)!] (20). The only temperature dependent quantity on the right hand side of Equation (20) is o. By the theory of rate processes this depen- dence is given as o = Doexp(-Ah+/RT) (21). where Ah+ is the activation enthalpy corresponding to an individual, molecular level state transition, R is the gas constant, and T is the absolute temperature. Equation (20) becomes nAh+ RT lnLl=K- (22): l/(n—1)!]. The quantity nAh+ is the "effective" where K = ln[p:mtn- activation enthalpy determined at the macroscopic level from the experimental data. Comparing Equations (5) and (7) with Equation (22) shows that the sequential chain model is actually a derivation of the power law: .1. n—l e-AH /RT tn—l .1. I u(t) = At = A0 ]e-nAh /RT tn—l n = [p m/(n-l)! o (23). The constant A0 = pgm/(n—l)!, while the activation enthalpy for death equals the sum of the activation enthalpies for the rate limiting .1. steps, or AH' = nAh+. It was already stated that the activation enthalpy for the death of Drosophila melanogaster was 190 kcal/mol and the value of the constant n was 5.5 i 6.6%1. According to the sequential chain model, the activation enthalpy corresponding to an individual state transition, Ahf, would be approximately 3A.5 kcal/mol. The fact that the activation enthalpies of the individual steps sum 7 in this manner is not the consequence of any physical or chemical characteristic of the underlying molecular process; it is due, instead, to statistics which sample the extreme rather than average properties of a system23. Although it was assumed here that the activation enthalpies, Ah+, for the individual steps were equal, they must be in fact at least roughly equal in the sequential chain model. It was shown23 that if the activation enthalpy of one step was significantly greater than the activation enthalpy of the other steps, then the resulting survivor curves would be inconsistent with the experimental data1 for Drosophila. If the hypothesis that protein denaturation causing thermal death in single cells3 is extended to the sequential chain model for multicellular organisms, the death of a multicellular organism should involve n + 1 protein denaturations each with activation enthalpy of about AH+/n, where n and AH+ are obtained from the survivorship curves. For example, the death of Drosophila should involve six or seven protein molecules each having activation enthalpies of about 3A.5 kcal/mol for thermal denaturation. THE CORRELATION BETWEEN THE MAXIMUM LIFE SPAN OF MAMMALS AND REPAIR OF DNA DAMAGED BY ULTRAVIOLET RADIATION2 The accumulation of damage in the DNA of cells increases with the age of the organism from which the cells were taken. Damage in tissues from aging mice was assayed by measuring with autoradiography the ability of DNA from these tissues to act as primers for ig_zi££g. nucleotide incorporation catalyzed by calf thymus polymeraseeh’zs. The result that DNA from old tissues acted as better primers than from young ones indicated that DNA from old cells contains relatively large numbers of strand breaks. 8 Measurements of alkali-labile bonds (single—strand breaks) in the DNA of old and young cells supported this interpretation. Since chick red cells did not repair x-ray breaks in their DNA, the DNA of chick red cells had more breaks than that from lymphocytese6. Further- more, the DNA from old red cells had more breaks than the DNA from young ones. This same study also showed that old rat muscle cells did not repair x-ray breaks as well as young onese6. However, nondividing cells, such as rabbit retinal27 or dog neuronal cells28, were able to repair ionizing radiation breaks as well as fibroblasts. Furthermore, rat retinal ganglion cells treated with the chemical carcinogen A-nitroquinoline l-oxide ig;xi££g_showed no age-associated changes in the levels of DNA repair measured by unscheduled DNA synthesiszg. On the other hand, singly isolated beating heart cells from newborn rats performed unscheduled DNA synthesis after ultraviolet irradiation, while this same repair synthesis could not be induced in beating cardiac cells isolated from adult rats30. Also there was a decrease in DNA repair capacity during successive subcultures of primary abdominal rat fibrOblasts treated with A-nitroquinoline l-oxide3l. Not only did the capacity for excision repair of ultraviolet radiation damage to DNA in primary cultures of mouse embryonic cells decrease with successive transfers ig_zi§§9, but also this repair capacity decreased in cells taken from later stages of development32. Moreover, hepatic cells of old mice were able to rejoin gamma radiation-induced DNA scissions as quickly as those of young mice, but only the radiation- induced DNA breaks, and not the age-associated scissions, were rejoined33. It was also shown that frequencies of mitomycin-C induced sister 9 chromatid exchanges declined with age in mouse and rat bone marrow cells ig_zigg_suggesting an altered response to DNA damage with aging The premature aging syndromes of Hutchinson-Gilford (pro- geria), Werner, and Rothmund Thompson are thought to be repair 35-37 deficient There is evidence of abnormal enzyme structure/ function in cells with these premature aging syndromes which seem 38’39. Although it was to mimic the normal cells when they senesce shown that fibroblasts isolated from patients suffering from progeria were deficient in the ability to repair DNA strand breaks induced by gamma radiationho, repair replication was normal after ultraviolet irradiation in the progerioid cellshl’"2. Although these studies seem to indicate that there is a general decline in DNA repair with the age of the animal, there might not necessarily be an age—related decline in DNA repair of a specific cell type. Furthermore, a correlation was observed between DNA repair and maximum life span of an animal. In one study fibroblasts from seven mammalian species that had completed about one twentieth of their life span were subjected to several fluences of ultraviolet radiation and then allowed to incorporate 3H-Thymidine for various periods of timee. The ability of the cells to perform unscheduled DNA synthesis (a measure of excision repair) was measured by autoradiography. Both the initial rate and the maximum incorporation of 3H—Thymidine increased with the maximum life span of the species (shrew, mouse, rat, golden hamster, cow, elephant, and human). The maximum extent of unscheduled 10 DNA synthesis was approximately proportional to the logarithm of the maximum life span of the species. Investigators who have employed ultraviolet irradiation to human cells in tissue culture agree that, although there is an age related decline in the cells' ability to perform repair syn- thesis, this decline is not the primary cause of cellular aging and eventual deathh3-"6. However, these same studies disagree as to when this failure becomes evident as the cells are progressive- ly subcultured. Some studies have indicated that this failure does not become apparent until very late in the ig_zi§§9_life spanh3, possibly not until the last passagehh. Other studies indicated that human diploid fibroblasts exhibit a reduced capacity to repair damaged DNA relatively early in their life spanh5’"6. Major decreases were observed in DNA repair of WI-38 cells after approximately 60% of their life span had been completedh6. There was also a strong correlation between cells unable to carry out semi-conservative DNA synthesis for division and those unable to perform repair synthesis A more recent study measured unscheduled DNA synthesis after ultra- violet irradiation in both confluent and arrested human diploid 1+7 fibroblasts In this study confluent cultures exhibited identical levels of unscheduled DNA synthesis at all ig_xi§§g ages. Cells arrested by lowering the serum concentration of the incubation medium exhibited similar levels of excision repair as did confluent cells during the initial one—third of the cells' characteristic ig_xi££2_ life span. However, the arrested cells exhibited a 30% to 50% increase in the amount of detectable DNA repair during the final two- thirds of the life span. 11 Certainly there is much more to aging than just the failure of an excision repair system for the removal of ultraviolet radiation- induced pyrimidine dimers. In some systems excision repair of ultra— violet induced dimers has been shown to be fairly independent of the overall aging process. Individuals with xeroderma pigmentosum are A1,A8,A9 O C I O O O O I SO defective in eXCISlon repair , have skin abnormalities , and develop potentially lethal skin cancer if exposed to sunlightsl. Some of these individuals also have ocular, nervous, or endocrine disorders and often their life span is decreasedEO. However they do not seem to age in other ways or have a life span shorter than a mouse, despite observations that cells from these individuals do less excision repair than mouse fibroblasts after ultraviolet irradia- tion2’"l’"8’"9. Also xeroderma pigmentosum fibroblasts do not go h through less passages than normal ones in culture 3’52. On the other hand, fibroblasts from individuals who age prematurely (progeria) do 53,5A not go through as many passages as do normal cells A1,A2 , but show normal levels of excision repair Furthermore, a correlation between DNA excision repair and life span does not necessarily prove that less DNA excision repair causes aging. There are also correlations between life span of mammals and other parameters (brain weight, body weight, and metabolic rate)55. However there is no evidence that any of these parameters determines the life span of a mammal. It is possible that the failure of any cellular repair process does not cause, but rather is a consequence of, the aging of a multicellular organism. Certainly it is very improbable that the amount of excision repair of pyrimidine dimers induced by ultraviolet irradiation is the most important determinant 12 of the maximum life span of a mammal, because there will always be some damage to DNA that will not be repaired via an error—free excision process even in normal cells. It is more likely that the amount of excision repair of ultraviolet radiation-induced pyrimidine dimers is only a correlate of the maximum life span potential of an organism. A multicellular organism might have many processes that are tuned so that all of them fail at the time the organism senesces. Nevertheless, the study of the excision repair of dimers in DNA may be very important in understanding the aging process. First of all, excision repair of pyrimidine dimers is the only molecular repair process discovered to date that has been correlated with the maximum life span of an organismg. This is certainly more significant than the correlations with gross parameters such as brain and body weight. The significance is that the rate of the aging process and the maximum life span of a multicellular organism may be determined by its individual cells, since each cell carries information that enables an investigator to predict the life span of the organism from which it was taken by its extent of excision repair of ultraviolet radiation-induced dimers. Moreover, defects in excision repair may cause aging and death in individual cells. Certainly, unrepaired damage of DNA could be detrimental to the cell2. Furthermore, the excision repair system in cells most likely involves proteins. These repair proteins probably thermally denature, causing less overall repair in the cells. A thermodynamic analysis can be applied to these repair protein denaturations. Finally, the observation that excision repair capacity decreased in mouse embryonic cells taken from later l3 stages of development32 suggests that the amount of repair is 56 influenced by cellular differentiation . THE HYPOTHESIS THAT DENATURATION OF DNA REPAIR PROTEINS IS THE RATE LIMITING STEP IN THE THERMAL DEATH OF SINGLE CELLS The compensation law (3) involving protein denaturation and thermal cell death leads to the hypothesis that protein denatur— ations are the rate limiting steps in the death of single cells3 The observations that both DNA damage and cell death increase with A3-A6 the passage number of the cells in tissue culture or the age of the animal from which the cell was taken2h-"2, and the correla- tion between life span and DNA repair2, lead to the hypothesis that decreased DNA repair causes cell death. This would be a composite decrease of all DNA repair, which may include, but not necessarily be restricted to, the excision repair of ultraviolet radiation- induced pyrimidine dimers. From these two hypotheses it follows that thermal denaturation of repair proteins may be the rate limiting step in the death of single cells. If the hypothesis that repair protein denaturation causing cell death is correct, then repair protein denaturation would be an activated process and a thermodynamic analysis could be applied to it. It would then be expected that a small increase in temperature would cause a large number of repair proteins to denature, resulting in a large decrease in repair. However, there are at least five possibilities for this expected observation with a small increase in temperature: (1) repair proteins may denature and repair ineffectively if not at all, (2) the manufacturing rate of the repair proteins may decrease, (3) repair proteins may convert into damaging proteins, (A) processes which damage may have their rates increased more than 1A the rate of repair, and (5) proteins in close proximity to the repair site may denature and interfere with the repair process. It will be shown in Chapter v (p. 158) that only the first, (1), and the last, (5), of these possibilities are consistent with the results of the experiments that were conducted in the present investigation. There may be many causes of the thermal death of single cells other than repair protein denaturation. For example, the denaturation of a lysosomal protein might cause a leak in a lysosome, allowing a toxic substance, contained in the lysosome, to escape and kill the cell. Even if repair protein denaturation is not the sole cause of cell cleath, or even the most important cause, the study of repair protein denaturation is important in understanding cellular aging and death. Assuming that there were other causes of cell death and these causes could somehow be eliminated, it would be unlikely that repair protein denaturation could be prevented. Eventually cell damage would accumulate, leading to cell malfunction, and would ultimately result in cell death. Furthermore the mechanism of repair protein denaturation leading to cell death may be similar to other causes of cell death. EXPERIMENTS THAT WERE CONDUCTED IN ORDER TO SUPPORT THE HYPOTHESIS THAT DENATURATION OF DNA REPAIR PROTEINS IS A RATE LIMITING STEP IN THE THERMAL DEATH OF SINGLE CELLS There are many repair mechanisms within a cell. For example, DNA can be damaged by ultraviolet irradiation, x-rays, gamma rays, and chemicals, and can be repaired by different mechanisms. It is not certain if any of these mechanisms are important for determining the life span of an organism. 15 However the measurement of repair of damage caused by ultra— violet radiation has several advantages over the measurement of repair caused by other damages. First of all the effects of ultra- violet irradiation on DNA can be well quantitated. Furthermore, the excision repair of DNA damaged by ultraviolet radiation has 57-61 been analyzed by a number of different techniques , including the actual removal of cyclobutyl pyrimidine dimers (excision), repair replication (the incorporation of radioactive label into parental DNA during repair), unscheduled DNA synthesis (the syn- thesis of DNA during non-S periods of the cell), the photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and endonuclease site specificity (the measurement of single strand breaks in the DNA at dimer sites). Finally, only repair of DNA damaged by ultraviolet irradiation has been correlated with the life span of an organism2. The experiments that were conducted measured DNA repair of damage caused by ultraviolet irradiation by the techniques of un- scheduled DNA synthesis, autoradiography, and endonuclease site specificity. In the experiments utilizing unscheduled DNA synthe- sis, confluent mammalian fibroblasts were incubated for three days at conditions unfavorable for semi-conservative DNA replication. The cells were then exposed to ultraviolet radiation and then allowed to incorporate 3H-Thymidine. The cells were incubated at different temperatures for various periods immediately before ultra- violet irradiation. Excision repair or unscheduled DNA synthesis was quantitated by the amount of 3H-Thymidine incorporated during l6 repair. Less 3H-Thymidine incorporation was observed with increased incubation temperature before ultraviolet irradiation. The experiments involving autoradiography were identical to the unscheduled DNA synthesis experiments, except that excision repair was quantitated by tracks on a photographic emulsion. This experi- ment was performed to show that the decreased excision repair with increased pre-UV incubation temperatures that was observed in the unscheduled DNA synthesis experiments was due to decreased excision repair in all the cells rather than to the thermal death of a large portion of the cells. The experiments involving endonuclease site specificity were identical with the other two experiments except that the cells were radioactively labeled before confluence, instead of during repair. After repair, the DNA was extracted from the cells and treated with an endonuclease from M. luteus. The endonuclease makes single strand breaks in the DNA at the ultraviolet induced dimer sites. Then by ultracentrifuging the DNA on sucrose gradients, the number of dimers could be quantitated. This experiment was done to show that increased incubation temperature before ultraviolet irradiation thermally impaired the endogenous excision repair mechanism, rather than hinder the transport and phosphorylation of exogenous 3H-Thymidine into 3H—dTTP in the DNA inside the nucleus of the cells. CHAPTER II THE EFFECT OF TEMPERATURE ON THE REPAIR OF ULTRAVIOLET INDUCED PYRIMIDINE DIMERS IN THE DNA OF MAMMALIAN CELLS AS MEASURED BY UNSCHEDULED DNA SYNTHESIS INTRODUCTION "Unscheduled DNA synthesis" induced by ultraviolet radiation was first demonstrated in HeLa cells62. The enzymatic "excision repair" of ultraviolet radiation induced pyrimidine dimers in DNA was later shown in HeLa cells63. Actual "repair replication" or "non-semiconservative DNA synthesis" had been reported in HeLa cells 6A,65 66,67 after treatment with ultraviolet radiation , x-rays or alkylating agents68. "Unscheduled DNA synthesis" has been correlated with "repair replication" in several mammalian cell lines after 69 .70 ultraviolet irradiation Repaired DNA following ultraviolet 65,71 irradiation can serve as a template for normal DNA synthesis Also in mammalian cell DNA, pyrimidine dimers and N-acetoxy-2- acetylaminofluorine lesions are substrates for excision repair 63.72-75 enzymes "76-79 Hydroxyurea can inhibit "semi-conservative DNA synthesis "63 without affecting "excision repair or "repair replication"80. It has commonly been used to reduce the amount of normal "semi-conservative DNA synthesis" in order to detect the otherwise masked amounts of "unscheduled DNA synthesis" caused by damaging DNA with various —87 agents81 . Similarly, cells maintained in a medium deficient in 17 l8 arginine also decreased the amount of "semi-conservative DNA synthesis"88 enabling measurement of "unscheduled DNA synthesis"89. Hydroxyurea allows the detection of 5-fold to 9-fold increases in 3H-Thymidine incorporation ("unscheduled DNA synthesis") in the essentially non-dividing human lymphocyte system following treatment with chemical carcinogen582. It is less effective in normally 90 rapidly dividing cells Because "repair replication" appears to occur in the absence of protein synthesis in both HeLa and Chinese hamster cellsgl, a sensitive assay was developed for quantitating "unscheduled DNA synthesis" in rapidly dividing eukaryotic cells by combining the two methods of inhibition of "semi-conservative DNA 92 synthesis" When transformed human amnion (AV3) cells were main- tained in a culture medium containing hydroxyurea and deficient in arginine, a lO-fold to 20-fold increase in "unscheduled DNA synthesis" was measured following treatment with ultraviolet radiation of N-acetoxy-2-acetylaminofluorine92. This technique would enable the detection of different "DNA excision repair synthesis" after ultra- violet irradiation with small differences in the temperature of incubation before ultraviolet irradiation. MATERIALS AND METHODS £9113. Chinese hamster lung fibrOblasts (V-79) and normal human skin fibroblasts (736 NF) were used in the experiments. Both cell cultures were obtained from J. E. Trosko, Human Development Department, Michigan State University, East Lansing, MI., and were grown under humidified 5% co2 in air. 19 Permanent stock cultures of the Chinese hamster cells utilized a "C-15" medium (see Appendix A) supplemented with 5% fetal calf serum. Permanent cultures of human fibroblasts utilized a "D" medium (see Appendix A) supplemented with 10% fetal calf serum. Growing cell cultures for experiments utilized "D" medium supple- mented with 5% fetal calf serum for V-79 cells and 10% fetal calf serum for human fibroblasts. The confluent cells utilized for experiments were maintained in "D" medium without arginine or isoleucine (see Appendix A) and supplemented with 5% dialysed fetal calf serum for V-79 cells and 10% dialysed fetal calf serum for human fibroblasts. All media were supplemented with penicillin (100 units/ml), streptomycin (100 ug/ml), and mycostatin (100 units /ml) . Experimental Culture Procedure (Figure l) Cells were inoculated into either 60 mm plastic Petri dishes or in 25 cm2 plastic flasks (Falcon Co., Oxnard, Calif. or Corning Glass Works, Corning, NY) and allowed to grow in "D" medium to heavy confluent densities. This took between 1 to A days for V-79 cells and between 1 to 2 days for human fibroblasts. At confluence, the "D" medium was replaced with a "D" medium deficient in arginine and isoleucine supplemented with dialysed fetal calf serum (5% for V-79 cells, 10% for human fibroblasts). After 2A hours, this medium was decanted and replaced with fresh "D" medium deficient in arginine and isoleucine, supplemented with the same concentration of dialysed fetal calf serum, and incubated for A8 hours. At this point, hydroxyurea was added to a final concentration of 5 mM, If used, 2O oczuoooea 95:30 3:85:33 ._ 839m 2520: my 352. m: £222.71» >2 1 cu + * non .2.u + o: I DOWNIOV IIJ, DLOI AWOL..- 345 my 5338 5:62: 3.5 2.2ng V 3:223 196.23 00:03:50 5:232: IIWI Illllllll..+..l III nlllllw 00mm _ a .1, Ooh” ill rl—llllllllll_.lll.|AI+I.A'UoFm _ e e rhlllllLllllL 9.? 8—5. 950... '— ITmSo; km 2 N .l _ 23; N» LII mm 2 vm m2; BHOLVHBdWSL 21 cycloheximide”wasadded at this point to a final concentration of 5 ug/ml. Ultraviolet irradiation of the cells was done after one hour of incubation in the presence of hydroxyurea. Cells were incubated at various temperatures ranging from 33°C to A3°C for various spans of time up to 5 days immediately prior to ultraviolet irradiation,1flnxnmfllsome or no portion of the growing period in "D" medium, and through some or no portion of the confluent period in "D" medium deficient in arginine and isoleucine. For the remainder of the time, the cells were incubated at 37°C. Ultraviolet Irradiation and Repair The medium was decanted from the flasks or Petri dishes and saved just prior to ultraviolet irradiation. The tops were removed from the flasks or dishes and the cells were exposed to 25A nm ultraviolet radiation, delivered from one germicidal lamp (General Electric, G15 T8) mounted in a sterile transfer hood. The incident dose rate, measured with a short wave Blak-Ray meter (Ultraviolet Products, San Gabriel, Calif.) was approximately 1.0 W/m2. Since the cells were exposed to the ultraviolet radiation for 15 seconds, the ultra- violet dose was 15 J/m2. While one of the plates was being irradiated, the remainder of the plates were kept on ice (A°C). [Me-3H]-Thymidine (3H—TdR; New England Nuclear, Boston, Mass.; A0 Ci/mmol) was added to the spent, decanted medium at 5 uCi/ml medium. This radioactive medium was then added to the cells immediately after ultraviolet irradiation. The cells were allowed to incorporate 3H-Thymidine from this medium for at least 2 hours at 37°C. In one experiment cells were incubated in this medium for 2 hours at Al°C. 22 For long periods of 3H-Thymidine incorporation, hydroxyurea was added every 2A hours to a concentration of 5 mM, After this post-treatment incubation period, the medium was decanted and the cells were washed twice with cold phosphate buffered saline. Their detachment from the plates was facilitated by a rubber policeman in phosphate buffered saline. The detached cells were collected by centrifugation with the phosphate buffered saline decanted. Analysis of "Unscheduled DNA Synthesis" The pellets were usually frozen at -20°C. After thawing, the pellets were resuspended in cold 10% trichloroacetic acid (TCA). After centrifugation, the tubes were inverted to drain off the TCA. At this point the pellets were dissolved in 0.3 M_KOH and incubated for 1 hour at 37°C to hydrolyse the RNA93. After incubation, the KOH solution was cooled and a solution was added containing enough HCl to neutralize the KOH, with TCA to be at a final concentration of 5%. Following centrifugation, the tubes were inverted to drain. The pellets were next resuspended to cold 5% TCA. The tubes were then centrifuged and inverted to drain. The tubes were again resuspended in 5% TCA and incubated for 20 minutes at 90°C to extract the DNAgh’QS. DNA was quantitated in a diphenylamine reagentg6 containing 2 g diphenylamine, 5.9 ml 61% perchloric acid, and 0.5 ml acetaldehyde (16 mg/ml) in 100 ml acetic acid. This solution was incubated for 18 hours at room temperature and compared to a highly polymerized DNA standard (Sigma Chemical Co., St. Louis, Mo.) with a Gilford spectro- meter. Radioactivity in the DNA extracts was measured in a scintil- lation fluid containing 21 g PPO, 1.1A g dimethyl POPOP, and 23 150 g naphthalene per 3 liters of p-dioxane. A Packard Tri-Carb liquid scintillation spectrometer (Model 3320) was used with discriminators set between 50 and 1000 divisions and gain at 52.0%. The counting efficiency for tritium was 51.5% measured with an internal standard. RESULTS 1. The effect of 2 hours of different pre-UV incubation temperatures on "unscheduled DNA synthesis" in V-79 cells. Investigations were conducted on Chinese hamster lung fibro- blasts (V-79) to determine if 2 hours of pre-ultraviolet (UV) hy- perthermia and hypothermia affected subsequent "unscheduled DNA synthesis". The V—79 cells were inoculated into 25 cm2 plastic flasks and were allowed to grow to confluence for 1 day at 37°C. After a 3 day incubation period at 37°C in a medium deficient in arginine and isoleucine, hydroxyurea was added to each flask to a concentration of 5 mM, The flasks weretightly sealed and were exposed for 2 hours to various temperatures before being ultraviolet irradiated at 15 J/m2. Water baths set at 37°C, AO°C, and A3°C each contained A flasks, while 3 flasks were placed on ice (A°C). After the 2 hour temperature treatment, 2 flasks from each of the thermal treatment groups were UV irradiated, while the remaining flasks were not subjected to ultraviolet irradiation. After this point, all of the flasks were allowed to incorporate 3H-Thymidine for 2 hours at 37°C. The specific activity indicating the amount of "unscheduled DNA synthesis" and the amount of DNA on each plate are given in Table 1. Only 0 to 32 ug of DNA were isolated in flasks that were 2A Table l. The effect of 2 hours of various temperature treatments before ultraviolet irradiation on 3H-Thymidine incorporation into the DNA of Chinese hamster lung fibroblasts MEASUREMENT UV DOSE ’TEMPERATURE BEFORE INCORPORATION A3°C A0°c 37°C h°c Weight of DNA isolated 15 J/m2 32 202 212 158 (pg/flask) 0 172 215 105 none 10 175 198 215 O 200 232 Specific activity 15 J/m2 3 170 168 12A (dpm/ug DNA) - 1A9 176 99 none 10 68 A8 56 - 56 68 Average specific activity difference — 98 11A 56 Average specific activity 0.3 2.6 3.0 2.0 ratio 25 incubated at A3°C for 2 hours, while the other flasks contained roughly 200 pg of DNA, ranging from 105 to 232 ug DNA. Cells that were treated for 2 hours at A°C, 37°C, and AO°C, but were not subjected to ultraviolet irradiation incorporated 3H-Thymidine for 2 hours at a specific activity of roughly 60 dpm/ug DNA, ranging from A8 to 68 dpm/ug DNA. The difference between the average specific activity of cells that were ultraviolet irradiated and the average background specific activity of control cells not exposed to ultraviolet irradiation yields the specific activity attributed only to excision repair of ultraviolet induced pyrimidine dimers. Fcr cells treated in AO°C water bath, 98 dpm/ug DNA of H—Thymidine was incorporated for excision repair and the ultraviolet irradiated cells had 2.6 times more 3H—Thymidine incorporation than non-irradiated cells. For cells treated for 2 hours in a 37°C water bath, 11A dpm/ug DNA of 3H—Thymidine was incorporated for excision repair and the irradiated cells had 3.0 times more 3H—Thymidine incorporated than non-irradiated cells. For cells kept 2 hours on ice, 56 dpm/ug DNA of 3H—Thymidine was incorporated for excision repair and the irradiated cells had twice as much 3H—Thymidine incorporation as non-irradiated cells. The results do not show a significant effect on the amount of post-UV "unscheduled DNA synthesis" caused by a 2 hour period of pre- UV hyperthermia. The 2 hour pre—UV treatment on ice appears to reduce the amount of "unscheduled DNA synthesis". Pre-UV hyperthermia at A3°C for 2 hours appears to cause significant cell detachment. 26 2. The effect of 8 and 21 hours of Al°C pre-UV incubation on "un- scheduled DNA synthesis" in V-79 cells. Since no effect on "unscheduled DNA synthesis" was observed with a 2 hour treatment at AO°C before UV irradiation, this period of hyperthermia was extended, with the temperature raised to Al°C, to determine if this harsher treatment would have an effect. Chinese hamster lung fibroblasts (V-79) were inoculated into 60 mm plasth: Petri dishes and were allowed to grow to confluence for 3 to A days at 37°C. The dishes were then incubated for 3 days in a medium deficient in arginine and isoleucine. Some of the dishes were incubated at Al°C for the final 8 hours of this 3 day period, while others were incubated at Al°C for the final 21 hours of this 3 day period. During the remainder of the 3 day period, the dishes were incubated at 37°C. Some of the other dishes were incubated only at 37°C throughout the entire 3 day period. After the 3 day period, hydroxyurea was added to a concentration of 5 mM and all the dishes were incubated for 1 hour at 37°C. At this point, some of the cells were exposed to 15 J/m2 of ultraviolet irradiation. Finally all of the cells were allowed to incorporate 3H—Thymidine for 2 hours at 37°C. The average amount of DNA contained in the dishes was 280 ug.. There was no significant difference in the amount of DNA isolated from dishes receiving different treatments. Figure 2 shows the amount of 3H-Thymidine incorporation for cells that were allowed to grow for 3 days before being maintained in a medium deficient in arginine and isoleucine. For cells that were incubated at 37°C throughout the entire 3 day period in deficient medium, an average of 85 dpm/ug DNA of 3H—Thymidine was incorporated for ultraviolet irradiated cells Figure 2. The amount of 3H-Thymidine (5 uCi/ml medium) incorporated into the DNA of V-79 cells for 2 hours at 37°C as a func- tion of the previous duration of incubation at Al°C. 3H—Thymidine incorporation followed the Al°C incubation period, hydroxyurea (5 mM) treatment for 1 hour, and 25A nm ultraviolet irradiation at 15 J/m2 (hollow circles) or non—irradiation (solid circles) 5;; 29 and A0 dpm/ug DNA for non-irradiated cells. This means that 2.1 times as much 3H—Thymidine was incorporated into the DNA of irradiated cells than was incorporated into the DNA of non-irradiated cells and A5 dpm/ug DNA of 3H-Thymidine was incorporated in the excision repair of ultra- violet radiation induced dimers. For cells that were incubated at Al°C for the final 8 hours in deficient medium before hydroxyurea treatment, an average of 10A dpm/ug DNA of 3 H-Thymidine incorporation was observed in UV irradiated cells, while an average of 38 dpm/ug DNA was observed in non-irradiated cells. Thus, irradiated cells incorporated 2.7 times as much 3H-Thymidine as non-irradiated ones and 66 dpm/ug DNA was incor- porated during excision repair. For cells incubated at Al°C for 21 hours before hydroxyurea treatment, an average of 72 dpm/ug DNA— t 2 F. 0 < 0 E 50 ES LIJ O. a) O 31 J 1 O 8 2| DURATION OF INCUBATION AT 4I°C (hours) Figure 3. 32 treatment, an average of 66 dpm/ug DNA of 3H-Thymidine was incor- porated in irradiated cells and an average of 16 dpm/ug DNA in non- irradiated cells. Irradiated cells incorporated A.1 times as much 3H—Thymidine as non-irradiated cells and 50 dpm/ug DNA was used in excision repair. The results show that a 21 hour period at A1°C before UV ir- radiation appears to reduce the amount of "unscheduled DNA synthesis" after UV irradiation. However there appears to be some variation in the amount of "unscheduled DNA synthesis" between the two experiments depicted in Figures 2 and 3 for cells that were incubated at A1°C for O and 8 hours before hydroxyurea treatment. This variation might be dependent on when the Petri dishes were initially seeded and growth time before being maintained in deficient medium. 3. The effect of 3 days of A1°C pre—UV incubation on "unscheduled DNA synthesis" in V—79 cells. The period of pre-UV hyperthermia was extended to 3 days to determine if this would cause any further reduction in "unscheduled DNA synthesis" after UV irradiation. Furthermore, some of the cells were exposed to a higher temperature during the period of pre—UV treatment with hydroxyurea, while other cells were, in addition, incubated at the raised temperature during 3H—Thymidine incorporation to determine if these treatments affected the amounts of "unscheduled DNA synthesis". Chinese hamster lung fibroblasts were inoculated into 60 mm plastic Petri dishes and were grown to confluence at 37°C for 2 to 3 days. At confluence, they were maintained in a medium deficient in arginine and isoleucine for 3 days either at 37°C or at A1°C. At this point hydroxyurea was added and the cells were incubated for 33 1. hour at either 37°C or A1°C. After some of the plates were ultra- violet irradiated, all of the cells were allowed to incorporate 3H-Thymidine for 2 hours at 37°C or at A1°C. The amount of "unscheduled DNA synthesis" in cells which took 3 days to reach confluence is shown in Figure A. All of these plates were incubated with hydroxyurea at 37°C for 1 hour and allowed to incorporate 3H-Thymidine for 2 hours at 37°C. The amount of DNA isolated ranged from 176 pg to 188 pg, averaging 185 pg, from cells that were always incubated at 37°C. Cells treated with ultraviolet 3H-Thymidine, radiation incorporated, on the average, 19A dpm/pg DNA of Iwhile non-irradiated cells incorporated A0 dpm/pg DNA. Irradiated cells incorporated A.9 times as much 3H-Thymidine as non-irradiated cells and 15A dpm/pg DNA was used in excision repair. The amount of DNA isolated from cells incubated at A1°C for _3 days in deficient medium ranged from 116 pg to 151 pg, averaging 13A pg. On the average, irradiated cells incorporated 95 dpm/pg DNA, while non-irradiated cells incorporated 16 dpm/pg DNA. Irradiated cells incorporated 6.0 times as much 3H-Thymidine as non-irradiated cells and 79 dpm/Ug DNA was used in excision repair. Figure 5 shows the amount of "unscheduled DNA synthesis" in cells which took 2 days to reach confluence. For cells which were incubated in deficient medium for 3 days ("pre-HU") at 37°C, incubated with hydroxyurea ("HU") at 37°C for 1 hour, and allowed to incorporate 3H-Thymidine ("repaired") at 37°C for 2 hours, 19A to 251 pg of DNA was isolated per plate, averaging 236 pg. Irradiated cells averaged 107 dpm/pg DNA of 3H-Thymidine incorporation, while non-irradiated cells incorporated 2A dpm/pg DNA. Irradiated cells incorporated Figure A. 3A The amount of 3H-Thymidine (5 pCi/ml medium) incorporated into the DNA of V-79 cells for 2 hours at 37°C as a func- tion of the previous duration of incubation at A1°C. 3H-Thymidine incorporation followed the A1°C incubation period, hydroxyurea (5 mM) treatment for 1 hour, and 25A nm ultraviolet irradiation at 15 J/m2 (hollow circles) or non-irradiation (solid circles) Figure 5. 36 The amount of 3H-Thymidine (5 pCi/ml medium) incorporated into the DNA of v—79 cells for 2 hours at 37°C following 25A nm ultraviolet irradiation at 15 J/m2 (hollow circles) or non-irradiation (solid circles) as a function of the temperatures of (a) the 72 hour incubation in deficient medium before hydroxyurea was added ("pre-HU"), (b) the 1 hour incubation with hydroxyurea (5 mM) before 3H—Thymidine incorporation ("HU"), and (c) the 2 hour 3H-Thymidine incorporation ("repair") 37 I I l I 2 8 Z 0 6‘ 0100 - \ 3 o \ ° 5. \ ,° '0 V \ /o > \ 8 / t \ / > ,t / 2 a \ Z / o 50- \ / E V 0 m o. a) . K\ o \ o I, \‘o—___._./// O O o J I I l "pre-HU" 37°C 4I°c 4|°C 4I°c "I-Iu" 37°C 37°C 4|°C 4I°C "repair" 37°C 37°C 37°C 4I°c TEMPERATURES Figure 5. 38 A.5 times more than non—irradiated ones and 83 dpm/pg DNA was due to excision repair. For cells "pre-HU" incubated at A1°C, "HU" incubated at 37°C, and "repaired" at 37°C, DNA was isolated at 111 to 173 Mg per plate, averaging 153 pg. Irradiated cells averaged 62 dpm/pg DNA incor- poration, while non-irradiated cells incorporated 12 dpm/pg DNA. Irradiated cells incorporated 5.2 times more than non-irradiated ones and 50 dpm/pg DNA was due to excision repair. For cells "pre-HU" incubated at A1°C, "HU" incubated at A1°C, and "repaired" at 37°C, DNA was isolated at 13A to 189 pg per plate, averaging 162 pg. Irradiated cells averaged A3 dpm/pg DNA incor- poration, while non—irradiated cells averaged 11 dpm/pg DNA. Ir— radiated cells incorporated 3.9 times more than non—irradiated ones and 32 dpm/pg DNA was due to excision repair. For cells "pre-HU" incubated at A1°C, "HU" incubated at A1°C, and "repaired" at A1°C, DNA was isolated at 90 to 129 pg per plate, averaging 10A pg. Irradiated cells averaged 88 dpm/pg DNA incor— poration, while non-irradiated ones averaged 18 dpm/pg DNA. Ir- radiated cells incorporated A.9 times more than non—irradiated ones and 70 dpm/pg DNA was due to excision repair. Therefore a 3 day period at A1°C before UV irradiation sig- nificantly reduced the amount of "unscheduled DNA synthesis". Also more 3H-Thymidine was incorporated in a 2 hour post—UV period at Al°C than at 37°C when the cells were incubated at A1°C for 3 days before ultraviolet irradiation. The amount of "unscheduled DNA synthesis" either decreased or remained at approximately the same level when the temperature of the pre—UV incubation with 39 hydroxyurea was raised from 37°C to A1°C. Non-irradiated controls measuring residual "semi-conservative DNA synthesis" did not demon— strate any significant change in specific activity with the temperature raised in the one hour pre—UV incubation with hydroxyurea. This indicates that the hyperthermia did not break down a sufficient amount of hydroxyurea that would result in an increase in "semi-conservative DNA synthesis". A. The effect of cycloheximide and 3 days of A1°C pre-UV incubation on "unscheduled DNA synthesis" in V-79 cells. Cycloheximide, an inhibitor of protein synthesis, was added to V-79 cells one hour before UV irradiation to determine if this treat— ment would further reduce the amount of "unscheduled DNA synthesis". This would be expected if the cells that did not receive cycloheximide were synthesizing additional DNA excision repair proteins during UV irradiation and 3H-Thymidine incorporation. Chinese hamster lung fibroblasts were allowed to grow to con- fluence at 37°C for 2 days. The cells were incubated at 37°C or at Al°C for 3 days in a medium deficient in arginine and isoleucine. At this point hydroxyurea was added to each of the dishes at a concen- tration of 5 mM and cycloheximide was added to some of the dishes at a concentration of 5 pg/ml medium. The cells were incubated for one hour at 37°C. After some of the cells were UV irradiated at 15 J/m2, all of the cells were allowed to incorporate 3H-Thymidine for 2 hours at 37°C in the spent medium containing hydroxyurea and sometimes cycloheximide. The results are shown in Figure 6. Cells incubated for 3 days in deficient medium at A1°C and then treated with cycloheximide contained 50 to 8A pg DNA per plate, Figure 6. "A0 The amount of 3H-Thymidine (5 pCi/ml medium) incorporated into the DNA of V-79 cells for 2 hours at 37°C as a func- tion of previous durations of incubation at A1°C. 3H-Thymidine incorporation followed the A1°C incubation period, treatment for 1 hour with hydroxyurea (5 mM) and with (hollow symbols) or without (solid symbols) cyclo- heximide (5 pg/ml), and 25A nm ultraviolet irradiation at 15 J/m2 (squares) or non-irradiation (circles) Al n Inga-u oo\ \ \\ \ O 30- 0 l0- quo 9:22: t._>:o< gunman 72 DURATION OF INCUBATION AT 4I°C(hours) Figure 6. ,0 A2 averaging 65 pg. Irradiated cell 3H-Thymidine incorporation averaged 18.5 dpm/pg DNA, while non-irradiated cells averaged 8.A dpm/pg DNA. Irradiated cells incorporated 2.2 times more than non-irradiated cells and 10.1 dpm/pg DNA was due to excision repair. Cells incubated for 3 days in deficient medium at A1°C, but not treated with cycloheximide, contained 50 to 90 pg DNA per plate, averaging 72 pg. Irradiated cells averaged 17.2 dpm/pg DNA incor- poration, while non-irradiated cells averaged 7.0 dpm/pg DNA. Irradiated cells incorporated 2.5 times more than non-irradiated cells and 10.2 dpm/pg DNA was due to excision repair. Cells that were always incubated at 37°C and treated with cycloheximide contained 66 to 91 pg DNA per plate, averaging 71 pg. Irradiated cells averaged 3A.8 dpm/pg DNA incorporation, while non- irradiated cells averaged 19.8 dpm/pg DNA. Irradiated cells incorporated 1.8 times more than non-irradiated cells and 15.0 dpm/pg DNA was due to excision repair. Cells always incubated at 37°C and not treated with cycloheximide contained 70 to 91 pg DNA per plate, averaging 8A pg. Irradiated cells averaged AA.2 dpm/pg DNA incorporation, while non-irradiated cells averaged 19.A dpm/pg DNA. Irradiated cells incorporated 2.3 times more than non-irradiated ones, and 2A.8 dpm/pg DNA was due to excision repair. The results show that cycloheximide did not alter the amount of "unscheduled DNA synthesis" in cells pre-UV incubated for 3 days at either 37°C or at A1°C. A3 5. The effect of incubation at A1°C for various spans of time before ultraviolet irradiation on "unscheduled DNA synthesis" in V-79 cells. An experiment was conducted to determine if the amount of "unscheduled DNA synthesis" decreased regularly with increasing time of pre—UV hyperthermia. Chinese hamster lung fibroblasts were inoculated into 60 mm plastic Petri dishes and were grown to confluence at 37°C. At confluence the cells were maintained for 72 hours in a medium deficient in arginine and isoleucine. The dishes were incubated at 37°C for the first 0, 12, 2A, 36, A8, 60, and 72 hours of this 72 hour period. The cells were then incubated at A1°C for the remain- ing time of the 72 hour period. After this 3 day period, the cells were treated with hydroxyurea and incubated at 37°C for 1 hour. At this point some of the plates were exposed to 15 J/m2 of ultraviolet irradiation and all of the cells were allowed to incorporate 3H-Thymidine at 37°C for 2 hours. The results are shown in Figure 7. As shown in the top of the Figure, the DNA isolated was less than 100 pg per plate and usually greater than 50 pg per plate. There is a slight tendency for the amount of DNA isolated to decrease with greater amounts of time the cells were incubated at A1°C. There does not appear to be a pattern with the amount of DNA isolated and whether or not the cells were exposed to ultraviolet irradiation. There also is a slight tendency for less 3H—Thymidine incor- poration in non-irradiated cells with greater periods of incubation at A1°C. The amount of incorporation decreased from an average of 5A dpm/pg DNA for non—irradiated cells incubated always at 37°C to Figure 7. AA The amount of DNA isolated (top) and 3H-Thymidine (5 pCi/ml medium) incorporated into the DNA (bottom) of V-79 cells for 2 hours at 37°C as a function of the previous duration of incubation at A1°C. 3H-Thymidine incorporation followed the A1°C incubation period, hydroxyurea (5 mM) treatment for 1 hour, and 25A nm ultra- violet irradiation at 15 J/m2 (hollow circles) or non- irradiation (solid circles) A5 T IOO -. . - o 0 /° ‘~ __ “.3 so _ C9 ‘ \_ , $\\-\-,‘&-_$_ 00 *‘W E4o~ o ° ' - i=3: . ' 5020- - 352 I I I I I I I O I I I l I I O A I— _. < I50 \ Z \ O \O O 3‘ \\ 0 O \ v IOO - °\ - O >- \ F \ S r: \K 0 /\ < \\9’/ \ ° 0 50 -x\ 0 \ - E ‘K 0 8 8 a_ C U) \I\‘ o O '/ \.~-\_-_._/ . . O I I I I I I 0 I2 24 36 48 60 72 DURATION OF INCUBATION AT 4|°C (hours) Figure 7. .3 A6 an average of 25 dpm/pg DNA for cells incubated at A1°C for 3 days. Cells that were exposed to ultraviolet radiation showed a much greater decrease in 3H-Thymidine incorporation with greater periods of incubation at A1°C. Incorporation decreased from 162 dpm/pg DNA for irradiated cells always incubated at 37°C to 50 dpm/pg DNA for irradiated cells that were previously incubated at A1°C for 3 days. For cells never incubated at A1°C, irradiated cells incorporated 3.0 times more 3H—Thymidine than non—irradiated cells, while for cells incubated at A1°C for 3 days, irradiated cells incor- porated 2.0 times more than non-irradiated cells. Figure 8 shows the amount of 3H-Thymidine incorporated in excision repair as a function of the time exposed at A1°C immediately prior to UV irradiation. Each point represents the difference between the specific activity of the average amount of 3H-Thymidine incorporated in ultraviolet irradiated cells and the average specific activity of the non—irradiated cells, resulting in the net specific activity attributed only to the excision repair of ultraviolet induced dimers. The results indicate that the net specific activity decreases from a value of 108 dpm/pg DNA for cells only incubated at 37°C to a value of 25 dpm/pg DNA for cells incubated at A1°C for three days immediately prior to ultraviolet irradiation. 6. The effect of incubation at 33°C for various spans of time before ultraviolet irradiation on 3H-Thymidine incorporation in V—79 cells. Since pre-UV hyperthermia appears to decrease the amount of subsequent 3H-Thymidine incorporation, experiments were conducted to Figure 8. A7 The net amount of 3H—Thymidine (5 pCi/ml medium) incor— porated into the DNA of V-79 cells for 2 hours at 37°C, which was used for the excision repair of ultraviolet radiation-induced dimers, as a function of previous dura- tions of incubation at A1°C. Each point is derived from the difference of the averaged values of points in Figure 7 between those representing ultraviolet irradiated cells and non-irradiated cells A8 I I I l I I I I40 " m " I20 " - < E o, ‘K 1 \ \ IOO - \ _ \ E \ a. . \ O 3 \\ >_ 80 - \ _ t \\ > I: \\ o .. _ <1 6° \ £2 \\ . E: 0 \ o UJ 40P- . \\ -( a. (D \ P \ m \o 2 zo~ _ o I I J J I I I 0 I2 24 36 48 60 72 DURATION OF INCUBATION AT 4|°C (hours) Figure 8. A9 determine if pre-UV hypothermia also affects the amount of 3H-Thymidine incorporation. Chinese hamster lung fibroblasts were inoculated into 60 mm plastic Petri dishes and were allowed to grow to confluence for 2A hours at 33°C and 37°C. At confluence the cells were maintained for 72 hours in a medium deficient in arginine and isoleucine. The cells which were grown at 33°C were incubated at 33°C throughout the entire 72 hour period. The cells which were grown at 37°C were incubated at 37°C for the first 0, 2A, A8, and 72 hours and incubated at 33°C for the remaining time of the 72 hour period in the deficient medium. In the final 1 hour of the 72 hour period, the cells were incubated with hydroxyurea at the same temperature at which they were incubated for the past 23 hours. All of the cells were then exposed to 15 J/m2 of ultraviolet radiation and then allowed to incorporate 3H-Thymidine for 2 hours at 37°C. The results for one of these experiments are shown in Figure 9. The amount of DNA isolated in this experiment averaged A03 pg, ranging from 3A7 to AA7 pg DNA per plate. The amount of 3H-Thymidine incor- poration was 22.1 dpm/pg DNA for cells incubated at 37°C for A days and was 7.2 dpm/pg DNA for cells incubated at 33°C for A days before ultra- violet irradiation. The results for a repeated experiment are shown in Figure 10. The amount of DNA isolated in this experiment averaged 360 pg per plate, ranging from 265 pg to A51 pg per plate. The amount of 3H-Thymidine incorporation decreased from a value of Al.0 dpm/pg DNA for cells incubated at 37°C for A days before ultraviolet Figure 9. 50 The amount of 3H-Thymidine (5 pCi/ml medium) incorporated into the DNA of V-79 cells for 2 hours at 37°C as a func- ion of previous durations of incubation at 33°C. H-Thymidine incorporation followed the 33°C incubation period, hydroxyurea (5 mM) treatment for 1 hour, and 25A nm ultraviolet irradiation at 15 J/m2 51 20- \ ._ .5- '\ / \ - SPECIFIC ACTIVITY ( dpm / pg DNA ) / \ \ / O I I I I I O 24 48 72 96 DURATION OF INCUBATION AT 33°C (hours) Figure 9. .3 "h: Figure 10. The amount of 3H—Thymidine into the DNA of V—79 cells tion of previous durations 3H—Thymidine incorporation period, hydroxyurea (5 mM) nm ultraviolet irradiation 52 (5 pCi/ml medium) incorporated for 2 hours at 37°C as a func- of incubation at 33°C. followed the 33°C incubation treatment for 1 hour, and 25A at 15 J/m2 53 I I I I I 40 -\ .- \ \\ o ‘2‘ \ Q \ O 3’30 — . \\ _ E \\ a. \ V \‘ o ‘7‘“ >. o I- _ o - .- 2 2 I'- O 4 9 '1': 8 IO — - O. U) o I I I l l 0 24 48 72 96 DURATION OF INCUBATION AT 33°C (hours) Figure l0. 5A irradiation to a value of 23.2 dpm/pg DNA for cells incubated at 33°C for A days before ultraviolet irradiation. While not conclusive, the results from these two experiments suggest that a period of pre—UV hypothermia might decrease the amount of 3H—Thymidine incorporation. 7. The effect of incubation at 33°C for various spans of time before ultraviolet irradiation on various periods of 5H-Thymidine incorporation in V-79 cells. An experiment was conducted to determine the effect of pre-UV hypothermia on the amount of 3H-Thymidine incorporation for extended lengths of time after UV irradiation. The procedure was identical to the previous two experiments in Section 6, except that 3H—Thymidine was incorporated at 37°C for 5, 10, and 20 hours after ultraviolet irradiation, rather than for 2 hours. The results are shown in Figure 11. Cells allowed to incorporate 3H-Thymidine for 5 hours had an average specific activity of 197 dpm/pg DNA, ranging from 1AA to 375 dpm/pg DNA. The amount of DNA isolated from these cells averaged 66 pg per plate, ranging from 50 to 102 pg per plate. Cells incubated for 10 hours after ultraviolet irradiation incor- porated 3H—Thymidine at an average of 300 dpm/pg DNA, ranging from 228 to 388 dpm/pg DNA. The amount of DNA isolated averaged 55 U8 DNA per plate, ranging from A1 to 71 pg per plate. Cells incubated for 20 hours after ultraviolet irradiation incor- porated 3H-Thymidine at an average of 518 dpm/pg DNA, ranging from 391 to 616 dpm/pg DNA. The amount of DNA isolated averaged A7 pg per plate, ranging from 23 to 81 pg per plate. The results indicate that even though 3H—Thymidine incorporation into DNA increased when allowed to proceed for longer periods of time, Figure 11. 55 The amount of 3H-Thymidine (5 pCi/ml medium) incorporated into the DNA of V—79 cells for various periods at 37°C as a function ofpmeviousdurations of incubation at 33°C. 3H-Thymidine incorporation for 5 hours (solid circles), 10 hours (hollow circles), and 20 hours (squares) followed the 33°C incubation period, hydroxyurea (5 mM) treatment for 1 hour, and 25A nm ultraviolet irradiation at 15 J/m2 S6 I I I I I 700- " I 2600- '- Z / O I //A //I g: _ / \ / 500- *~—/ \ I / " \ \/ E O. I 'U I “400-. _ .2 A ' s / \ ,0. I: o / \ / 0 300” / \ /- 9 / O ‘8’ . E 2 // 0200'- ‘- IéI-J O\ ///'\\ / a) \2’ \\../ 0 I00“ " o I I I I I O 24 48 72 96 DURATION OF INCUBATION AT 33°C (hours) Figure II. 57 the pre-UV hypothermia did not appear to affect the amount of 3H—Thymidine incorporation. 8. The effect of incubation at 33°C for 5 days before ultra- violet irradiation on various periods of 3H-Thymidine incor- pgration in V-79 cells. An experiment was conducted to determine the effect of pre-UV hypothermia for 5 days on 3H—Thymidine incorporation for durations up to 68 hours in Chinese hamster lung fibroblasts. Cells were inoculated into 60 mm plastic Petri dishes, were allowed to grow to confluence for A8 hours, and then were maintained in a medium deficient in arginine and isoleucine for 72 hours. During this entire 120 hour period, the cells were either incubated at 33°C or at 37°C. Hydroxyurea was added in the final hour of this 120 hour period. At this point, all the cells were exposed to ultraviolet radiation and were allowed to incorporate 3H-Thymidine at 37°C for 2, 5, 10, 20, 32, AA, 56, and 68 hours. The results for 3H-Thymidine incorporation up to 32 hours are shown in Figure 12. According to the top of the Figure, the DNA isolated decreased from an average of 32 pg per plate with 2 hours of 3H-Thymidine incorporation to an average of 2A pg per plate for 32 hours of incorporation. For cells incubated for 5 days at 33°C before ultraviolet irradiation, 2 hours of 3H-Thymidine incorporation resulted in a specific activity of an average of 360 dpm/pg DNA and this value increased to an average of 2218 dpm/pg DNA for 32 hours of incubation. For cells incubated for 5 days at 37°C before ultraviolet irradiation, the average specific activity increased from 112 dpm/pg DNA for 2 hours incorporation to 1920 dpm/pg DNA for 32 hours incor- poration. 58 Figure 12. The amount of DNA isolated (top) and 3H—Thymidine (5 pCi/ml medium) incorporated into the DNA (bottom) of V-79 cells at 37°C as a function of the duration of 3H-Thymidine incorporation, following a 120 hour incubation period at either 37°C (solid circles) or 33°C (hollow circles), hydroxyurea (5 mM) treatment for 1 hour, and 25A nm ultraviolet irradiation at 15 J/m2 59 4.3 . azompamom. - [/3/ ' ,n. l-_- I > 300 - / . \ ' - p l I 7 "— : I \8'\ O 2 / 0 // I \‘ 200 '- o/ ’. ‘3- 2 / ll. , ‘ . a . / I11 I00 / - a, / U) o I . I I I . I . I . I I I ‘0 A20 40‘ 60 80 HU l-lU HU ‘ INCORPORATION 0F 3H-THYMI Figure l3. HU DINE (hours) Figure 1A. 63 The net amount of 3H-Thymidine (5 pCi/ml medium) incor- porated into the DNA of V-79 cells at 37°C, which was used for the excision repair of ultraviolet radiation-induced dimers, as a function of the duration of 3H—Thymidine incorporation. Each point is derived from the difference of the averaged values of points in Figure 13 between those representing ultraviolet irradiated cells and non- irradiated cells, which were previously incubated for 120 hours at either 37°C (solid circles) or 33°C (hollow circles) 6A 300 - - . /l’7§:§¢ /0 4 fl‘ ’o’___.o/ s 20,- , x I»! _ g: / I \ /// \ E . 3 IOO 18 ' _ v / > \ t ' A Z O I I I I I . t, I I l I '5 (THU 4I-IU 4I-IU I \‘I‘HU 4 \ 0 E -IOO- \ - 0 m \ O. (I) '__ '200— .— [LI 2 600- \- I I I I I I I I I O 20 4O 60 80 INCORPORATION OF 3H-THYMIDINE (hours) Figure l4. 65 average value of 33 ug from plates allowed to incorporate for Al hours, and this value decreased to an average of 10 ug from plates allowed to incorporate for 87 hours. It appeared that the treat— ments of a 5 day incubation at 33°C, or ultraviolet irradiation, did not affect the amount of DNA eventually isolated, while the incorporation of 3H-Thymidine at 37°C resulted in decreasing amounts of DNA isolated with increasing times of incubation with the radio- active label. The bottom of Figure 13 shows the amount of 3H-Thymidine incor- poration per unit weight of DNA as a function of the length of the period of incorporation. For cells incubated at 37°C for 5 days before ultraviolet irradiation, 3H-Thymidine incorporation increased from an average of 8A dpm/ug DNA for 2 hours of incorporation to an average of Shh dpm/ug DNA for Al hours of incorporation, and then decreased to an average of 232 dpm/ug DNA for 87 hours of incorporation. For cells incubated at 37°C for 5 days, but not exposed to ultraviolet irradiation, the specific activity increased from an average 20 dpm/ug DNA for 2 hours incorporation, to an average of 259 dpm/ug DNA for Al hour incorporation, and to an average of 565 dpm/ug DNA for 87 hour incorporation. For cells incubated at 33°C for 5 days before ultra— violet irradiation, the specific activity mean values increased from 62 dpm/ug DNA for 2 hour incorporation to 518 dpm/ug DNA for Al hour incorporation, and stayed relatively constant to 510 dpm/ug DNA for 87 hour incorporation. For cells incubated at 33°C for 5 days, but not UV irradiated, the specific activity mean values increased from 13 dpm/ug DNA for 2 hour incorporation to 266 dpm/ug DNA for Al hour 66 incorporation, and stayed relatively constant to 2A0 dpm/ug DNA for 87 hour incorporation. Figure 1A shows the net specific activity attributed only to excision repair of ultraviolet induced dimers as a function of the period of 3H—Thymidine incorporation, which is derived from the dif- ference of the mean specific activities between ultraviolet irradi- ated cells and non-irradiated cells. For cells incubated at 33°C for 5 days, this amount of "unscheduled DNA synthesis" due to ultraviolet irradiation increased from A9 dpm/ug DNA for 2 hour incorporation to 282 dpm/ug DNA for 29 hour incorporation, decreased slightly to a minimum of 216 dpm/ug DNA for 51 hour incorporation, and returned to 270 dpm/ug DNA for 87 hour incorporation. Cells incubated at 37°C for 5 days resulted in the amount of "unscheduled DNA synthesis" increasing from 6A dpm/ug DNA for 2 hour incorporation to 285 dpm/ug DNA for Al hour incorporation. This value decreased to approximately zero at 51 hour incorporation. After 63 hours, more 3H—Thymidine was incorporated into the DNA of non-irradiated cells than in cells that were exposed to ultraviolet irradiation. The above experiment was repeated except that 3H—Thymidine was incorporated at 37°C for 2, 5, 16, 30, A1, 5A, 66, 78, 93 and 116 hours after ultraviolet irradiation. Hydroxyurea was added at 21, A5, 68, and 96 hours after ultraviolet irradiation, as well as 1 hour before irradiation, in increasing increments of 5 mM each. The results are shown in Figures 15 and 16. Although not shown, the amount of DNA isolated averaged 6A mg for plates allowed to incorporate 3H-Thymidine for 2 hours, ranging from AA to 88 pg per plate. DNA isolated averaged A8 pg for plates 67 with Al hour incorporation, ranging from 38 to 60 pg per plate. Finally, DNA isolated averaged 21 ug for plates with 93 hour incor— poration, ranging from 8 to 30 pg per plate. Again there was no correlation between the amount of DNA isolated and prior temperature, or whether or not the cells were irradiated. Figure 15 shows the amount of 3H-Thymidine incorporation per unit weight of DNA as a function of the length of time the 3H-Thymidine was allowed to incorporate. For cells incubated at 33°C for 5 days before ultraviolet irradiation, the mean specific activity was 162 dpm/pg DNA for 2 hour incorporation and increased to 22A2 dpm/ug DNA for Al hour incorporation. This increased slightly to 2303 dpm/ug DNA for 93 hour incorporation. For non-irradiated cells incubated at 33°C for 5 days, the mean specific activity was 65 dpm/ug DNA for 2 hour incorporation and increased to 13AO dpm/Ug for Al hour incorporation. This decreased slightly to 1282 dpm/ug DNA for 93 hour incorporation. For cells incubated at 37°C for 5 days before ultraviolet irradi- ation, the mean specific activity was 19A dpm/ug DNA for 2 hour incor- poration and increased to 171A dpm/ug DNA for Al hour incorporation. The mean specific activity remained fairly constant and then decreased to 922 dpm/ug DNA for 93 hour incorporation. The mean specific activity for non-irradiated cells, incubated at 37°C for 5 days before 3H-Thymidine incorporation was 5A dpm/ug DNA for 2 hour incorporation and increased to 916 dpm/ug DNA for Al hour incorporation. This decreased to 712 dpm/ug DNA for 93 hour incorporation. Figure 16 shows the amount of "unscheduled DNA synthesis" attri- buted only to ultraviolet induced excision repair. The amount of "unscheduled DNA synthesis" in cells incubated for 5 days at 33°C 68 Figure 15. The amount of 3H-Thymidine (5 uCi/ml medium) incorporated into the DNA of V-79 cells at 37°C as a function of the duration of 3H-Thymidine incorporation, following a 120 hour incubation period at either 37°C (circles) or 33°C (squares) and 25A nm ultraviolet irradiation at 15 J/m2 (hollow symbols) or non-irradiation (solid symbols). Hydroxyurea ("HU") was added at 21, A5, 68, and 96 hours after ultraviolet irradiation, as well as l hour before irradiation, in increasing increments of 5 mM_each 69 2500 SPECIFIC ACTIVITY (dpm/ug ONA) 0| 8 O - ’ O 20 4O 60 80 I00 IZO A A A A A I-IU HU HU HU HU INCORPORATION OF 3H-THYMIOINE (hours) Figure l5. Figure 16. 70 The net amount of 3H—Thymidine (5 uCi/ml medium) incor- porated into the DNA of V-79 cells at 37°C, which was used for the excision repair of ultraviolet radiation- induced dimers, as a function of the duration of 3H—Thymidine incorporation. Each point is derived from the difference of the averaged values of points in Figure 15 and Figure 17 between those representing ultraviolet irradiated cells and non-irradiated cells, which were previously incubated for 120 hours (Figure 15) at either 37°C (solid circles) or 33°C (squares) or were previously incubated for 72 hours (Figure 17) at either 37°C (hollow circles) or at AO°C (triangles) 71 r I I I I T I I I I I - -I IOOO *- \ -* g h- \ ’ | q 0 I 3’ 800 — \ - \ ‘ ~ I I \ E I \‘d I \ o. — I _ P \ o / , x f; 600 - I / a ->— /’O~.\ / | l: r- ‘{ \o‘\/J - 0 <2 . / 2 400- // - 9..- ,/ I 8 " ' ‘o// a (0’5 ’— I— 200— /’A"\n.\ _ 52’ 'f’\ / “X W s _ \ —1 ' \ O I I I J I L J ‘I 1 I I O 20 4O 60 80 IOO INCORPORATION 0F 3H-THYMIOINE (hours) Figure l6. 72 was 97 dpm/ug DNA for 2 hours, 902 dpm/ug DNA for Al hours, and 1021 dpm/ug DNA for 93 hours. In cells always incubated at 37°C, "unscheduled DNA synthesis" was 1A0 dpm/ug DNA for 2 hour incor- poration, 798 dpm/ug DNA for A1 hours, and 210 dpm/ug DNA for 93 hours. The results from both experiments show that pre—UV hypother- mia does not appear to affect the amount of "unscheduled DNA syn- thesis" after UV irradiation when 3H-‘I'hymidine was allowed to in- corporate into the DNA for periods up to Al hours. However, hypo- thermia for 5 days before 3H-Thymidine incorporation of non-irradiated cells either resulted in an increase, or no change, of incorporation of the radioactive label. This suggests that 5 day hypothermia some- times, but not always, results in an increase in subsequent residual "semi-conservative DNA synthesis". After Al hours, 3H—Thymidine up- take into DNA varied with different treatments, but the results are difficult to interpret. 10. The effect of incubation at AO°C for 3 days before ultraviolet irradiation on variousgperiods of "unscheduled DNA Synthesis"- in V-79 cells. An experiment was conducted to determine the effect of pre-UV hyperthermia on the amount of "unscheduled DNA synthesis" for various durations after ultraviolet irradiation. Chinese hamster lung fibroblasts were inoculated into 60 mm plastic Petri dishes and were allowed to grow to confluence at 37°C. At confluence, the medium was changed to one deficient in arginine and isoleucine. At this point, the cells were incubated for 72 hours at either 37°C or AO°C. Hydroxyurea was added after this period at a concentration of 5 my and the cells were incubated at 37°C for l 73 hour. At this point, at least two plates in each temperature group were exposed to ultraviolet radiation and the DNA in all of the plates was allowed to uptake 3H—Thymidine at 37°C for either 2, 5, 16, 29, Al, 51, 63, 75, 87, or 99 hours. Hydroxyurea was added after 18, A2, and 66 hours of 3H—Thymidine uptake, in additions of 5 mfi_each. The results are shown in Figures 16 and 17. The amount of DNA isolated per plate is illustrated on the top of Figure 17. In cells that were always incubated at 37°C, the average amount of DNA isolated per plate was 108 pg after 2 hours of incor— poration of 3H-Thymidine, 7A pg after Al hours, and 11 pg after 99 hours. For cells incubated at AO°C for 72 hours, the average amount of DNA isolated per plate was 93 pg after 2 hours and 30 pg after Al hours of 3H-Thymidine incorporation. Cells that were incubated at AO°C for 3 days and were allowed to incorporate 3H-Thymidine at 37°C for 51 hours or greater were almost cxmnpletely detached from the plates. The bottom of Figure 17 shows the amount of 3H—Thymidine incor- poration per weight of DNA as a function of the length of the incor- poration period. For irradiated cells, always incubated at 37°C, the mean amount of 3H-Thymidine incorporated per pg DNA was 132 dpm after 2 hours incorporation, 856 dpm after A1 hours, and 1552 dpm after 87 hours. For non-irradiated cells, always incubated at 37°C, mean 3H-Thymidine incorporation per pg DNA was A8 dpm after 2 hours incor- poration, 36A dpm after A1 hours, and 930 dpm after 87 hours. For cells incubated at AO°C for 3 days before ultraviolet irradiation, the mean amount of 3H-Thymidine incorporation per pg DNA was 52 dpm after 2 hours incorporation, 372 dpm after A1 hours, and A5 dpm after 75 hours. For non-irradiated cells incubated at AO°C for 3 days, the Figure 17. 7A The amount of DNA isolated (top) and 3H-Thymidine (5 pCi/ml medium) incorporated into the DNA (bottom) of V-79 cells at 37°C as a function of the duration of 3H-Thymidine incorporation, following a 72 hour incuba- tion period at either 37°C (circles) or AO°C (triangles) and 25A nm ultraviolet irradiation at 15 J/m2 (hollow symbols) or non—irradiation (solid symbols). Hydroxy- urea was added at 18, A2, and 66 hours after ultraviolet irradiation, as well as 1 hour before irradiation, in increasing increments of 5 mg each 75 WEIGHT OF DNA ISOLATED (I19) I20 A '1 b 3 _ 80— ‘7’" Q - _ .W c- 0 ,£\ a _ LA ‘ \ : I . \K 4° "3" ‘\ ' . \ ‘ '- A >\ 5 \ q l600- g_\ - P \ .. o I \\ .. / 200- o / \ _ 8 0 § SPECIFIC ACTIVITY (dpm / pg DNA) O 20 4O 60 80 IOO INCORPORATION OF 3H-THYMIDINE (hours) Figure l7. 4-3 76 mean 3H-Thymidine incorporation per pg DNA was 11 dpm after 2 hours incorporation, 171 dpm after Al hours, and 8A dpm after 75 hours. Figure 16 shows the amount of "unscheduled DNA synthesis" attributed only to the excision repair of ultraviolet induced dimers. This is the difference of the mean specific activities of 3H—Thymidine incorporation into DNA in ultraviolet irradiated cells and non- irradiated cells. The amount of "unscheduled DNA synthesis" in cells always incubated at 37°C was 85 dpm/pg DNA after 2 hours of 3H-Thymidine incorporation and A92 dpm/pg DNA after Al hours. The amount of "unscheduled DNA synthesis" was saturated after this point with a specific activity of approximately 500 dpm/pg DNA. The amount of "unscheduled DNA synthesis" in cells incubated at AO°C for 3 days was A1 dpm/pg DNA after 2 hours and saturated at 225 dpm/pg DNA at 29 hours of incorporation. After Al hours this net specific activity decreased, and reached zero at 75 hours of incorporation. The results indicate that pre—UV hyperthermia produced a de- crease in both the initial rate and maximum extent of "unscheduled DNA synthesis" after UV irradiation. This hyperthermia also result- ed in a faster rate of cell detachment from the plates during the period of 3H-Thymidine incorporation. 11. The effect of incubation at A1°C for 3 days before ultra- violet irradiation on 2 and 2A hours of 3H-Thymidine incor— poration in human skin fibroblasts. Human cells were utilized in an experiment to determine the effect of pre—UV hyperthermia on the amount of subsequent "unscheduled DNA synthesis". The different cell type was utilized to show that a reduction of "unscheduled DNA synthesis" with pre-UV hyperthermia is not necessarily restricted to V—79 cells. 77 Human skin fibroblasts, at passage 20, were inoculated into 60 mm plastic Petri dishes, at a concentration of about 106 cells per dish, and were allowed to grow at 37°C for 36 hours to conflu— ence. The confluent cells were incubated in a medium deficient in arginine and isoleucine, for 72 hours, at either 37°C or A1°C. After this period, hydroxyurea was added at a concentration of 51m! and all the plates were incubated at 37°C for 1 hour. At this point, most of the plates were exposed to ultraviolet irradiation, and all of the cells were then allowed to incorporate 3H-Thymidine at 37°C for either 2 or 2A hours. The amount of DNA isolated per plate averaged 19.5 pg, ranging from lA.2 to 26.9 pg. The amount of DNA isolated per plate did not appear to be affected by temperature, ultraviolet irradiation, or the period of 3H-Thymidine incorporation. Figure 18 shows the amount of 3H-Thymidine incorporation as a function of the length of time of incorporation. For cells incubated at 37°C before ultraviolet irradi— ation, the specific activity increased from an average value of 1530 dpm/pg DNA for 2 hour incorporation to AA3O dpm/pg DNA for 2A hour incorporation. For cells incubated at Al°C for 3 days before ultra- violet irradiation, the specific activity increased from average of 8A0 dpm/pg DNA for 2 hour incorporation to A210 dpm/pg DNA for 2A hour incorporation. Non-irradiated cells incorporating 3H-Thymidine for 2 hours showed a specific activity of approximately 100 dpm/pg DNA. Like the experiments utilizing V-79 cells, the above experiment demonstrates that pre-UV hyperthermia results in a significant decrease in subsequent "unscheduled DNA synthesis" in human skin fibroblasts. Figure 18. 78 The amount of 3H-Thymidine (5 pCi/ml medium) incorporated into the DNA of human skin (736-NF) fibroblasts at 37°C as a function of the duration of 3H-Thymidine incor- poration, following a 72 hour incubation period at either 37°C (circles) or A1°C (triangles), hydroxyurea (5 mg) treatment for 1 hour, and 25A nm ultraviolet irradiation at 15 J/m2 (hollow symbols) or non-irradiation (solid symbols) 79 é \ g \ \\ (dpm/pg DNA) \ é \ \ S SPECIFIC ACTIVITY O I 02 24 INCORPORATION 0F 3I-I-THYNIIOINE (hours) Figure l8. 8O 12. The effect of incubation at 37°C and Al°gfor l, 8, and 2A hours in deficient medium on subsequent "unscheduled DNA synthesis" in human fibroblasts. In all of the preceding experiments involving the effect of hyperthermia on subsequent "unscheduled DNA synthesis" (Sections 1 - 5, 10, and 11), cells were incubated at the higher temperature for various durations within the 3 day period while being maintained at confluence in deficient medium. During this 3 day period in deficient medium, the cells were proceeding from a state of normal "semi-conservative DNA synthesis" to a state of very limited "semi- conservative DNA synthesis". Many different effects of temperature can take place during this period, which may influence the amount of subsequent "unscheduled DNA synthesis". Therefore an experiment was conducted in which the cells were subjected to a period of hyper- thermia following the 3 day period in deficient medium. Human skin fibroblasts, at passage 22, were inoculated into 60 mm plastic Petric dishes at a concentration of approximately 106 cells per dish, and were allowed to grow at 37°C for Al hours to confluence. At confluence, the cells were maintained at 37°C for 72 hours in a medium deficient in arginine and isoleucine. After this period, the cells were incubated for an additional period of 1, 8, or 2A hours at either 37°C or Al°C. Hydroxyurea was added at a concentration of 5 mM_at the beginning of the final 1 hour of this additional period. At this point half the plates were exposed to ultraviolet radiation, and all the cells were allowed to incor- porate 3H-Thymidine at 37°C for 2 hours. The amount of DNA isolated per plate averaged 16.7 pg, ranging from 11.8 to 23.6 pg. The amount of DNA isolated did not depend on incubation temperature or exposure 81 to ultraviolet radiation, but it did decrease slightly with increas- ing duration of incubation before 3H-Thymidine incorporation. Figure 19 shows the amount of 3H-Thymidine incorporation as a function of the length of the additional period the cells were maintained at 37°C or A1°C in the deficient medium after the initial 72 hour period at 37°C. Cells incubated at 37°C before irradiation had mean specific activities of 2800, 2850, and 1900 dpm/pg DNA, while cells incubated at A1°C before irradiation had mean specific activities of 2800, 2500, and 1870 dpm/pg DNA, for respective additional incubation periods of 1, 8, and 2A hours after the initial 72 hour period at 37°C. Non-irradiated cells always had specific activities of nearly 100 dpm/pg DNA. The results of this experiment show no significant effect of pre- UV hyperthermia, for periods up to 2A hours, on the amount of subsequent "unscheduled DNA synthesis". However there appeared to be a decrease in "unscheduled DNA synthesis" with increasing duration of pre—UV incubation in deficient medium regardless of the temperature. DISCUSSION Ultraviolet irradiated cells always had more 3H—Thymidine incor- poration than non-irradiated cells when they were allowed to incor- porate for A1 hours or less. When Chinese hamster lung fibroblasts, always incubated at 37°C, were allowed to incorporate 3H—Thymidine for 2 hours, irradiated cells showed from 2.1 (Figure 2) to A.9 (Figure A) times more incorporation than non-irradiated ones. Likewise, when human skin fibroblasts, always incubated at 37°C, were allowed to incorporate 3H-Thymidine for 2 hours, irradiated cells showed 15 (Figure 18) to 35 (Figure 19) times more incorporation than non-irradiated cells. This is consistent with previous experiments of J. E. Trosko (personal Figure 19. 82 The amount of 3H-Thymidine (5 pCi/ml medium) incorporated into the DNA of human skin (736-NF) fibroblasts at 37°C as a function of previous durations of incubation at either 37°C (circles) or A1°C (triangles), after an initial 72 hour incubation period at 37°C. Just prior to 3H—Thymidine incorporation, the cells were treated with hydroxyurea (5 mM) for 1 hour and either exposed (hollow symbols) or not exposed (solid symbols) to 25A nm ultraviolet irradia- tion at 15 J/m2 83 24 DURATION OF INCUBATION (hours) O Mu—flfi—l—r \ “'0 D l O "3~\ \ \M\\ 0 F.——*_..:-_—:;:.__.-.:: 8 m _ LW . 2 m 220 03:33 >.:>_.8< ocauam m Figure l9. 8A 92 communication) and published results . This indicates that the tech- nique used is sensitive enough to detect "unscheduled DNA synthesis" involved in the excision repair of ultraviolet induced damage. These results also indicate that the amount of "unscheduled DNA synthesis" is much greater in human skin fibroblasts than in Chinese hamster lung fibroblasts for the given ultraviolet radiation dose of 15 J/m2. Although Chinese hamster cells that were incubated at A3°C for 2 hours were almost completely detached, cells incubated at AO°C for 2 hours incorporated only slightly less 3H-Thymidine than cells that were always incubated at 37°C (Table 1). This decrease did not appear to be significant. Also, V-79 cells incubated at Al°C for 8 hours showed an insignificant increase (Figure 2) or decrease (Figure 3) of 3H-Thymidine incorporation.with respect to cells always incubated at 37°C. Although this deviation appears insignificant, it might depend on whether the growth time for the cells to reach confluence was 3 days (Figure 2) or A days (Figure 3). The same cells incubated at A1°C for 21 hours always had less 3H-Thymidine incorporation for 2 hours than cells constantly maintained at 37°C. Although this decrease was insignificant for the experiment displayed in Figure 2, cells incubated at A1°C for 21 hours in the experiment shown in Figure 3 resulted in a net specific activity of 2 hour incorporation due to UV irradiation alone of 50 dpm/pg DNA, which is 67% of the net specific activity of 75 dpm/pg DNA observed for cells that were only incubated at 37°C. Hyperthermia for 3 days prior to UV irradiation always resulted in significantly less 3H-Thymidine incorporation. This was demonstrated in 6 experiments illustrated in Figures A - 8 and 16 - 18. Chinese 85 hamster cells incubated at A1°C for 3 days resulted in the net specific activity being between 23% (Figure 8) and 60% (Figure 5) of the value derived from cells only incubated at 37°C with 2 hour incorporation. The same cells incubated at AO°C for 3 days had A9% of 3H—Thymidine incorporated for 2 hours as in cells only incubated at 37°C (Figure 16). Human skin fibroblasts incubated at A1°C for 3 days had a net specific activity which was 50% of that from cells only incubated at 37°C (Figure 18). Chinese hamster cells, incubated at A1°C for 3 days before hydroxyurea treatment and allowed to incorporate 3H—Thymidine at 37°C for 2 hours, gave a net specific activity of 50 dpm/pg DNA when incubated with hydroxyurea at 37°C for 1 hour, and 32 dpm/pg DNA at A1°C. This difference does not appear to be significant. At least, incubation at Al°C for 1 hour does not appear to break down the hydroxyurea, resulting in more 3H-Thymidine incorporation from "semi-conservative DNA synthesis". The same cells, incubated at A1°C for 3 days and at A1°C for 1 hour with hydroxyurea, gave a net specific activity of 32 dpm/pg DNA when 3H-Thymidine was incorporated for 2 hours at 37°C after ultraviolet irradiation, and 70 dpm/pg DNA when incorporation was done for 2 hours at A1°C after irradiation. It is well known that the rates of many enzymatic reactions increase with higher temper- ature. The observed increase in specific activity, when 3H—Thymidine was incorporated at the higher temperature, might be due to an increased rate of enzymatic processes involved in the excision of ultraviolet induced dimers. As was previously discussed, a signifi- cant reduction in 3H-Thymidine incorporation was observed only when 86 cells were pre—UV incubated at A1°C for periods longer than 21 hours. Certainly any thermal impairment of the excision process, such as denaturation of the excision repair enzymes, would be negligible during the 2 hour incorporation period at A1°C. As shown in Figure 6, cycloheximide does not appear to affect the amount of 3H-Thymidine incorporated into the DNA, for irradiated or non-irradiated cells, incubated at either 37°C or A1°C for 3 days. Apparently maintenance of V—79 cells in a medium deficient in arginine and isoleucine is enough to halt protein synthesis. Cycloheximide is not needed to prevent repair proteins from being resynthesized and was not used in subsequent experiments. No definite conclusions could be reached from experiments involving incubation of v—79 cells at 33°C for periods up to 5 days before 3H-Thymidine incorporation (Figures 9 - 16), except that this period of pre-UV hypothermia does not appear to affect the net amount of "unscheduled DNA synthesis" attributed only to the excision repair of ultraviolet induced damage, when incorporation was conducted for Al hours or less. This hypothermia sometimes increases (Figures 12 - 15), decreases (Figure 10), or apparently has no definite effect (Figures 9, 11, and 16) on total incorporation of 3H-Thymidine at 37°C for 2 hours. Since this data is inconsistent, it could only be concluded that hypothermia before ultraviolet irradiation had no significant effect or that the effects are too complicated to be interpreted. According to Figures 1A and 16, net 3H-Thymidine incorporation for excision repair is saturated at approximately 27 hours. Saturation was observed with other cell types by other investigatorsg. However, as shown in Figure 16, cells incubated at AO°C for 3 days were saturated 87 with 3H-Thymidine at 16 hours, while the cells that were used in the same experiment, but were only incubated at 37°C, saturated at Al hours. In this experiment, there was less DNA isolated at saturation, than at times before saturation, for cells incubated at both temperatures (Figure 17). This suggests that cells were detach- ing, and perhaps dying, before 3H-Thymidine saturation. From the top of Figure 17, it appears that cells incubated at AO°C for 3 days were detaching (or dying) at a much faster rate than cells only incubated at 37°C. This observation supports the hypo— thesis that thermal denaturation of the repair processes is the rate limiting step in the death of single cells. The argument goes as follows. Incubation at AO°C for 3 days denatures the repair processes so that the cells could perhaps repair only half as well as cells that were always incubated at 37°C. The denaturation of a repair process would be a "rate limiting step", since it takes 3 days, a relatively long time, to accomplish. Next, environmental insults, such as ultraviolet irradiation, damage the DNA. These are obviously not "rate limiting steps", since they occur within a fraction of a second. After 2 hours of incorporation of 3H4mumfidineat 37°C, almost the same amount of DNA was isolated for cells incubated for 3 days at either 37°C or AO°C, but cells incubated at AO°C incorporated only A9% of 3H—Thymidine as cells only incubated at 37°C. After 2 hours, the cells did not accumulate enough unrepaired damage, or the damage did not have a sufficient period of time to cause cell mortality, yet the excision repair of UV induced dimers in cells incubated at AO°C was only half as efficient as repair in cells incubated at 37°C. 88 With further incubation at 37°C with 3H-Thymidine, enough unrepaired damage (not necessarily UV induced) would accumulate, within a suffi- cient period of time, to express its lethality. Cells that were incubated at AO°C would incorporate less 3H-Thymidine, and accumulate unrepaired damage at a faster rate, causing an increased death rate, during the incorporation period, over cells that were only incubated at 37°C. This is observed in the top of Figure 17, if the amount of DNA isolated per plate can be interpreted as a measurement of cell survivorship. The figure also indicates that the cell death rate is not significantly enhanced by prior ultraviolet irradiation, since irradiated cells and non-irradiated cells, kept under otherwise identi- cal conditions, contain almost equal amounts of DNA. This suggests that ultraviolet irradiation is not a major cause of the observed loss of cells. Other damaging causes might be the exposure to A°C (on ice) before 3H-Thymidine incorporation, continued exposure to radioactive thymidine, or continued maintenance in a spent arginine and isoleucine deficient medium supplemented with dialysed fetal calf serum. These damages are probably repaired by mechanisms other than the pyrimidine dimer excision repair system. Presumably these other mechanisms can be thermally impaired by 72 hour incubation at AO°C." The hypothesis that the denaturation of repair proteins is the rate limiting step in the thermal death of single cells is still consistent with the above argument and observations in Figures 16 and 17. Even though the major cause of cell death is probably not the denaturation of the repair enzymes that excise ultraviolet induced pyrimidine dimers, measuring the repair of these enzymes can be used to monitor the repair by hypo- thesized repair enzymes that cause cell death if thermally denatured. 89 Probably exposure to AO°C or greater, over a 3 day period, causes thermal damage to many systems within the cell. These may include repair systems so essential to the cell that the denaturation of repair proteins, contained within these systems, may cause cell death. However, if thermal cell death caused decreased repair, which is the converse of the above hypothesis, entirely different results would have been observed. First of all a significant amount of cells would have been expected to detach during 3 days of incubation at AO°C as for the cells that were incubated at A3°C for 2 hours (Table 1). This is not observed, since even after 2 hours of 3H-Thymidine incorporation, plates incubated at either 37°C or AO°C contained equal amounts of DNA (Figure 17). However, if in this case "dead" cells did not detach after 3 days at AO°C and if the "dead" cells had not incorporated 3H-Thymidine, while the "live" cells incorporated as much 3H-Thymidine as cells only incubated at 37°C, then the average specific activity for cells incubated at AO°C should be significantly less than for cells only incubated zit 37°C. However, the cells incubated at AO°C began to detach after 2 hours of 3H—Thymidine incorporation. The "live" cells have no reason to detach at this point and the "dead" cells, which are not metabolizing, are not expected in) maintain their adhesion to the surface. While the "dead" cells detach, the surface of the plate should contain a greater percentage of "live" cells which have as much 3H—Thymidine incorporated into their DNA as cells only incubated at 37°C. With increasing time of incubation with 3H—Thymidine, the DNA isolated should have had greater specific activities, eventually reaching the specific activity of the cells incubated at 37°C, 90 when the "dead" cells had completely detached. This is clearly not what is observed. Between 16 and Al hours of 3H—Thymidine incorpora- tion, the period of greatest cell detachment for cells that were incubated at AO°C for 3 days, the specific activity for these cells remained constant, while the specific activity for cells only incubated at 37°C continued to increase. Therefore, the lower specific activity observed in cells incubated at AO°C for 3 days relative to cells always incubated at 37°C appears to be attributable to thermal impairment of the excision repair process rather than to an increasing percentage of dead cells. As mentioned earlier in the Discussion section, pre-UV hyper- thermia for 3 days always significantly reduced the amount of sub- sequent 3H-Thymidine incorporation into the DNA of both Chinese ham- ster cells (Figures A - 8, l6, and 17) and human skin fibroblasts (Figure 18). Since the period of pre—UV hyperthermia is relatively long, the rate limiting step in the thermal inhibition of 3H-Thymidine incorporation into DNA should be an activated process. The results of the experiment shown in Figure 19 suggest that there may be another time dependent process involved in the inhibition of 3H-Thymidine incorporation. However this process is not an activated process, since an increase in temperature from 37°C to A1°C would not cause any signif— icant further inhibition of 3H-Thymidine incorporation. Following a 3 day period of confluence in deficient medium at 37°C, human skin fibro- blasts, taken from the experiments that are illustrated in Figure 19, were further incubated at either 37°C or Al°C for various periods of time prior to ultraviolet irradiation. Although there was no signif- icant effect with temperature on the subsequent incorporation of 91 3H-Thymidine, a significant reduction in the uptake of this radio- active label was observed with increasing durations of pre-UV incubation, regardless of the temperature. No significant effect with temperature was expected since the period of hyperthermia only extended up to 2A hours. Significant decreases in 3H-Thymidine incor- porationwnnuaobserved only with a 3 day period of pre-UV hyperthermia in previous experiments. This thermally activated process of 3H—Thymidine incorporation may involve the denaturation of a protein, since protein denaturation is an activated process3. 0n the other hand, the time dependent, but temperature independent, inhibition of 3H-Thymidine incorporation in the experiment of Figure 19 is not an activated process, and probably does not involve a protein denaturation. This process of inhibition may involve another process involved in the transport and phosphorylation of exogenous 3H-Thymidine to 3H—dTTP into the DNA of the cell, such as the permeability of the cell membrane to 3H—Thymidine. This might involve a time dependent, but temperature independent,clogging of the membrane with material, resulting in a decrease in permeability of the membrane to 3H—T'hymidine. SUMMARY Confluent mammalian fibroblasts were incubated for three days at conditions unfavorable for semi—conservative DNA replication. After exposure to 15 J/m2 of ultraviolet radiation, the cells were allowed to incorporate 3H—Thymidine. The cells were incubated at different temperatures for various durations immediately before ultraviolet irradiations. Excision repair was quantitated by the amount of 3H-Thymidine incorporated into the DNA after ultraviolet irradiation. 92 Ultraviolet irradiated cells always had more 3H—Thymidine incor- poration than non-irradiated cells. Hyperthermia at AO°C or A1°C, for 3 days before ultraviolet irradiation, always resulted in significantly less 3H—Thymidine incorporation following irradiation. However, after Chinese hamster cells were incubated at A1°C for 3 days and then UV irradiated, more 3H—Thymidine was incorporated into DNA for 2 hours at A1°C than was incorporated for 2 hours at 37°C, indicating that incorporation of the label occurred at a faster rate during hyper- thermia. Furthermore, incubation at A1°C for one hour does not appear to break down hydroxyurea. Cycloheximide does not appear to affect the amount of 3H—Thymidine incorporation. However, no definite conclusions could be reached from experiments involving incubation of V-79 cells at 33°C for periods up to 5 days before 3H—Thymidine incorporation. Net 3H-Thymidine incorporation for excision repair appears to be saturated at approximately 27 hours. Chinese hamster cells that were incubated at AO°C for three days before 3H-Thymidine incorporation detached (died) at a faster rate during the labeling period than cells only incubated at 37°C. Ultraviolet irradiated cells did not appear to detach at a faster rate than non—irradiated cells. These observations are consistent with the hypothesis that thermal denaturation of the repair process is the rate limiting step in the death of single cells. The results expected if the converse of this hypothesis were valid are not observed. CHAPTER III THE EFFECT OF TEMPERATURE ON THE REPAIR OF ULTRAVIOLET INDUCED PYRIMIDINE DIMERS IN THE DNA OF MAMMALIAN CELLS AS MEASURED BY AUTORADIOGRAPHY INTRODUCTION Autoradiography is a technique used to quantitate the amount of radioactivity present within individual cells or cell organelles. The radioactive cellular material is usually fixed on a microscope slide, exposed with photographic emulsion, developed, and stained. During exposure, nuclear disintegration materials produce "grains" or "tracks" detected in the emulsion. The number of "grains" can be counted in each cell or organelle, which gives the relative amount or radioactivity for each unit. Autoradiography is usually used to study DNA excision repair in more detail. It is still considered a method of "unscheduled DNA synthesis" because radioactive thymidine is commonly utilized to be incorporated into the DNA when "semi-conservative DNA synthesis" is repressed. Rather than measure the bulk "excision repair" for an entire plate of cells, the autoradiography of "unscheduled DNA synthesis" can be used to measure the relative amounts of "excision repair" within each individual cell or nucleus. This technique was utilized to render more support for the hypothesis that denaturation of the DNA repair proteins is the rate limiting step in the death of single cells. 93 9A MATERIALS AND METHODS galls. Chinese hamster lung fibroblasts (V-79) were used in the exper- iment. The cell culture was obtained from J. E. Trosko, Human Development Department, Michigan State University, East Lansing, MI., and was grown under humidified 5% C02 in air. Permanent stock cultures of the Chinese hamster cells utilized a "C-15" medium (see Appendix A) supplemented with 5% fetal calf serum. Growing cell cultures, for the experiment, utilized "D" medium (see Appendix A) supplemented with 5% fetal calf serum. The confluent cells utilized for experiments were maintained in "D" medium without arginine or isoleucine (see Appendix A) and supplemented with 5% dialysed fetal calf serum. All media were supplemented with penicillin (100 units/ml), streptomycin (100 pg/ml), and mycostatin (100 units/ml). Experimental Culture Procedure (Figure l) Cells were inoculated into 60 mm plastic Petri dishes (Corning Glass Works, Corning, NY) and allowed to grow in "D" medium for 70 hours to heayy'confluent densities. Cells were maintained for 72 hours at confluence, at either 37°C or A1°C, in a "D" medium deficient in arginine and isoleucine and supplemented with 5% dialyzed fetal calf serum. This medium was changed to fresh deficient medium after 2A hours of confluence. After the 72 hour period, the cells were incubated at 37°C for 1 hour with 5 mM_hydroxyurea. Ultraviolet Irradiation and Repair The medium was decanted from the Petri dishes and saved just prior to ultraviolet irradiation. The tops were removed from the dishes and 95 the cells were exposed to 25A nm ultraviolet radiation, delivered from one germicidal lamp (General Electric, G15 T8) mounted in a sterile transfer hood. The incident dose rate, measured with a short wave Blak-ray meter (Ultraviolet Products, San Gabriel, Calif.) was approx- imately 1.0 W/m2. While one of time dishes was being irradiated, the remainder of the dishes were kept on ice (A°C). [Me—3H]-Thymidine (3H-TdR; New England Nuclear, Boston, Mass.; AO Ci/mmol) was added to the spent, decanted medium at 5 pCi/ml medium. This radioactive medium was then added to the cells immediately after ultraviolet irradiation. The cells were allowed to incorporate 3H-Thymidine from this medium for 2 hours at 37°C. After this post-treatment incubation period, the medium was decanted and the cells were washed twice with cold phosphate buffered saline. Their detachment from the dishes was facilitated by a rubber policeman in phosphate buffered saline. The detached cells were collected by centrifugation with the phosphate buffered saline decanted. Autoradiography After centrifugation, the pelleted cells were fixed overnight, without disrupting the pellet, in methanol/acetic acid (3:1 v/v). After fixation, the fixative was decanted and the cell pellets were disrupted in methanol/acetic acid (3:1). Next, one or two drops of the cell suspension were added to wet microscope slides covered with distilled water. This allowed the cells to spread in a thin flat film over the slides. After drying overnight, the cells were dipped in undiluted NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). After an 8 day exposure period, the emulsions were developed for 2 minutes in Kodak 96 D19 developer, rinsed in double distilled water for 0.5 minute, fixed with Kodak rapid fix for 2 minutes, and rinsed 3 times with double distilled water for 7 - 10 minutes each time. Staining The slides were stained with hematoxylin and eosin. After development, the slides were rinsed in distilled water for 2 minutes, stained with hematoxylin for 5 minutes, dipped in distilled water, and dipped in tap water. After rinsing with 35%, 50%, 70%, and 95% ethanol for 2 minutes each, the slides were stained with eosin for 2 minutes. At this point, the slides were dipped in 95%, another 95%, and 100% ethanol, and rinsed in two solutions of xylene for A minutes each. Finally cover slipes were mounted on the slides with Permount. RESULTS As shown in the autoradiographs in Figure 2%) and Figure 21, cells that were only incubated at 37°C (Figure 20) appear to contain many more grains per nucleus than cells that were incubated at A1°C for 3 days (Figure 21). Figure 22 shows the results of counting the number of grains per nucleus for 700 cells in each treatment group. This histogram shows the number of nuclei having the number of grains indicated on the x-axis. This indicates that cells only incubated at 37°C tended to contain significantly more grains per nucleus than cells incubated at A1°C for 3 days. The total amount of grains counted was 15,5A8 in the 700 cells that were only incubated at 37°C and 5,321 in the 700 cells that were incubated at A1°C for 3 days. This resulted in an average of 22.2 grains per nucleus for cells only incubated at 37°C and 7.6 grains per nucleus for 97 Figure 20. Autoradiograph of V—79 nuclei after 72 hour incubation at 37°C, treatment for 1 hour with hydroxyurea (5 mM), ultra- violet irradiation (25A nm) at 15 J/m2, and exposure to 3H-Thymidine (5 pCi/ml medium) for 2 hours at 37°C 98 Figure 20. Figure 21. 99 Autoradiograph of V-79 nuclei after 72 hour incubation at A1°C, treatment for 1 hour with hydroxyurea (5 mM), ultra- violet irradiation (25A nm) at 15 J/m2, and exposure to 3H-Thymidine (5 pCi/ml medium) for 2 hours at 37°C r u .m. F Figure 22. 101 Distribution curves representing the number of cells with indicated grains per nucleus. V-79 cells were incubated at either 37°C (solid circles) or Al°C (hollow circles) for 72 hours in a medium deficient in arginine and isoleucine, treated for 1 hour with hydroxyurea (5 mM), ultraviolet irradiated (25A nm) at 15 J/m2, and exposed to 3H-Thymidine (5 pCi/ml medium) for 2 hours. Seven hundred cells were counted in each treatment group. The absence of a data point means there were no cells with that grain number 102 70+— A 60 II - NUMBER OF CELLS 0‘ O I TL. I N O F A I IO ‘1‘ - AH I O I I; A"; x 7.; .. A.“ LA. Inz‘ . . .~. 2 I O 20 4O 60 80 IOO GRAINS / NUCLEUS Figure 22. 103 cells incubated at A1°C for 3 days. If each cell contained an average of 6 pg DNA97 , the specific activities were 321 grains per minute per pg DNA for cells only incubated at 37°C and 110 grains per minute per pg DNA for cells incubated at A1°C for 3 days. Nuclei from cells that were only incubated at 37°C contained, on the average, 2.92 times more grains than nuclei from cells incubated at A1°C for 3 days. DISCUSSION Cells only incubated at 37°C incorporated an average of roughly 3 times more 3H-Thymidine in their nuclei than cells incubated at A1°C for 3 days before ultraviolet irradiation. This result can be explained by two hypotheses: Either the 3 days incubation at Al°C causes the cells to incorporate 33% as much 3H-Thymidine as cells only incubated at 37°C or 67% of the cells incubated at 37°C were killed by exposure at A1°C for 3 days before ultraviolet irradiation. Figure 23 shows the experi- mental data again for cells incubated at A1°C for 3 days before ultra- violet irradiation as well zus the expected results from the cells incubated at 37°C if either of the hypotheses were correct. If the cells that were incubated at A1°C for 3 days incorporated 33% as much 3H-Thymidine as cells only incubated at 37°C, the sum of the total number of cells only incubated at 37°C containing either 3n - 1, 3n, or 3n + l grains per nucleus should equal the total number of cells containing n grains per nucleus. These hypothetical results are produced from each group of 3 adjacent points on Figure 22 for cells only incubated at 37°C. As shown in Figure 23, these hypothetical results closely match the experimental data. If incubation at A1°C for 3 days before ultraviolet irradiation killed 67% of the cells that would have been alive if they were only Figure 23. 10A Distribution curves representing the numbers of cells with indicated grains per nucleus. The solid circles represent the experimental data from Figure 22 for V-79 cells which were incubated at A1°C for 72 hours before ultraviolet irradiation. The hollow circles represent the expected values if the cells incor— porated 33% as much 3H-Thymidine as cells that were incubated at 37°C for 72 hours before ultraviolet irradiation. The triangles represent the expected values if 67% of the cells died and incorporated no 3H—Thymidine, while the remaining 33% of the cells incorporated as much 3H-Thymidine as cells that were incubated at 37°C for 72 hours before ultraviolet irradiation 105 _ d 1 _ r TI rl Tl I s D Au we I II-.. a . c . I. - 6 nor... IIIOIII IIOIIIIII A I. an.-u.--.mi-ol. : , ., 0 O O m 9 8 7 6 5 4 3 3.58 no 59232 I A We?” AA] e MI 40 GRAINS / NUCLEUS A \L ._ k r 80 IOO 60 20 Figure 23. 106 incubated at 37°C, the A67 of the 700 total cells should contain no grains in their nuclei since they are "dead" and therefore should not incorporate 3H-Thymidine. The 233 remaining cells should be distributed like the cells incubated only at 37°C, shown in Figure 22, since these cells should incorporate as much 3H—Thymidine as cells only incubated at 37°C. The number of cells containing a given number of grains per nucleus in the hypothetical results should be 33% of the number of cells containing the same number of grains per nucleus as for cells only incubated at 37°C. As shown in Figure 23, these hypo- thetical results do not fit the experimental data. The results of this experiment strongly support the hypothesis that the denaturation of the DNA repair enzymes is a rate limiting step in the death of single cells. Incubation at A1°C would cause a sufficient number of DNA repair proteins to denature so that only 33% of the 3H-Thymidine would be incorporated into the DNA of these cells as compared to cells that were only incubated at 37°C. The actual results obtained would be expected, with the cells dying at a later time. SUMMARY Chinese hamster lung fibroblasts were incubated at either 37°C or Al°C for 3 days before ultraviolet irradiation. Incorporation of 3H-Thymidine for 2 hours was measured by autoradiography. The results indicate that nuclei from cells incubated only at 37°C each contain approximately 3 times as much 3H—Thymidine as cells incubated at A1°C for 3 days. This supports the hypothesis that the denaturation of the DNA repair proteins is the rate limiting step in the thermal death of single cells. CHAPTER IV THE EFFECT OF TEMPERATURE ON THE REPAIR OF ULTRAVIOLET INDUCED PYRIMIDINE DIMERS IN THE DNA 0F MAMMALIAN CELLS AS MEASURED BY ENDONUCLEASE SITE SPECIFICITY INTRODUCTION An endonuclease from Micrococcus luteus has been used to monitor the progress of DNA excision repair in ultraviolet irradiated human cells 61. This endonuclease is utilized in a sensitive enzymatic assay for quantitating the occurrence of pyrimidine dimers in human DNA irradiated i§_zivg, The endonuclease purified from.M, luteus selectively produces single-strand breaks at dimer—containing sites (or nuclease- susceptible sites) in UV—damaged native DNA during the ip_vi§£g assay. The amount of endonuclease-induced single-strand breaks is determined by subsequent sedimentation of the endonuclease-treated DNA through alkaline sucrose gradients. The number of UV-induced pyrimidine dimers is proportional to the number of single—strand breaks produced by the endonuclease. Cells contain a number of enzymatic mechanisms to repair chemical 58,98-100 and physical damage to their DNA One mechanism is the excision repair of ultraviolet-induced pyrimidine dimers. This mechanism involves four (perhaps five) general steps: (the action of N-glycosidase), incision, excision, polymerization, and ligation. The effect of temperature on excision repair in mammalian cells was measured by using the endonuclease from.M, luteus. An effect of temperature observed with the use of this 107 108 technique implies that heat must significantly affect, at least, the incision step involved in excision repair. MATERIALS AND METHODS Cefi Chinese hamster lung fibroblasts (V—79) and the normal human skin fibroblasts Rid Mor CRL 1220 were obtained from R. B. Setlow, Biology Department, Brookhaven National Laboratory, Upton, N.Y. These cells were grown under humidified 10% C02 in air. The Rid Mor cells were at passages 1A through 21 and were transferred at a 3:1 split ratio. Normal skin fibroblasts (736 NF) were obtained at passage 18 from J. E. Trosko, Human Development Department, Michigan State University, East Lansing, MI., and were grown under humidified 5% C02 in air. W Growing cell cultures used for both stocks and experiments utilized a "D" medium (see Appendix A) supplemented with 5% fetal calf serum for the Chinese hamster cells and 10% fetal calf serum for the human fibroblasts. The confluent cells utilized for the experiments were maintained in "D" medium without arginine or iso— leucine (see Appendix A) supplemented with 5% dialysed fetal calf serum for the V-79 cells and 10% dialysed fetal calf serum for both strains of human fibroblasts. All media were supplemented with penicillin (160 units/ml) and streptomycin (160 pg/ml). Experimental Culture Procedure Approximately 100,000 to 200,000 cells were inoculated into 60 mm plastic Petri dishes (Corning Glass Works, Corning, N.Y.) 109 containing 6 ml of "D" medium supplemented with fetal calf serum. The cells were allowed to grow at 37°C for 2A hours or less until they were almost at confluence. At this point, the cells were either labeled with 3H-Thymidine at 0.1 to 0.3 pCi/ml (6.7 Ci/mmol, New England Nuclear, Boston, Mass.) or with lhC-Thymidine at()JX3pCi/m1 (5O mCi/mmol, New England Nuclear, Boston, Mass.) at 37°C. The V-79 cells were labeled for 18 hours, while the human fibroblasts were labeled for A8 hours. After this labeling period, the plates were at heavy confluent densities, containing either A x 106 Chinese 5 human fibroblasts. At this point, the hamster cells or A x 10 medium was changed to a "D" medium deficient in arginine and iso- leucine, supplemented with dialysed fetal calf serum at 5% (for V-79 cells) or 10% (for human cells). The cells were maintained in this deficient medium at either 37°C or at A1°C between 2A and 1AA hours. After the initial 2A hours of the incubation period, the medium was changed to fresh deficient medium” After this incubation period, the cells were sometimes incubated at 37°C for 1 hour with hydroxyurea added to a final concentration of 5 mM, Ultraviolet Irradiation and Repair The medium was decanted from the plates and saved just prior to ultraviolet irradiation. The tops were removed from the dishes and the cells were exposed to 25A nm ultraviolet radiation. The V-79 cells were always irradiated at a dose of 2.5 J/m2, while the human cells were always irradiated at a dose of 20 J/m2. Immediately after ultraviolet irradiation, the cells were incubated at 37°C in the spent, decanted medium for O, 6, or 2A hours. 110 Cell Lysis and Phenol Extraction of the DNA After the post-treatment incubation period, the medium was discarded and the cells were washed twice with either cold phos- phate buffered saline (for V-79 cells) or an EDTA-containing saline solution (for human cells). Cell detachment from the plates was facilitated by a rubber policeman in 2.5 ml of the same solu— tion. The cell suspension was transferred to centrifuge tubes. Sometimes suspensions of cells that were labeled with different isotopes were combined at this point. After centrifugation, the tubes were inverted to drain. The pellet was resuspended in a solution containing 0.1 M_Tris-HCl (pH 8), 0.2 M_NaCl, and 0.5 M_EDTA. Up to 1 ml of this solution was used to resuspend one plate of V-79 cells, while up to 2 ml was used for V-79 cells from two plates containing different radioactive isotopes. For human fibroblasts, 0.15 ml of the solution was used for cells from one plate, while 0.3 ml was used for cells from two plates containing different label. The cells were then pipetted to smaller tubes; 10% Sarkosyl (Ciba Geigy Corporation, Ardsley, N. Y.) in the same solution was added to the cell suspension to a final concentration of 0.33%; pronase (Calbiochem, La Jolla, Calif.) was added to give 0.13 mg/ml, and the cell suspension was incubated at A5°C for 10 minutes to lyse the cells. After this period, the lysed cells were usually frozen at —20°C. After thawing, the lysed cells were incubated at 37°C for one hour. If necessary, the lysed cells from two tubes each containing cells from only one plate were combined before this incubation lll period. An equal amount of phenol, neutralized at pH 8 and equili- brated with endonuclease buffer [0.02 M_Tris-HC1 (pH 8), 0.0A M_NaC1, 1 mM_EDTA], was added to the lysate, and the samples were gently rotated for l to 2 hours at room temperature. The DNA solution was separated from the phenol by centrifugation at room temperature. The upper aqueous phase containing DNA was collected and extracted twice with an equal volume of ether to remove most of the phenol. The DNA solution was dialysed overnight against 500 m1 of endonuclease buffer at A°C. The Endonuclease Assay The endonuclease used was a crude extract equivalent to Fraction III of Carrier and SetlowlOl. Five microliters of the extract (5 mg protein per ml) was added to each 100 pl of the DNA solution. Incu- bation at 37°C for 20 minutes was sufficient to take the enzymatic reaction to completion. Usually either 100 pl or 200 pl of the DNA was used in the endonuclease assay. The reaction was terminated by removing the mixture with a wide—tipped micropipet and layering it on top of a 5% to 20% alkaline sucrose gradient containing 0.5 M_NaCl; with 0.2 ml of a lysing solution, containing 0.5 M NaOH and 0.05 M EDTA layered on its top, and a 60% sucrose cushion added to its bottom. The DNA was sedimented at 20°C in an SW 60 rotor of a Beckman L5-50 ultracentrifuge at 50,000 rpm for 60 to 120 minutes. An SW 56 rotor was used for the DNA from human 736 NF cells. Fractions were collected starting from the bottom of the gradient and the acid- insoluble radioactive material was placed in vials containing a scintillation cocktail of 12 g Permablend (90% PPO and 10% M2-P0POP) per 3.8 liters toluene and was counted in a scintillation counter. 112 The labeled DNA from V-79 and Rid Mor cells was counted in either a Beckman or Packard counter in the laboratory of R. B. Setlow, Biology Department, Brookhaven National Laboratory, Upton, N.Y. The labeled DNA from 736 NF human fibroblasts was counted in a Packard Tri-Carb liquid scintillation spectrometer (Model 3320), with discriminators set between 250 and 500 for tritium and between 200 and 1000 for th. The gain was set at 100% for tritium and 6.85% for 1’4C. The distribution of counts was converted to average molecular weights by a computer program as described elsewhere72 and in Appendix B. The amounts of radioactivity per gradient were between 3,000 and 12,000 cpm of tritium and between 2,000 and 7,000 cpm of th from V-79. There was between 800 and 8,000 cpm of each isotope in DNA from human cells in each gradient. Calculations of Endonuclease—Sensitive Sites The weight—average molecular weight, Xm.n,°m. M 1 1 1 w _ Zm.n. 1 1 was used rather than the number—average molecular weight, 2mini Mn = 2n. ’ 1 because Mn is very sensitive to fluctuations in the amount of DNA near the top of the gradient. It was assumed that the breaks were distributed randomly and therefore Mn = Mw/2' The number of breaks per unit weight of DNA is the reciprocal of Mn. The change in l/Mn as a result of endo- clease treatment should represent the number of endonuclease-sensitive sites per dalton of DNA. 113 RESULTS Experiments Involving Chinese Hamster Lung Fibroblasts The use of M, luteus endonuclease provides a sensitive means to detect pyrimidine dimers in DNA induced by ultraviolet radiation. The DNA from Chinese hamster lung (V-79) cells was always incubated at confluence at either 37°C or A1°C for 72 hours before ultraviolet irradiation in a medium deficient in arginine and isoleucine. After this 72 hour period, the cells were incubated for 1 hour at 37°C with hydroxyurea, added at a concentration of 5 mM, The DNA from the V-79 cells was always sedimented at 50,000 rpm for 60 minutes. In one experiment, V-79 cells were incubated for 3 days at 37°C and then were either exposed to 2.5 J/m2 UV radiation or not exposed. After irradiation, the UV irradiated cells were mixed and lysed immediately with non-irradiated cells. The resulting sedimentation data of Figure 2A indicates that the MV for UV ir- radiated cells is 23.A9 x 106 daltons and for non-irradiated cells is 6A.A3 x 106 daltons. The number of endonuclease sites per dalton that were produced by 2.5 J/m2 UV radiation is given by 6 2(1/23.A9 - l/6A.A3))c10- = 5.A x 10-8. When the same experiment was repeated without using the endonuclease, the MV for UV irradiated cells was 61.75 x 106 daltons and for non-irradiated cells was 62.A8 x 106 daltons. The number of breaks per dalton that were produced by 6 = O.A x 10‘8. 2.5 J/m2 UV irradiation was 2(1/6l.75 - 1/62.A8) x 10- Thus, ultraviolet radiation alone produces an insignificant number of breaks in DNA when this is compared to the number of endonuclease sensitive sites it produces. Figure 2A. 11A Sedimentation profiles of extracted DNA from V-79 cells after treatment with M, luteus endonuclease. Values of MV were 6A.A3 x 106 (Fractions 1 to 27) for non-irradiated cells (solid circles) and 23.A9 x 106 (Fractions 5 to 28) for cells ultraviolet irradiated at 2.5 J/m2 (hollow circles) 115 I I I I I I4 - 5 I2 - _ 3; IO P - > i: I 3 8 _ N, \ .. g N / IN; < I A ’ ‘ a: I \ ,4 \ _ I \ E 6 ' \‘x I A .- 3 I, ‘17; \ a: I K, 3‘ 4 - I A .. I I \ A II ’5‘ k \ I ‘\ 2 I- ”J p’}! A.‘ \W’O‘G- J 09/11 vaflwn o Owl I I I I ' I O 5 IO IS 20 25 30 FRACTIONS <——- SEDIMENTATION Figure 24. 116 In another experiment, the V-79 cells were incubated for 3 days at 37°C, and subsequently exposed to 2.5 J/m2 ultraviolet radiation. After UV irradiation, the cells were either lysed immediately for 10 minutes at A5°C, and then frozen, or were incubated for 6 hours at 37°C before being lysed and frozen. After thawing, the lysed cells were mixed and the remainder of the procedure was carried out. The resulting sedimentation data of Figure 25 indicates that cells lysed immediately after UV irradiation had Mw of 20.77 x 106 daltons and that cells that were incubated for 6 hours at 37°C, after UV irradiation, had Mw of 26.50 x 106 daltons. The number of endonuclease sites per dalton that were removed during the 6 hour incubation period is 6 = 2.1 x 10'8. 2(1/20.77 - 1/26.5o) x 10' In order to determine the effect of temperature on the amount of endonuclease sites removed, the cells were incubated at either 37°C or A1°C for 3 days before UV irradiation. After UV treatment at 2.5 J/m2, the cells were incubated at 37°C for 6 hours. After this 6 hour incubation period, the cells were mixed together and the rest of the procedure was carried out. The resulting sedimentation data of Figure 26 indicates that Mw is 23.18 x 106 daltons for cells incubated for 3 days at A1°C and 25.37 x 106 daltons for cells incubated for 3 days at 37°C. The difference in the number of endo- nuclease sites removed per dalton during a 6 hour incubation is 6 = 0.7A x 10-8. In an identical experiment, 6 = 0.90 x 10'8. 2(1/23.18 — 1/25.37) x 10“ this difference was 2(1/25.53 - l/28.85) x 10- Another identical experiment conducted without endonuclease, but with the same samples as in the experiment just mentioned, gave the 6 difference 2(51.25 — 1/53.28) x 10' = 0.15 x 10'8. Therefore the Figure 25. 117 Sedimentation profiles of extracted DNA from V—79 cells after treatment with M, luteus endonuclease. Cells were exposed to 2.5 J/m2 ultraviolet radiation. Values of MV were 20.77 x 106 (Fractions 2 to 26) for DNA extracted immediately after irradiation (solid circles) and 26.50 x 106 (Fractions 2 to 26) for DNA extracted 6 hours after irradiation (hollow circles) 118 I4- — I2? " )- I:l0- g - > E f; < _ _ c5’8 ’I \I s 7‘4 ‘\ e— I,’ 3A _ '2 If ‘\ m # \ 32 ,1’ \p 33 4" If '- O 5 IO I5 20 25 30 FRACTIONS ‘ <-— SEDIMENTATION Figure 25. lo Figure 26. 119 Sedimentation profiles of extracted DNA from V-79 cells after treatment with M, luteus endonuclease. Cells were exposed to 2.5 J/m2 ultraviolet radiation and the DNA was extracted 6 hours after irradiation. Values of MV were 25.37 x 106 (Fractions 1 to 25) for cells that were incubated at 37°C for 72 hours before irradiation (solid circles) and 23.18 x 106 (Fractions 1 to 25) for cells that were incubated at thC for 72 hours before irradia- tion (hollow circles) I4 I2 5 PERCENT RADIOACTIVITY O 120 #‘x (x _. 1:, ,4 \\ _ 2,, \qu II; .. if} Hg .. Mg 1 l I I l 1 o 5 no :5 20 25 30 FRACTIONS <-—— SEDIMENTATION Figure 26. 121 difference in the removal of endonuclease sites between cells incubated for 3 days at 37°C and ones incubated for 3 days at thC, 8 = 0.75 x 10’8. Thus, in two per dalton, is (0.90 - 0.15) x 10‘ experiments, pre-UV hyperthermia results in the inhibition of subsequent removal of endonuclease sensitive sites that are produced by ultraviolet radiation. Experiments Involving Human Skin Fibroblasts When human skin fibroblasts were used, the pre-UV incubation period was varied between 1 and 6 days, while the cells were main- tained at confluence in a medium deficient in arginine and isoleu- cine. After this period, hydroxyurea was either added at a concen- tration of S mM_for a period of one hour at 37°C, or not used. Sedimentation of DNA was always at 50,000 rpm, but the period varied from 60 to 120 minutes. Figure 27 shows the sedimentation data for the Rid Mor cells that were incubated for 3 days at 37°C and treated with hydroxyurea before they were either exposed to 20 J/m2 of ultraviolet radiation or were not irradiated. The number of breaks produced per dalton by UV radiation and endonuclease action was 2(1/7.06 - l/Sl.75) x 10-6 : 2h.5 x 10-8. When the identical experiment was run with the same samples, but with the endonuclease left out, the number of breaks per dalton produced by the UV irradiation was 2(l/h5.32 - l/h7.15) - -8 x 10 6 = 0.17 x 10 . Therefore, the number of endonuclease sites —8 produced per dalton by UV radiation was (2h.5 - 0.17) x 10 = 2h.3 x 10'8. In another experiment, the Rid Mor cells were incubated for 3 days at 37°C and treated with hydroxyurea before UV irradiation Figure 27. 122 Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M. luteus endonuclease. Values of Mw were 51.75 x 106 (Fractions 1 to 29) for non-irradiated cells (solid circles) and 7.06 x 106 (Fractions 1h to 29) for cells ultraviolet irradiated at 20 J/m2 (hollow circles) 123 l4— l2P _ _ p m 8 6 >._._>_._.o._._>_._.o. I— 3 ac ,fly’hX ‘ Q \ 3‘: I It ,_ ., .. ii I‘M \ _ Z [I g] ‘3‘} k ‘3 9‘4 J \ 33 4 _. f f IR \ .. a. I ‘R \ I" \ \ / I It 2.. p/N y xxx _ cod 5" 23551 0W1 I I I I I o 5 IO I5 20 25 so 35 FRACTIONS <—SEDIMENTATION Figure 34. Figure 35. inc Sedimentation profiles of extracted DNA from human skin fibroblasts (Rid Mor) after treatment with M, luteus endonuclease. Confluent cells were incubated at A1°C for 1AA hours in medium deficient in arginine and isoleucine before exposure to 20 J/22 ultraviolet radiation. Values of MV were h.0h x 10 (Fractions 5 to 31) for DNA extracted immediately after irradiation (solid circles) and 5.20 x 106 (Fractions 2 to 31) for DNA extracted 2h hours after irradiation (hollow circles) 1141 '4 _ I I I l I I L I2 — - 1 3:- IO — e 2 [-— , ‘4’ e _ II \\ _ ‘5’ M i E d ‘2. \ E 6 b ’I III - :3 II I‘ 95 ’ i). O. 4 — fl; \h\ — \\ 35% ‘ I I I l I I5 20 25 3O 35 FRACTIONS é— SEDIMENTATION Figure 35. d 1h2 F.0H Hm.» mm.m m a: mZIwmw Fm m.am ma.» 20.: H a: mZIwmw mm o.HH om.m :0.: m a: so: cam mm w.om :w.w Ho.: m a: 902 cam :m m.om Hm.» NH.: H a: no: dam mm 3.:m mm.» 20.: w am so: cam mm 0.2m mm.» no.2 m Pm so: oflm Hm w.~m mm.» ww.m a Pm so: cam om mason :m soapwfiowhafl soapwflvmaafl ca pmaow>wapaz pmaow>wppad mGOpHmU gouge ampmw Amhwcv Aoov woa pom mhdon 2m hampmfiwoaaw mawp myopwammsop cm>oamp mmpflm oofipowaPXm cowpomapxm mummaoscovom 3 GOprfivwapw pmaofi>mhpad vamp mo Amoopamw woav Z apnwfims maommp coanoa mmhp cw popsdz amazomaoa mmwam>w pnwfloz coflpwnoocfl HdwpflqH Hamo madmwm mumcHnoapHa case amass Ga ©m>oamh mmpfim m>flpflmcom mmwmaoscowsm .m manme lh3 When the above experiment was repeated with 736-NF human fibro- blasts, pre-UV incubation at Al°C for 6 days resulted in cell detach- ment from the plates. The 105 minute sedimentation data for 736-NF cells, pre-UV incubated at Al°C for 1 and 3 days, are shown in Figures 36 and 37. The number of endonuclease sites removed per dalton in 2h hours at 37°C is given in the last two rows of Table 2. Increasing the pre-UV period of hyperthermia from 2h hours to 72 hours resulted in a decreased removal of sites after UV irradiation. A final experiment determined the influence of hydroxyurea on the number of endonuclease sensitive sites. Rid Mor cells, incubated for 3 days at 37°C or Al°C, were either untreated or treated with hydroxyurea at 5 mM_for one hour at 37°C before UV irradiation at 20 J/m2. All DNA was sedimented for 120 minutes. When cells were pre—UV incubated at 37°C, the number of endonuclease sensitive sites per dalton that the untreated cells had over the hydroxyurea treated 6 cells was 2(1/h.20 - l/h.2l) x 10- = 0.1 x 10—8. When cells were pre-UV incubated at Al°C, this value was 2(1/5.26 - 1/5.18) = 0.6 x 10-8. When cells that were pre-UV incubated for 3 days at 37°C were post-UV incubated for 2h hours at 37°C in the same pre-UV medium, the difference in the number of endonuclease sites per dalton between untreated cells 6 8 = 13.7 x 10' . In a repeated experiment, this value was 2(1/9.h2 - l/6.15) x 10-6 = and hydroxyurea treated cells was 2(1/7.87 - 1/5.1l) x 10- ll.3 x 10-8. This averages to 12.5 x 10-8. The inhibition that hydroxy- urea has on the number of endonuclease sites removed per dalton during 2h 8 = 12.6 x 10'8. Thus hours after UV irradiation is (12.5 + 0.1) x 10- hydroxyurea inhibits the removal, but not the production, of endonuclease sensitive sites in DNA. Figure 36. 1M Sedimentation profiles of extracted DNA from human skin fibroblasts (735—NF) after treatment with M, luteus endonuclease. Confluent cells were incubated at K1°C for 2h hours in medium deficient in arginine and isoleucine before exposure to 20 J/m2 ultraviolet radiation. Values of Mw were A.0h x 106 (Fractions 9 to 25) for DNA extract- ed immediately after irradiation (solid circles) and 7.12 x 106 (Fractions 1 to 25) for DNA extracted 2h hours after irradiation (hollow circles) lIIS I 3O W II I 25 \Il.‘ \ \e\ b\\ CF“ bIII IIIIMJ. I’ll-I'll 5 d, If! dill I‘ll Io/l 1) r 1W N... m u _ _ _ _ _ _ . O 4 2 O 8 6 4 2 O >._..>_._.o<0_omthd ca no GOHPw>HpU< #QmeGOU mpwm hwdmmm .HO #93054 ORPHOQ. fiOfiPdQfiOGH mmhfimfih PQHOQ aaoaoa ac soauapassa aosaoap obs .m canoe 155 1. k T ' student-)5] C The Equations (2h) are plotted in Figure 38 for temperatures (T) at 37°C, ho°c, and h1°c, with TC = 325°K and b = -6h.5 cal/mol °K, the constants for irreversible protein denaturation and single cell death. Since thermal inhibition of excision repair may in— volve irreversible protein denaturation and be related to cell death, this process of inhibition is expected to have the same constants. When Equation (1) of page 1 of this dissertation is integrated, it yields 1n (NO/N) = th (25). If T1 and T2 are two different temperatures, the difference between the rate constants at these temperatures is kD(Tl) - kD(T2) = (1/t) 1n [N(T2)/N(Tl)] (26). The rate constant differences, kD(T) - kD(37°C), are shown in Figure 38 for T = AO°C and T = Al°C. The data from all experiments showing an effect on repair with pre—UV hyperthermia are compiled in Table 3. The activation enthalpy, AH+, is read from Figure 38 for corresponding rate constant differences. The average activation enthalpy for V-79 cells is 179 kcal/mol, ranging from 162 to 213 kcal/mol. For V-79 cells, 16 out of 17 experiments lie within 179 i 17 kcal/mol. For human skin fibro- blasts, the average activation enthalpy is 199 kcal/mol, ranging from 180 to 235 kcal/mol. 156 EVIDENCE THAT THE DENATURATION OF THE DNA REPAIR PROTEINS IS A RATE LIMITING STEP IN THE THERMAL DEATH OF SINGLE CELLS The results from unscheduled DNA synthesis and autoradiography indicate that a significant decrease in the amount of excision repair precedes death in V-79 cells, following an incubation period for three days at either AO°C or Al°C. This suggests that the thermal impairment of a DNA repair process, such as excision repair, is the rate limiting step for the thermal death of V-79 cells. The results from endonuclease site sensitivity indicate that the thermal impairment of the incision step (perhaps the thermal denaturation of a dimer specific endonuclease) is the rate limit- ing step in the thermal damage of the excision repair process for both V-79 cells and normal human skin fibroblasts. Since the "repair ratios" and "thermal inhibition of repair" activation enthal- pies (Table 3) were roughly the same whether repair was measured by 3H—Thymidine incorporation or by endonuclease site specificity, it appears that the lower 3H-Thymidine incorporation into DNA, observed after UV irradiation following 3 day incubation at AO°C or at Al°C, is primarily due to the thermal impairment of the in- cision step of excision repair, rather than due to the thermal impairment of the mechanisms for transport and phosphorylation of exogenous 3H-Thymidine into the 3H-dTTP which is to be incor- porated into the damaged DNA. Other investigators measured thermal death in cells by the loss of the ability of a cell to proliferate sufficiently to form a grossly visible clone in l weekl6. They determined the activation enthalpy for the thermal death in a subline of the V strain Chinese 157 'hamster lung fibroblasts to be 185 kcal/mol. This value is very close to the activation enthalpies of "thermal inhibition of repair" for Chinese hamster lung fibroblasts (V-79) as compiled in Figure 38 and Table 3, suggesting that the thermal impairment of a DNA repair process, such as the incision step of excision repair, is a rate limiting step in the thermal death of Chinese hamster lung fibro- blasts. Although three day incubation of V-79 cells enhanced the rate of detachment (a characteristic of cell death) after this period, Figure 17 shows that ultraviolet irradiation at 15 J/m2 did not affect the rate of detachment. This implies that a temperature sensitive mechanism other than excision repair must be necessary to keep the cells attached to the surface of the culture dish. The thermal damage to this mechanism must be the rate limiting step for cell detachment since the period of hyperthermia precedes cell detachment. However, the activation enthalpies for the ther- mal damage of this mechanism and excision repair must be roughly the same since the period of hyperthermia is the same for the ef- fects of the thermal damage of these mechanisms to be expressed. Other investigators measured the effect of hyperthermia on the repair of x-ray damagelou-106. Although single— and double- strand breaks are not detectable after hyperthermia at temperatures between Al°C and h5.5°C107 , x-ray induced strand—break rejoining was found to be significantly inhibited by prior heating to these h temperatureslo . Furthermore, thymine base damage of the 5', 6'- dihydroxydihydrothymine (t') type is produced by hydroxyl radicals attacking the DNA during the x-irradiation of cellleB. The 158 excision repair of the t'-type product from x—irradiated DNA has 109 been shown in a number of cell lines Although it was shown that hyperthermia at h5°C did not produce an excess of t'-base damage in Chinese hamster ovary (CHO) cells and that 15 minute hyperthermia at h5°C before x-irradiation did not result in more base damage than in unheated cells, the excision of the t'-type products is inhibited by as much as 33% by preheating the CHO cells 105 at h5°C for 15 minutes before x-irradiation The activation enthalpy was calculated by using Equations (2h) and (28) and by assuming the compensation law. Using N(h5°C)/N(37°C) = 0.67, t = 15 minutes, and T = h5°C, and assuming kD(37°C) as negligible, the activation enthalpy for the inhibition of the excision of the t'-type products in CHO cells becomes 18h kcal/mol. This value is very close to the activation enthalpy for thermal death in Chinese hamster lung fibroblasts and for the inhibition of UV- induced dimer excision repair in V-79 cells. Other investigators did not find an effect on repair when V-79 cells were heated to h2°C for two hours before x-irradiationlO6. However when Equation (2h) was used with T = h2°C, t = 2 hours, and the activation enthalpy assumed to be 18h kcal/mol, N(h2°C)/N(37°C) became 0.95, an effect difficult to detect. It was mentioned in Chapter I (p. 13) that there were at least five possibilities which could account for the decrease in repair with hyperthermia. Possibilities (2), (3), and (h) are shown to be inconsistent with the results from the experiments involving excision repair of UV—induced dimers. Regarding possibility (2), the manufacturing rate of repair proteins did not decrease after 159 hyperthermia since cycloheximide, an inhibitor of protein synthesis, did not inhibit unscheduled DNA synthesis in UV-irradiated or non- irradiated V—79 cells that were incubated for 3 days at either 37°C or A1°C. However, the manufacturing rate of the repair proteins may have decreased during the period of hyperthermia before cycloheximide was added. Regarding possibility (h), processes which damage the DNA do not have their rates increased more than the rate of repair since a three day incubation at Al°C does not produce more endonuclease sensitive sites in the DNA of cells that were UV-irradiated or non- ‘irradiated. This result is also inconsistent with possibility (3); thus repair proteins are not converted into damaging proteins. How- ever, both possibilities (l) and (5) are consistent with the results of the experiments. Not only might a dimer-specific endonuclease denature and repair ineffectively or not at all (1), but proteins in close proximity to the pyrimidine dimer may denature and interfere with the incision step in the excision repair process (5). It was reported that exposing cells to temperatures greater than 37°C causes a temperature- and time—dependent increase in the protein-to-DNA ratio 109 of chromatin An excessive amount or protein adsorbed onto chromatin might render the DNA inaccessible to the function of the 105 excision enzymes . THE ACTIVATION ENTHALPIES OF THE DNA REPAIR PROTEINS DETERMINING THE MAXIMUM LIFE SPAN OF A MULTICELLULAR ORGANISM The V strain of Chinese hamster cells was originally isolated from the lung tissue of Cricetulus griseus, the Chinese striped hamsterllo. The life spans of mice and rats, which are closely related to the hamster, follow the power law of Equations (5) and (6) up to 160 the age of 1000 days, with n = 5 1. Hamsters usually live up to two years and hibernate during the winter allowing their body tem— peratures to fall to A°C. Also the species Cricetulus griseus is particularly susceptible to cancer and their cells tend to have greater amounts of chromosomal aberrations and polyploidylll. Thus Cricetulus griseus may spend only a year with a body temper- ature of 37°C. If the power law is to be valid over this year, pt should be less than one for t up to one year. This means that 0 should be less than 0.0027 day-l. If 0 equals k the rate constant for the D’ rate limiting steps leading to the death of the hamster, then accord- ing to Figure 38, the activation enthalpy (AH+) for the rate limit- ing steps should be greater than 193 kcal/mol. This value is high but within the range of activation enthalpies for thermal inhibition of excision repair as measured by unscheduled DNA synthesis in cells taken from Cricetulus griseus (V—79). Moreover, it is very close to the activation enthalpy of 196 kcal/mol for the thermal impairment of the incision step in excision repair as measured by endonuclease site specificity. Thus the thermal denaturation of a DNA repair protein, such as a dimer specific endonuclease, might be a rate limit- ing step in the death of Cricetulus griseus. CHAPTER VI CONCLUSIONS As a living cell procedes through time, thermal fluctuations will tend to denature its proteins. Some of the proteins that are associated with DNA are potentially lethal to the cell when denatured, particularly those involved in repair. Thus a protein near a damaged portion of the DNA may denature and inhibit the repair of the damage, or a protein involved in the repair process may denature preventing any further repair of the damage from taking place. Unrepaired damage of the DNA might either be lethal to the cell or be carried along as a mutation to its daughter cells if the cell is allowed to divide. Hyperthermia enhances the rate of these protein denaturations, consequently promoting cell death. One type of damage to DNA is exemplified by the production of pyrimidine dimers by ultraviolet radiation. In the excision repair of UV-induced dimers, the incision step is necessary before the other steps can follow. A protein denaturing near the dimer site might prevent the incision mechanism from producing a nick in the DNA near the dimer site; or a protein of the incision mechanism itself, such as a dimer specific endonuclease, might denature to a product unable to carry out incision at the dimer site. The dimer will persist until an intact incision mechanism is accessible to the dimer site. This might not occur until after the presence of the 161 162 dimer is expressed in a form that is lethal to the cell. For example, a dimer occuring at a segment of the DNA encoding for a protein might cause the segment to be mistranscribed. This might result in the production of a lethal protein or a useless protein in place of a protein that is necessary for cell survival. The dimer might also fall within a regulatory site interfering with cell proliferation. Since the effects of the presence of the dimer are immediate, the rate limiting step for these effects is the thermal denaturation of the protein that prevents the incision step in the excision repair of the dimer. By stimulating protein denaturation with hyperthermia, it was shown that the thermal impairment of the incision step was the rate limiting step in the thermal inhibition of the excision repair of UV—induced dimers for both V-79 cells and human skin fibroblasts. By using the compensation law, it was shown that the activation enthalpy for thermal hindrance of the incision step was roughly equal to the activation enthalpy for the death of V-79 cells. Furthermore, thermal hindrance of excision repair was observed to precede death in the Chinese hamster fibroblasts. Thus it might be concluded that the thermal denaturation of a protein associated with the incision step of excision repair of ultraviolet radiation-induced pyrimidine dimers is the rate limiting step for one of the processes leading to the death of V—79 cells. Unrepaired DNA in cells of multicellular organisms can perhaps lead to the death of the organism. This damage might transform a cell into a cancer cell; it might lead to the production of a substance that is lethal to the organism; or it might lead to the death of a few 163 cells that are necessary for the proper functioning of a control center within the organism. Thus the activation enthalpy for the denaturation of a protein associated with DNA repair might be an important factor for determining the life span of an organism. The evolution of a longer lived species might be accomplished by increasing the activation enthalpy for thermal denaturation of these DNA repair proteins and by increasing the replacement rate of these proteins. Hypothesized DNA repair proteins, that may denature on the order of the life span of g, griseus, have an activation enthalpy close to the activation enthalpy for the thermal inhibition of the removal of endonuclease sensitive sites, when the compensation law is used to calculate these activation enthalpies. This suggests that the thermal denaturation of a DNA repair protein, such as a dimer specific endo- nuclease, is the rate limiting step for one of the processes leading to the death of the Chinese striped hamster. CHAPTER VII RECOMMENDATIONS Further experiments can be conducted to relate the denatura- tion of DNA repair proteins and the thermal death of single cells. It was mentioned in Chapter V (p. 158) that two possibilities could account for the observed decrease in repair with pre-UV hyperthermia. One possibility is the thermal denaturation of a protein that is involved in the incision step of excision repair, such as a dimer specific endonuclease. Another possibility is the thermal denatura- tion of a protein in close proximity to the pyrimidine dimer site, which may interfere with the incision step of excision repair when denatured. Dimer—specific endonuclease from M, luteus can be heated at various temperatures before being assayed with UV irradiated DNA as a substrate. The first possibility is supported if the endonuclease pre-heated at higher temperatures made fewer breaks in the DNA during the assay. If the cells were treated with a protein inhibitor, such as cycloheximide throughout the entire period of hyperthermia, an investi- gator could determine whether the manufacturing rate of the repair proteins decreased at the higher temperature. Furthermore, if cells were heated at three or more different temperatures above 37°C before ultraviolet irradiation, an activation enthalpy for the thermal inhi- bition of repair could be obtained directly, without the benefit of the MA 165 compensation law. The duration of the pre-UV hyperthermia should be long enough to observe an effect and short enough as to not cause the lethality of the cells. Ideally, cells inoculated from the same original culture should be heated simultaneously in three or more separate incubators set at different temperatures. This is because there appears to be some variation in the amount of excision repair after ultraviolet irradiation that may depend on the history of the culture from which the cells were inoculated, when the inoculation took place, or the concentration of the cells during inoculation. More intensive studies of this above mentioned variation in excision repair should be conducted in order that this variation be controlled. Experiments can be conducted with other means of prevent- ing "semi-conservative DNA replication" such as lowering the serum concentration of the medium. Since the amount of excision repair determined by the dimer-specific endonuclease assay is not masked by "semi-conservative DNA synthesis", experiments can be conducted with confluent cells that are not required to be maintained in a medium that is deficient in arginine and isoleucine. Since incubation in deficient medium might tend to reduce protein synthesis, experiments can be conducted with cells in media containing arginine and isoleucine to determine if DNA repair proteins are replaced. If they are replaced, pre—UV hyperthermia might affect the rate and amount of replacement. The effect of pre-UV hyperthermia on excision repair can be observed in repair deficient mutants to determine if the repair proteins in the mutants are more sensitive to heat than those in normal cells. These experiments can be conducted with cells from other tissues and other organisms to determine if there is a variation in the thermal inhibition 166 of repair with different tissues and different organisms. A study can be conductedix>determine if repeated UV irradiation can enhance the amount of excision repair. The effect of hyperthermia with repeated UV irradiation of DNA on excision repair can be determined. Finally, more extensive studies can be conducted on the effect of pre-treated hyperthermia on repair of damage caused by treatments other than UV irradiation, such as x—ray, gamma ray, or chemical treatment. APPENDICES Appendix A 'The media used in the present investigation are designated as "C-15", "D", and "D" without arginine or isoleucine. Media "C—15" and "D" was prepared from a specially ordered 10 liter package of Eagle's minimal essential medium (EMEM) which had 50% increase of all the essential amino acids, except glutamine, 50% increase of all the vitamins, 100% increase of all the non-essential amino acids, but without glucose and phenol red (Gibco, Grand Island, NY). In addition, the 10 liter aqueous solution of "D" medium contained 1.1 g sodium pyruvate, 10 g glucose, h.87 g sodium chloride, 10 g sodium bicarbonate. Medium "C—15" was prepared in the same manner as "D" except that 15 g sodium bicarbonate was used. In addition, "C-15" medium contained 10 mg phenol red powder, 50 mg hypoxanthine, 50 mg thymidine, and 50 mg uridine. Medium "D" without arginine or isoleucine was prepared in exactly the manner as "D" medium except that the specially ordered package of EMEM was deficient in arginine and isoleucine. 167 Appendix B ‘The average molecular weights of DNA from Rid Mor cells were calculated from a