. 3,...— --—_ K“ ‘ ‘. .__ Le :5-535; , -. Y in; Michigan State Universi W masts .— This is to certify that the thesis entitled The Isolation and Partial Characterization of Chinese Hamster Fibroblasts with Altered UV Sensitivity and/or DNA Repair presented by Roger Alan Schultz has been accepted towards fulfillment of the requirements for , , netics Ph. D degree in Ge Major professor QM, c” M / Date W; ’5 /;fl) - / / 0-7639 brig .H ‘ ~a '.' :féli‘ii; -- 1f.- ~imvr.'~.«..' ' , Z '5] .1 E FNS: Zflurdupcrttu RETURN!“ LIBRARY MTERIALS: Place in book return to reuove charge from circulation record THE ISOLATION AND PARTIAL CHARACTERIZATION OF CHINESE HAMSTER FIBROBLASTS WITH ALTERED UV SENSITIVITY AND/OR DNA REPAIR BY Roger Alan Schultz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Interdepartmental Program 1980 ABSTRACT THE ISOLATION AND PARTIAL CHARACTERIZATION OF CHINESE HAMSTER FIBROBLASTS WITH ALTERED UV SENSITIVITY AND/OR DNA REPAIR BY Roger Alan Schultz The isolation and characterization of bacterial mutants with abnormal UV sensitivity or defects in DNA repair have contributed extensively to our present understanding of prokaryotic DNA repair mechanisms and their role in muta- genesis. A similar approach is currently proving produc- tive in the analysis of yeast and Drosophila DNA repair pro- cesses. Studies in mammalian somatic cells, however, have primarily been restricted to normal cells and cells from patients with one of a few known syndromes which exhibit abnormal responses to mutagens and/or altered DNA repair functions. Techniques have been reported whereby the induction, selection, and partial characterization of pre— sumed mammalian DNA repair mutants have been described. The success of these efforts has been limited by the rela- tive tedious nature of these selection techniques, insta- bility of mutants isolated, or the use of cell lines not easily adaptable to mutation assays. Reported here is the Roger Alan Schultz isolation and partial characterization of several Chinese hamster fibroblast cell lines with alterations in DNA repair and/or UV sensitivity. The isolation procedure consisted of (l) mutation induction by 5-bromodeoxyuridine-black light and ultraviolet light (UV) treatments; (2) incorporation of tritiated thy- midine in repair proficient cells at high temperature (38°C) following UV damage; (3) cold holding (4°C) of these cells to permit tritium-induced killing; and (4) recovery and testing of repair-deficient and UV sensitive cells which have survived and formed colonies at low temperature (34°C). Seventy-two surviving colonies were isolated from 2 x 107 cells plated for selection. Of the 72 colonies, 20 demon- strated potential interest and 4 were selected for extensive study. One mutant, identified as UVS-7, was found to be slightly more sensitive to UV, not sensitive to x-rays or N-acetoxy-Z-acetylaminofluorene (NAc-AAF) and exhibited a highly reduced level of unscheduled DNA synthesis (UDS), as compared to the parental line. Two additional lines, UVS-4O and UVS-44,were sensitive to UV, x-ray, N-methyl- N-nitro-N-nitrosoguanidine, and NAc-AAF but exhibited only slightly reduced UDS. A fourth line, UVr-23, had enhanced UDS, was resistant to UV, but exhibited no abnormal sensi- tivity to x-ray or NAc-AAF. Mutation studies revealed UV2-7 to exhibit a UV-induced hypermutability as compared to parental control cells at the three loci measured by Roger Alan Schultz resistance to ouabain, 6-thioguanine, and diphtheria toxin. Analysis of the spontaneous mutation rate to 6-thioguanine resistance demonstrated that the rate for UVS-7 was not at variance with that of the parental control cells. UV- induced mutation experiments also suggested hypermutability for UVs-40, hypomutability for UVr-23, and no significant deviation in the UV-induced mutability of UVS-44, as com- pared to parental control cells. The data confirm the contribution of deficient excision-repair to mutagenesis and support the existence of an alternative repair mechanism. Further characterization of the mutants discussed here or employment of the isola- tion technique to select new mutants should prove useful in the analysis of mammalian DNA repair processes and the details of mutation fixation. To Barbara for sharing so very much and having so much to share To my father and mother for love and support ii ACKNOWLEDGMENTS I wish to extend a special thanks to Dr. James E. Trosko and Dr. Chia-cheng Chang for their enormous contribu- tion to my general graduate training and the theoretical discussions and technical labors necessary to accomplish the work presented here. I also wish to thank Dr. James Higgins and Dr. Loren Snyder for the interest and time that they contributed to this project and my education, as mem- bers of my graduate committee. I also wish to thank Larry Yotti and Stephen Warren, as well as Marc Castellazzi, Stephen D' Ambrosio, Betty Dawson, Judy Funston, Thomas Glover, Carolyn Grossman, Carol Lassila, Philip Liu, Beth Rupp and Gen Tsushimoto. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . vii LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . .viii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . , . . . . . . . . . . . . . . . 5 The Use of Genetic Mutants to Examine DNA Repair and Mutagenesis . . . . . . . . . . . . . . . . . 5 Bacteria . . . . . . . . . . . . . . . . . . . . 5 Lower Eukaryotes . . . . . . . . . . . . . . . . l3 Mammalian Cells . . . . . . . . . . . . . . . . 16 Repair in Chinese Hamster Cell Strains . . . . . . . 22 Additional Factors Relevant to Mammalian DNA Repair and Mutagenesis . . . . . . . . . . . . . . 23 Isolated Mammalian Cell Mutants with Altered UV-Sensitivity and/or DNA Repair . . . . . . . . . 27 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . 32 Cell Strain . . . . . . . . . . . . . . . . . . . . 32 Culture Medium . . . . . . . . . . . . . . . . . . . 32 Culture Vessels and Incubation Conditions . . . . . 33 Cell and Colony Counts . . . . . . . . . . . . . . . 34 Plating Efficiency and Cell Survival Determi- nations . . . . . . . . . . . . . . . . . . . . . 34 Mutant Induction . . . . . . . . . . . . . . . . . . 34 Mutant Selection and Isolation . . . . . . . . . . . 36 UV Sensitivity . . . . . . . . . . . . . . . . . . 37 Unscheduled DNA Synthesis (UDS) . . . . . . . . . . 38 Sensitivity to Additional Mutagens . . . . . . . . . 39 Cell Growth Rate . . . . . . . . . . . . . . 40 Recovery to High Molecular Weight DNA Following UV-Irradiation . . . . . . . . . . . . . . . . . . 40 Chromosome Analysis . . . . . . . . . . . . . . . . 42 Establishment of Mutation Frequencies . . . . . . . 42 Determination of Spontaneous Mutation Rates . . . . 45 iv RESULTS . . . . . . . . . . . . . . . . . . . . Selection Protocol . . . . . . . . . . . . . Initial Estimates of UV Sensitivity . . . . Radiation Sensitivity . . . . . . . . . . . Sensitivity to Chemical Mutagens . . . . . . Unscheduled DNA Synthesis (UDS) . . . . . . Temperature Sensitivity Test . . . . . . . . Chromosome Examination . . . . . . . . . . Recovery to High Molecular Weight DNA Following UV-Irradiation . . . . .1. . . . . . . . . Cell Growth Rate . . . . . . . . . . . . . . UV-Induced Mutation Frequencies-~UVS-7 . . . UV-Induced Mutation Frequencies--UVS-40 . . UV-Induced Mutation Frequencies--UV -23 UV-Induced Mutation Frequencies--UVS-44 . . Retesting of Induced Mutants . . . . . . . . Spontaneous Mutation Rates . . . . . . . . . Stability of Mutants . . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . The Isolation Protocol and Its Success . . . Comparative Evaluation of UV Sensitivity and Unscheduled DNA Synthesis . . . . . . . . The Damage of UV Versus That of N-acetoxy-Z- acetylaminofluorene . . . . . . . . . . . UV-Induced Mutagenesis . . . . . . . . . . . Spontaneous Mutation Rate . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . . . . . LIST OF REFERENCES . . . . . . . . . . . . . . . Page 87 92 94 95 101 104 106 LIST OF TABLES Table Page 1. Drosophila Mutants of Meiotic and/or Mutagen-Sensitive Functions . . . . . . . . . . 15 2. Human Hereditary Disorders Which May Be Related to DNA Repair Deficiency . . . . . . . . 17 3. Selection of Mammalian Mutants . . . . . . . . . . 31 4. Initial Crude Repair . . . . . . . . . . . . . . . 51 5. DO Values for UV Exposure . . . . . . . . . . . . 55 6. Low Temperature Effects on UV-Survival . . . . . . 60 7. UV-Induced Mutagenesis for UVS-7 and Its Parental V79 Cells . . . . . . . . . . . . . . . 68 8. UV-Igduced Diphtheria Toxin Resistance for UV -7 and Its Parental Control . . . . . . . . . 7l 9. UV-Induced Mutagenesis for UVS-4O and Its Parental V79 Cells . . . . . . . . . . . . . . . 73 l0. Exprgssion of UV-Induced Mutagenesis for UV -40 and Its Parental Control . . . . . . . . 75 ll. UV-Induced Mutagenesis for UVr-23 and Its Parental V79 Cells--I§ Situ Expression . . . . . 77 12. UV-Induced Mutability of UVr-23 and Its Parental Control with Replated Expression . . . 79 13. UV-Induced Mutagenesis of UVS-44 and Its Parental Control . . . . . . . . . . . . . . . . 82 14. Spontaneous Mutation Rates . . . . . . . . . . . . 86 vi Figure 10. 11. 12. l3. 14. 15. 16. 17. LIST OF FIGURES Mutant Selection Protocol . . . . . . . . . Mutation Assay Measuring the Frequency of Diphtheria Toxin Resistance . . . . . . . Mutation Rate Methodology . . . . . . . . . Preliminary Range of UV-Sensitivity . . . . UV and X-Ray Survival for Selected Clones . N—acetoxy-Z-acetylaminofluorene Toxicity . . N-methyl—N-nitro-N-nitrosoguanidine Toxicity UV-Induced Unscheduled DNA Synthesis . . . . Low Temperature Repair . . . . . . . . . . . Chromosomes . . . . . . . . . . . . . . . . Relative Size of Irradiated DNA . . . . . . Gradiant Sedimentation Profiles . . . . . . Mutant Growth Rates . . . . . . . . . . . . Diphtheria Toxin Resistance for UVS—7 and Its Parental Control . . . . . . . . . . . Expr ssion of UV-Induced Mutagenesis for UV -40 and Its Parental Control . . . . . Expression of Ouabagn and 6-thioguanine Resistance for UV -7 and Parental Control Cultures . . . . . . . . . . . . . Spontaneous Mutation Frequencies in UVS-7 and Parental Control Cultures . . .'. . . vii Page 35 46 47 53 54 57 58 59 61 62 64 65 67 72 76 80 84 AT BrdU CAMP CHO dATP dCTP DT DT ER FA HAT HCR HGPRT Lf MNNG LIST OF ABBREVIATIONS apurinic/apyrimidinic ataxia telangiectasia 5-bromodeoxyuridine adenosine 3',5'- cyclic phosphoric acid Chinese hamster ovary deoxyadenosine 5'—triphosphate deoxycytidine 5'-triphosphate statistic representing the increase in dose necessary to reduce survival by l/e (.37) on the straight-line portion of a survival curve Diphtheria toxin Diphtheria toxin resistant Excision repair Fanconi's anemia S ug/ml hypoxanthine, 5 ug/ml thymidine, and 3.2 uM amethopterin host cell reactivation hypoxanthineguanine phosphoribosyltransferase joules floculating unit N-methyl N'-nitro-N-nitrosoguanidine statistic representing the multiple of 100% to which the straight-line portion would extrapolate at 0 UV exposure viii NAc-AAF OUR PBS XP N-acetoxy-2-acetylaminofluorene ouabain resistant phosphate buffered saline photoreactivation sister chromatid exchanges 6-thioguanine 6-thioguanine resistant tritiated thymidine unscheduled DNA synthesis ultraviolet-light xeroderma pigmentosum ix INTRODUCTION It is now recognized that repair processes play an essential role in the maintenance of genetic continuity, compensating for the instabilities of the DNA molecule. The DNA is constantly experiencing alterations of a spon- taneous or induced nature, which, if uncorrected or miscor- rected, may result in a mutation or a lethal event. The consequences of a lethal event are self-evident, however, those related to mutations, particularly as they occur in the somatic cell, are only beginning to be understood. Somatic mutations in mammals have now been implicated in a number of disease states including the carcinogenic (1, 2), atherosclerotic (3), and aging (2, 4) processes. In an effort to help delineate the molecular mech- anisms of mutagenesis, the past twenty years have seen a formidable effort put forth in the study of DNA repair. In prokaryotes, much has been learned and several mechanisms of repair have been identified. In addition to the repair of damaged DNA, prokaryotes have mechanisms of recombination that permit the "tolerance" of damage persistant through replication. In addition to these constituative mechanisms, inducible, "error-prone" functions have also been demon- strated. A significant portion of this knowledge has come from the isolation and characterization of specific mutants with alterations related to repair functions. Despite the plethora of information that has been acquired from bac- terial studies, the exact details concerning mutation fixa- tion remain to be ascertained. Studies in eukaryotes have been based primarily on the early observations in prokaryotes. Eukaryotic cells, particularly mammalian cells, have proven technically more difficult to handle. Researchers interested in the study of mammalian repair functions have been encouraged to focus on wild type cell lines and lines established from a few selected cancer-prone syndromes. These approaches have con- tributed to our understanding of mammalian repair and muta- genesis, suggesting the existence of some prokaryotic DNA repair and tolerance mechanisms in mammalian cells. Considerable differences exist between prokaryotic and eukaryotic organisms. Eukaryotic DNA is complexed with protein, arranged in nucleosome structures, and confined by a nuclear envelope. Furthermore, eukaryotic gene regulation is complex, and multicellular organisms experience a magni- tude of differentiated states at any given time. Such fac- tors are likely to influence the damage of eukaryotic DNA, as well as the mechanisms and efficiency of the repair of that damage. The identification of differences between prokaryotic and eukaryotic repair has been limited primarily to the inability to identify, convincingly, prokaryotic functions in eukaryotic organisms. Apurinic endonuclease activity has not been identified in Drosophila and questions remain regarding the existence of photoreactivation or a recombi— national postreplication repair in mammalian cells. While such negative data suggest differences between the repair functions of various organisms, they do not confirm such differences, nor do they elude to what alternative functions may exist in higher organisms. An obvious approach to this problem is the isolation and char- acterization of eukaryotic mutants. Such an approach has proven successful with Drosophila, as a number of repair mutants have been isolated and are now being characterized in detail. Differences, however, are also likely to exist between Drosophila repair functions and those in mammalian cells. Recently, techniques have been reported whereby the induction, selection, and partial characterization of pre- sumed mammalian DNA repair mutants have been described. Due to the relatively tedious nature of these selection tech- niques, the instability of the mutants isolated, or use of cell lines not easily adaptable to mutation studies, these attempts have had limited success. This dissertation reports an attempt at a new selec- tive procedure for isolating mammalian cell mutants with potential alterations in DNA repair functions. Several relevant Chinese hamster fibroblast cell lines have been isolated and partially characterized. This characteriza- tion includes selected studies of spontaneous and UV- induced mutagenesis. LITERATURE REVIEW The Use of Genetic Mutants to Examine DNA Repair and Mutagenesis Bacteria A great deal has been learned in the last two decades regarding prokaryotic DNA repair and mutagenesis. Much of this knowledge, including the initial suggestion that repair functions existed, was a consequence of the study of mutants with defects relevant to these functions. Mutants have contributed both to the discovery of new repair functions and to the understanding of the biological conse- quences of functions previously defined. Before discussing some specific contributions made by the study of such mutants, clarity is served by reviewing the mechanisms of prokaryotic DNA repair that are currently understood. Review and discussion will be kept brief. Photoreactivation (PR) is one of the simplest of all repair functions. It involves the enzymatic repair of cyclobutane dimers through a light-dependent reaction. The reaction results in the restoration of DNA to an undamaged state without breakage of the sugar-phosphate backbone (5). The reaction is limited to action on pyrimidine dimers. Excision—repair (ER) does not require light and is not restricted to action On pyrimidine dimers. Excision- repair may be classified in two forms, nucleotide ER and base ER. Nucleotide excision-repair involves (l) the incision of DNA near a recognized site of damage; (2) excision of the damaged base or bases plus a number (>20) of additional nucleotides or small oligonucleotides (long patch); (3) polymerization of new DNA using the undamaged strand as a template; and (4) ligation. The incision step appears to function differently in different organisms, and is not yet completely understood. Some orga- nisms which exhibit radiation-resistance, such as Micrococcus luteus, posses a small protein with incision specificity for pyrimidine dimers (6). Most organisms, however, appear to rely on a series of gene products, all of which are neces- sary for incision to occur. This is exemplified by the uvrA, uvrB, and BEES gene products of E. 991i, which will be discussed later. Base excision-repair (7) involves the generation of an apurinic or apyrimidinic (AP) site by the enzymatic or spontaneous removal of a damaged or mispaired base, incision by an apurinic endonuclease, excision of the AP site and a few additional bases (short patch), polymeriza- tion, and ligation. Post-replication repair is a process whereby bac- teria avoid the lethal effects of persistant damage by leaving gaps in daughter strands opposite to such damage, and later filling these gaps by recombinational exchanges with a sister strand. The enzymatic details of this repair are not entirely clear, but will be eluded to later. The process results in the repair of the gaps left by replica- tion past the damage, but as it does not result in the repair of the damage itself, the process is often referred to as a "tolerance" function, rather than repair. Bacteria also possess an inducible, highly error- prone form of repair, designated the "SOS function." This function appears to work by filling the forementioned daugh- ter strand gaps through the random insertion of bases. The induction of this function appears to be related to the presence of DNA degradation products occurring in response to inhibited DNA replication (8). The literature on bacterial repair mutants is far too extensive to be reviewed in detail here. A few key examples and details will be cited to illustrate the tre- mendous contribution that mutant isolation and analysis has made to this field. The review of Hanawalt gt 31. (9), may be considered the most extensive to date on dark-repair in both bacteria and mammalian cells. Table l.of that review illustrates both the vast quantity and variability of factors that may influence repair and mutagenesis. More than fifty different genes have been implicated in E. coli K-12 repair processes, and the precise function of many of these remain unclear. . In 1978, Paul Howard Flanders presented a histori- cal perspective on DNA repair. In his presentation (10), he pointed out that it was the study of a UV-sensitive mutant cell line of Escherichia coli, E. £2li,§sv that first brought to light the existence of DNA repair func- tions for him and other researchers. Ruth Hill had observed that this mutant was incapable of reactivating ultraviolet light (UV)-irradiated bacteriophage. As the mutant was itself UV-sensitive, the likely explanation was that the mutant was incapable of repairing UV-induced damage. Howard-Flanders proceeded to isolate the g. 391$ K-lZ mutants which map to the genes 2335, gyrg, and 3359 (ll). Shortly thereafter, Howard—Flanders and Clark began working with £225- mutants, which were found to be highly sensitive to both UV and ionizing radiation. They recognized this sensi— tivity to be a consequence of deficient recombination (12, 13). Double mutants possessing both 215- and reg- phenotypes were determined to be highly sensitive to UV. It is now recognized that a single pyrimidine dimer in the DNA of these double mutants can prove lethal (14). The initial isolation of these two classes of mutants, 235— and 539-, has stimulated an enormous amount of research examining DNA repair and its relation to muta- genesis. It has been determined that neither uvrA- nor EXEE- mutants are capable of incision (15, 16) and although 3359- strains do perform incision, the rate appears slower than in wild type cells (17). There is additional evidence that the defect is related to incision, as dimer-specific T4 endonuclease V can be used to restore viral reactivation ability to these mutants (18). Complementation studies have indicated that the BXEé gene product is separable from a product related to gyrg and EYES (19). The latter may represent the formation of a complex. The use of 21;. mutants was responsible for the first identification of postreplication-repair. Rupp and Howard-Flanders (20) noted that when mutants were exposed to low doses of UV, and then permitted to incorporate 3H- thymidine, the labeled DNA sedimented more slowly than the DNA from similarly treated, but unirradiated controls. Allowing the irradiated cells a 70 minute chase time in unlabeled medium, resulted in a return to control sedimenta- tion characteristics. The observation came to be recognized as "postreplication-repair." The history of reg- mutants has been elusive and the details of postreplication-repair have remained unclear. The rggA gene product has been suggested to play an important function in the "cutting" of the undamaged homologous DNA for recombination (10). There is preliminary evidence that Eggg- mutants may be defective in a step that permits the generation of recombinational intermediates, and thus 10 prevents the completion of exchange (21). Advances in cur- rent technology will be necessary to resolve £395 function. In addition to the need for recombination in postreplication- repair, as illustrated by the need for the £295 gene product, additional gene products are required which are not involved in normal recombination. These include the product of the Egg: gene. In addition to the forementioned repair, the £29_A and lgx_§ genes appear to be coordinated in an inducible postreplication-repair function. The inducible responses have been summarized by Hanawalt g£_al. (9) and include inhibition of cell division, inhibition of post-irradiation DNA degradation, induced bacterial mutagenesis, and the induction of viral reactivation and mutagenesis by pre- irradiation of the host cell (Weigle reactivation and muta- genesis (22). For review, also see Witkin (23)). A mOdel suggesting coordination between £§g_A and l§x_A for induc- tion proposes that the le§_A gene product is a repressor, controlling the £§g_A gene product (a protease activity) at a low, constitutive level (24, 25). At this low level, rec A product is capable of performing functions necessary for constitutive postreplication-repair. Upon induction, rec A product inactivates the lgx_§ repressor and other repressors controlling SOS functions. One proposed conse- quence of these changes is the modification of a polymerase to permit error-prone "transdimer synthesis" (26). 11 There also appears to be an inducible nature to long patch excision-repair. This mechanism has been demonstrated to be dependent on rec A (27), requires protein synthesis as demonstrated by chloramphenicol inhibition, and is induced by UV (28). This form of excision-repair is pre- sumed to be mutagenic. The observation that long patch repair increases with dose in a nonlinear fashion would support the induction hypothesis. However, the observation that mutation fixation can occur during the inhibition of protein synthesis (29) raises some doubt. An attempt to unravel the enzymology of the excision and resynthesis steps of excision-repair have proven diffi- cult, due to the apparent multiplicity of enzymes available for each task (30). Studies employing mutants defective in any of a variety of exonuclease activities have demon- strated the role of at least three exonuclease activities in excision (9). To complicate matters, mg; and dn§_§ mutants exhibit normal UV-sensitivity, yet a reduced rate of excision, and a significant hypermutability (31, 32). The examination of E. 9911 mutants deficient in polymerase I, II, or III, have suggested that all three polymerases are involved in repair processes (9). Initial observations of pol A mutants (polymerase I-deficient) appeared to indi- cate enhanced repair (33). This observation is currently thought to reflect the involvement of polymerase I in short patch repair. The pol A mutants are therefore thought to 12 conduct an abnormal amount of long patch repair, in order to compensate for reduced short patch repair. This led researchers to observe a greater extent of repair, even though an equal or smaller number of damaged sites were being removed. A requirement for polymerase I and/or polymerase III has been demonstrated for postreplication- repair, as the single mutants pgl_A and pgl_g are capable of repairing daughter gaps, whereas the double mutants are not. In addition to the examination of bacterial mutants that contribute directly to repair functions, there is a class of mutants that exhibit indirect effects. These mutants alter the physiological state of the cell in such a manner, that the repair functions and mutagenesis are affected. An example of such a mutant is ggg’, isolated by Hochhauser and Weiss (34). These cells are deficient in dUTPase and consequently have elevated dUTP pools. The cell misincorporates large levels of uracil into the DNA. The mutants generally exhibit an inordinately large number of Okazaki fragments, representing the incorporation of uracil followed by the base excision of that base. Through the generation of double mutants gut- mutants have been use- ful in identifying the function of uracil DNA glycosylase (Egg gene), polymerase I and ligase in the base excision- repair of misincorporated uracil (7). In addition, the hypermutability of a ung- mutant is not enhanced by the 13 generation of a gag“ ggp' double mutant (35). The g3;- mutants were therefore useful in demonstrating that the mdsincorporation of uracil does not substantially contribute to mutagenesis. It appears, rather, that cytosine deamina- tion is the major cause of the mutator phenotype of 223- mutants. One additional aspect of the utilization of genetic mutants to study DNA repair and mutagenesis in prokaryotes has come through the employment of plasmids called R factors which carry antibiotic resistance genes. Ames and co- workers have develOped a bacterial assay to detect mutagens (36). The assay examines the conversion from histidine requirement to prototrOphy in strains of Salmonella typhimurium in which conversion is specifically dependent on base-pair substitution or a frameshift event. Sensitivity to mutagens in these lines can be enhanced by the addition of a mutation which eliminates excision-repair. In addition, McCann E; El; (37) have demonstrated that the inclusion of R factor in these strains enhances the mutagenicity of cer- tain mutagens. While the precise details of R factor action remain uncertain, the mutagen specificity suggests the involvement of a rec-dependent, error-prone process. Lower Eukaryotes The use of mutants has also been helpful in the study of repair and mutagenesis in lower eukaryotes. UV-sensitive mutants have been isolated in Saccharomyces cerevisiae (38), l4 and mutants sensitive to both x-rays and UV have been iso- lated in Saccharomyces (39), Neurospora crassa (40), Schizosaccharomyces pombe (41), and others (42). Many of these mutants have "rec-like" characteristics suggesting the existence of recombination functions in lower eukar- yotes (42). Additional Neurospora crassa mutants have been isolated with defects in endo-exonuclease activity, which are mutagen-sensitive (43). Mutant studies in Dictyostelium discoideum have resulted in the identification of nine com- plementation groups (44). Through the analysis of double mutants, at least two repair pathways have been identified, one of which is similar to bacterial excision-repair. Recently attention has focused on the identifica- tion of Drosophila mutants with relevance to DNA repair. It was soon learned that some mutants which had been selected on the basis of mutagen sensitivity (mpg) shared characteristics with others selected for analysis as meiotic mutants (gel) (45). This initial observation suggested the existence ofounctions common to both DNA repair and meiotic recombination. Mutants have now been isolated that are specific to repair functions, meiotic recombination, and both repair and meiotic recombination. A few representa- tive mutants illustrating this fact are presented in Table 1. In addition to the excision and postreplication- repair functions identified in these mutants, 15 .hVImv mmocoummmm on Howmm + + + + HmEuoz cOAOBOHHdmuumom . eoa AH. was I I I I pwospmm m mwmnwmm + + + + cmosomm :oAUBOAHmmuumom mwumma + + + + coospom :oflmwoxm wummm mmm m2: >mmlx >3 coflumcflneoowm cmwwmmmm ucmusz >ua>fluflmcmm Onuoflmz m>flwommmo .mcofluocsm O>HuflmcomIcmwmusz HO\Ucm owuoflmz mo mucous: pawnm0mounll.a magma l6 photoreactivation has been demonstrated in Drosophila tissue culture cells (48) and larvae (49). The generally ubiqui- tous activity of uracil DNA glycosylase has remained unde- tectable in Drosophila cell lines, embryos, larvae, pupae, or adult flies (50). Mammalian Cells The major contributions to the present understanding of mammalian repair functions and mutagenesis have come through the study of human syndromes which exhibit mutagen sensitivity or a high incidence of cancer associated with altered repair function. Several such syndromes are listed in Table 2. The disease that has proved most beneficial in the study of mammalian repair is xeroderma pigmentosum (XP). Many reviews on XP have been published, including those by Cleaver (51) and Friedberg 33.31. (52). XP patients exhibit a severe photosensitivity of the skin and a high incidence of Skin cancer, restricted to exposed areas of the body. The first reported evidence that XP cells were hypersensitive to UV dates back to the work of Gastler in 1964 based on studies of total protein (53). Since then, many laboratories have demonstrated reduced XP colony forming ability following exposure to UV (54, 55, 56). This UV sensitivity has been correlated with an inability to reactivate UV damaged viral DNA (57), com- parable to the observations made with E. Eel; EYE E mutants (18). 17 Table 2.--Human Hereditary Disorders Which May Be Related to DNA Repair Deficiency. Disorder Inheritance Ataxia telangiectasia Bloom's syndrome Cockayne's syndrome Dyskeratosis congenita Fanconi's anemia Progeria Retinoblastoma Xeroderma pigmentosum (Classical XP) Xeroderma pigmentosum (XP variant) Autosomal recessive Autosomal recessive Autosomal recessive X-linked recessive Autosomal recessive Autosomal recessive (?) Autosomal dominant (chromosome 13) Autosomal recessive Autosomal recessive (?) 18 Sensitivity to additional mutagens has been exam- ined. XP cells have been found sensitive to a wide variety of mutagens. These studies are summarized elsewhere (52). Slight XP sensitivity to x-rays and N-methyl—N-nitro-N— nitrosoguanidine (MNNG) have been observed by Lytle EE 3;. (58) and Stich £3 3;. (59), respectively. Maher and McCormick (60) have suggested that these sensitivities are not significant, and proposed that XP cells be used to identify those mutagens which cause major distortion in the DNA, possibly requiring a long patch repair which is not present in XP. Other mutagens (e.g., x-ray, MNNG) which cause. strand breaks and minor base changes, they propose, are repairable in XP and may represent damage repaired by a short-patch mechanism. Biochemical studies have clearly demonstrated reduced excision-repair in some XP cells. Cleaver demonstrated a reduction in repair synthesis in response to UV in XP cells (61). Setlow 23 El- (62) and Cleaver and Trosko (63) sub- sequently demonstrated that XP cells were not capable of excising pyrimidine dimers. A wide variety of techniques have now been used to examine many XP cell lines for excision-repair capacity. The data, in conjunction with complementation studies, indicate that XP is genetically heterogenous with at least eight complementation groups. Groups A-G are all excision-deficient to some degree, whereas the group classified as the XP variant exhibits 19 normal levels of excision-repair. Groups A-G are defective in ER to varying degrees, with only slight variation within any given group. Observations suggest that the degree of ER reduction does not correlate with UV-sensitivity (52). Sensitivity does appear to correlate with the degree of neurological involvement seen in the individual patients. Despite normal ER, variant XP cells do exhibit a sensitivity to UV and some chemical mutagens (64) and a reduction in host cell reactivation (65, 66). In addition, XP variant cells exhibit a slower return to high molecular weight DNA following UV-irradiation than that observed in normal cells (67). This observation presumably demonstrates defective postreplication-repair in variant XP cells. Studies of the UV and chemical-induced mutagenesis of XP cells have demonstrated all groups examined (including variant) to be hypermutable when mutation frequencies are examined per unit dose (56, 68). For classical XP cells, this observation corresponds to that in EXE- bacteria, where it has been assumed that reduced ER results in.a greater dependence on error-prone postreplication-repair. For the variant XP cells, this observation is not easily explained by any currently understood mechanism of repair, but may reflect dependence on a yet undefined error-prone postreplication-repair, or the existence in variant cells of an error-prone physiological state. Classical XP cells exhibit normal mutability when mutations are considered per 20 unit survival (68), whereas XP variant cells may demonstrate a slight hypermutability based either on unit dose or unit survival (60). Ataxia telangiectasia (AT) is another genetic dis- order that has contributed to the study of mammalian repair. Although heterogeneity has been found in this disorder, all lines tested were sensitive to ionizing radiation (52). Using extracts from Micrococcus luteus known to catalyze strand breakage of y—irradiated DNA, some AT cell lines have been shown to exhibit a reduced ability to remove enzyme-sensitive sites following y-irradiation (68). It has not yet been possible to conclude that this apparent repair deficiency is the basic defect of AT. AT cells also exhibit chromosome instability. Fanconi's anemia (FA) is another human syndrome which is associated with chromosomal instability and pre- disposition to cancer (69). Data examining the cytotoxicity, removal of damage, and mutability (70) in response to cross- linking agents suggest that FA is deficient in a short-patch repair function (69). Crosslink damage is repaired by a sequence of events involving the first half-excision from one strand, strand displacement and recombination, and final excision of the link to the second strand. FA cells appear to be defective in the first step of crosslink removal and not defective in the removal of thymine dimers. Classical XP cells are capable of first half—excision, so the 21 mechanism appears not to be related to the long-patch defect of XP. The data indicate that the first half-excision and excision of thymine dimers require different mechanisms. Dyskeratosis congenita represents another example of a syndrome with altered crosslink repair, as measured by an excessive induction of sister chromatid exchanges (SCE) following psoralen-UV light treatment (70). Bloom's syndrome fibroblasts should also prove useful in the study of repair mechanisms, exhibiting enhanced spontaneous levels of SCEs (71). Retinoblastoma may also prove useful, demon- strating a relationship between x-ray sensitivity and a deletion in a specific region of chromosome 13 (72). Cockayne's syndrome represents an additional example of a condition that exhibits specific sensitivity (damage requiring long-patch repair) without the identification of a precise repair defect (52). Additional syndromes have been cited (52). Collectively, the many mammalian syndromes studied with potential importance to the mechanisms of DNA repair and mutagenesis, suggest the existence of a wide variety of relevant genotypes. Technical limitations, including poor plating efficiency and limited lifespan, have complicated the study of many of these syndromes. Conflicting results have been published regarding characteristics of many of these lines (52), which likely reflect genetic hetero- geneity, alterations in individual culture conditions, or 22 significant limitations of the tecnhiques employed. There- fore, although many unique strains have been examined, the delineation of repair and mutagenesis mechanisms in mammalian cells is far from complete. Repair in Chinese Hamster Cell Strains In Chinese hamster cells, Elkind (73) observed that x-ray exposure was less toxic if administered at lower dose over a longer period of time. This was one of the first re- ported observations that inferred the existence of mammalian DNA repair. Excision-repair has now been confirmed in mam- malian cells. Chinese hamster cells, however, have much lower levels of excision-repair than normal human cells (74). Trosko E; El; (75) demonstrated the difficulty in detecting dimer removal in hamster cells. ‘Hamster cells have been shown to conduct levels of repair replication equivalent to those of human cells at times shortly after UV (76). Additional differences between normal human and hamster excision-repair are seen in studies examining the repair of NAc-AAF-induced damage. Data indicate that the repair of UV and NAc-AAF are additive in human cells (77) but not in hamster cells (78). The data suggest that in human cells, different repair pathways are necessary to repair the two types of damage. This disagrees with the previously reported observations for XP cells (60) and with the results of similar experiments reported by Brown gplgl. (79). The results in human cells are unclear and those in hamster cells suggest sbmilar repair pathways for the two 23 types of damage. In addition to the observations related to excision repair, the postreplication-repair phenomenon has been observed in Chinese hamster cells (80). Additional Factors Relevant to Mammalian DNA Repair and Mutagenesis Although the study of the aforementioned syndromes has suggested the existence of base and nucleotide excision— repair and postreplication-repair in mammalian cells, the data are far from complete. Disagreement has arisen in regard to the interpretation of certain data. Lehman (81) has demonstrated the time-dependent closure of daughter strand gaps in mammalian cells, suggesting postreplication- repair. Setlow and D'Ambrosio (82) have demonstrated the enhancement of such a phenomenon by a split—dose protocol and suggested this represented evidence of an inducible mammalian repair function. Cleaver gg 31. (83) and Painter (84) have argued that their data may be reinterpreted to indicate that gaps formed are only the consequence of delays in normal synthesis, and not at all related to a repair function. To date this argument has been unresolved. An additional discrepancy has arisen in the litera— ture in regard to the identification of mammalian photore- activation. For many years it was assumed that mammalian cells did not possess this function (85, 86). Recently, Sutherland and Oliver were able to demonstrate a culture condition dependent photoreactivation in human cells (87). 24 Mortelmans £5.31. have examined these specific conditions and suggest that the observations may represent the action of "nonspecific photosensitizers" rather than an actual PR enzymatic event. The specificity of the conditions under which PR can be observed in mammalian cells also raises questions regarding its function 12 3139. Confusion also exists in the literature regarding the demonstration of recombinational events in mammalian cells in conjunction with repair events. A number of laboratories have demonstrated such events (88, 89), but Cleaver g; 3;. (75) remain skeptical, proposing that arti— facts make data interpretation difficult. They suggest that small daughter strands may exist at the time of irradia- tion, permitting the induction, rather than the transfer, of dimers into those strands. Meneghini and Menck (90) used two techniques, (1) synchronized cells blocked with hydroxyurea; and (2) the incorporation of S-bromodeoxyuridine (BrdU) until the time of irradiation, to eliminate artifacts in the measure of parental strand dimers transferred to daughter strands. The measurements revealed a 10-20 percent transfer. This would be considerably less transfer than that observed in bacterial cells. Additional mechanisms must be involved in the bypass of dimers. One additional area of uncertainty in mammalian repair that has received considerable attention is the question of inducible functions. This has been reviewed 25 by Hanawalt g; 3;. (9). Observed results with three experi- mental techniques support the existence of an inducible function in mammalian cells. The first is the Observation of Weigle reactivation and Weigle mutagenesis, which involves the irradiation of a host cell to stimulate the reactivation and mutagenesis of an irradiated virus. Weigle reactiva- tion has been observed in mammalian cells for Herpes virus (91), Kilham rat virus (92), adenovirus (93), and SV40 (94). Weigle mutagenesis was noted to occur with Herpes (95). Reactivation in mammalian cells has been demonstrated to be inhibited by cycloheximide treatment (92) suggesting a dependence on protein synthesis. Split-dose enhancement of daughter strand gap closure has been interpreted as evidence for induction of a repair function. D'Ambrosio and Setlow (82, 96) have demonstrated that a small inducing-dose of UV several hours before a large damaging dose can stimulate the recovery of DNA to full size quicker than the recovery observed without stimu- lation. This effect can be inhibited by cycloheximide (82). The observations of Chang 3; 3;. (97), using a split-dose protocol, revealed reduced mutagenesis with Chinese hamster cells. With the addition of caffeine and cycloheximide, they observed enhanced mutagenesis, and suggested the exis- tence of an error-free postreplication-repair in Chinese hamster cells to account for the results. 26 A third line of evidence supporting inducible mam- malian repair is the ability to enhance the repair of alkylation damage. Pretreatment with low doses of an alkylating agent stimulates the repair of damage caused by a subsequent large dose. This procedure has been used in whole animals to enhance the removal of 06 methyl guanine from the DNA of rat liver (9). Painter (84) has suggested that the split-dose work might be reinterpreted, and that it does not support the inducibility of a repair function. He proposes that the initial dose results in a reduction in DNA synthesis and potentiates the occurrence of a higher molecular weight species than would be seen in unirradiated controls. This species would lead to an earlier production of large molec- ular weight DNA in an enhanced postreplication-repair proto- col. Another hypothesis used to discount the data in sup- port of inducible mammalian repair is derived from the work of Loeb and coworkers. Using an ig‘yiggg assay to examine the fidelity of DNA replication of an amber mutant of ¢Xl74 (am3¢X174) with E. coli polymerase I, Weymouth and Loeb 2 2 (98) demonstrated that the substitution of Mn+ for Mg+ , with specific nucleotides, cOuld enhance the error frequency of the polymerase. An additional experiment (99) demon- strated that (l) Co+2 for Mg+2 could produce similar results, (2) the error rate was dependent on the nucleotide dCTP 27 and dATP levels, and (3) the relative increase in dATP levels was directly proportional to an enhanced error rate. The data are consistent with the 13 2332 work of Peterson 23 3;. (100) which demonstrates that a 3- to lO-fold increase in mutagenesis is observed in the presence of specifically altered nucleotide pools. Recently, the isolation of a hypermutable cell strain with an inherently altered dCTP level (101) and a similar strain with normal polymerase activity (102) have been reported. These further support the la XEEEQ observations. The data suggest that the physiological state under which normal enzymes must function can drastically alter the fidelity with which they act. It seems likely that most repair enzymes will have restricted environments in which they can efficiently function. The UV exposure used for the induction of the split-dose phenomenon or the Weigle reactivation affects normal DNA synthesis and may affect additional cell functions leading to an altered physiOlogical state. This altered state may result in Weigle effects and enhanced removal of 06 methyl guanine. Isolated Mammalian Cell Mutants with Altered UV-Sensitivity and/or DNA Repair A number of attempts to isolate mammalian cell mutants using nonselective procedures, with the aid of mas- sive screening or replica plating techniques, have been reported (103, 104, 105, 106). These researchers have 28' chosen nonselective techniques either because of a lack of success with alternatives, or a concern over the harsh pro- cedures necessary for mutant enrichment (103). Sato and Hieda (105) utilized massive screening to identify a mutant mouse lymphoma cell line sensitive to methyl methane- sulfonate and x-ray, which was not altered in UV sensitivity. Kuroki and Miyashite (103) used Lederberg style replica plating which, although tedious, yielded a 100% plating efficiency. They used MNNG to provide a mutagenized popu- lation of mouse cells before selection and were able to iso- late seven UV-sensitive clones. Many of these mutants proved not to be stable. Stamato and Waldren (104) employed a nylon cloth replica plating technique to isolate a stable, UV-sensitive Chinese hamster ovary clone. This clone has not been characterized in great detail. Nonselective mutant procedures have also been used to examine the effects of temperature (107), viral transformation (108), and ploidy (109) on DNA repair and/or mutagen sensitivity. Procedures for general mammalian mutant selection or enrichment have been reviewed elsewhere (110, 111). These procedures have been used in an effort to isolate UV- sensitive and/or repair mutants. The most commonly employed technique involves the incorporation of BrdU and subsequent exposure to visible light as a means of lethal growth. The employment of this technique for the isolation of mutagen-sensitive or repair mutants requires 29 modifications of the techniques initially described by Puck and Kao (112) for the selection of nutritional mutants. Randtke g£_gl. (113) used such a technique to iso- late thirteen clones of Chang liver cells that were pre- sumed slow to progress into normal synthesis following a sublethal dose of UV-irradiation. Normal cells which do progress into synthesis incorporate BrdU and are killed when exposed to visible light. The hypothesis appeared cor- rect as unirradiated groups produced no survivors. One of the isolated clones was UV-sensitive. Isomura 23 El- (114) isolated two UV-sensitive clones using a similar approach with HeLa S3 cells. MNNG was used to mutagenize the initial population before selec- tion. The clones were found to be more sensitive to 4- nitroquinoline l-oxide, but not x-ray, than the parental HeLa cells. Klimek gE 21- (115), using similar selection, iso- lated both UV-resistant and UV-sensitive clones of HeLa S3 cells, which appeared to exhibit differential repair as measured by BrdU photolysis. A more recent enrichment technique, which should prove useful in'future studies, is the use of irradiated viruses to select UV-sensitive and repair mutants. Repair- proficient cells will reactivate the virus (host cell reactivation (HCR)) and be killed, whereas repair-deficient cells should survive. This technique has been used with 30 human FL cells and Herpes virus (116). The results were dramatic, with nineteen of 238 isolated clones exhibiting UV-sensitivity. Of four clones studied, two were stable for extended periods and only one of these two appeared to be HCR deficient when reexamined. Chinese hamster cells do not appear to incorporate the viruses commonly used for such studies (Dave Lytle, personal communication), so alter- native methods will have to be devised for selection with these cells. Recent experimentation in repair related research has seen a renewed interest in the isolation of mammalian cell mutants. The isolation of hamster mutants with defects related to postreplication-repair were recently reported (117). The cell lines exhibited hypersensitivity to a variety of mutagens. The selection procedure for these lines did not employ any major improvements upon old iso- lation techniques. In addition to these mutants, another laboratory has reported the isolation of seven CHO clones apparently excision-repair deficient and UV-sensitive (118). Two of the lines exhibit hypermutability. The lines were isolated by a unique technique that utilized the photographic examination of colony growth following UV exposure to deter- mine sensitivity. The most important mammalian mutants discussed here are summarized in Table 3. 31 manmusEummmm cofiumUHMHucopH UGOAOHMTU mm oasmmumouonm mad m>flufimcmmu>o + mcaumfia mafiaamm omo mad m>HuHmcomI>D OUHOASm HOHH> am unadm mm Genevaé mad m>fluflmcmmu>o Areas + Doom mm mama ucmHOmep mm «Ha m>HUHmcmmI>D Unmfla + Doom mm mama mag m>AUAmcmmI>o broad + scum maamo um>aa mcmno MOH O>HpfimcomI>D mafiumam moaammm 4m2m mmsoz aoa m>fluflmcmmu>2 mcflumam moaaamm Hmuomo moa m>HuHmcmmI>mqu o>fluumaomcoz mEosmE>H mmsoz mocmuowom muaumfiuouomumno msuwcnoma mafia HHOU pawns: :OwumHOmH .mucmusz amaamEEmz mo cowuomammII.m magma MATERIALS AND METHODS Cell Strain The parental cells used for the isolation of poten- tially mutant cell lines and used throughout these experi- ments for control values were V79 Chinese hamster lung fibroblasts. This transformed, aneuploid cell line was originally derived from a male Chinese hamster, Cricetulus griseus, 2n=22 (119). The cell line can be grown in mass culture attached to the bottom surface of plastic or glass flasks and plastic tissue culture dishes and is capable of forming colonies from a single cell in such vessels with nearly 100% plating efficiency. Culture Medium Cells were grown and experiments conducted in Eagle's Minimal Essential Medium (MEM) (120) with Earle's salts (GIBCO, Grand Island, New York), supplemented with a 50% increase in all essential amino acids except gluta- mine, 100% increase in all nonessential amino acids, 50% increase in all vitamins, and 1 mM sodium pyruvate. The concentration of bicarbonate was adjusted to 1.5 g/l. Sterilization was achieved by positive pressure filtration through nucleopore filters (Nucleopore Corporation, 32 33 Pleasanton, California). Sterile medium was stored in the dark, in a 4°C cold room. Prior to use, the medium was supplemented with 5% fetal calf serum (GIBCO, Grand Island, New York, Flow Laboratories, Inc., Rockville, Maryland, or Pel Freeze, Rogers, Arkansas) which had been stored at -20°C, thawed, and heat inactivated at 56°C for 25 minutes if not previously inactivated by the manufacturers. The medium was further supplemented with penicillin G (100 units/ m1) and streptomycin (100 ug/ml). Culture Vessels and Incubation Conditions Stock cultures were grown in sterile glass bottles or plastic flasks (Corning Glass Works, Corning, New York). When sufficiently dense, cells were subcultured with 0.01% crystalline trypsin (Sigma Chemical Company) in phOSphate buffered saline (PBS) without calcium and magnesium ions. Cultures were routinely subcultured one or two days prior to an experiment at sufficient density to ensure log phase cells at the time of experimentation. For most experiments, cells were grown in 9 cm plastic culture dishes (Falcon Plastics, Oxnard, California or Corning Glass Works, Corning, New York). Both stock cultures and experimental plates were incubated in water jacketed incubators which provide a stable temperature of 37°C (unless otherwise indi- cated), with humidified air and 5% C02. 34 Cell and Colony Counts Trypsinized cells in suspension were counted using a hemacytometer. Colonies developing from single cells were scored visually when sufficiently large, or after staining by rinsing with 0.85% saline, fixing with 95% ethanol, and staining with 2.5% Giemsa stain. Plating Efficiency and Cell Survival Determinations Plating efficiencies and replating efficiencies were determined by plating 200—300 cells in 10 ml of medium in a 9 cm plate, with 3 or 4 plates per treatment group. For replating efficiencies, plates were handled identical to treatment plates with the exclusion of selective medium. Cell survival was determined for a variety of muta- gens described below. In all cases, a sufficient number of cells was plated for each treatment group, to permit an estimated 50-300 colonies surviving per plate, with 3 to 4 plates per treatment group. Colonies were scored as previ- ously described. Mutant Induction The procedure for mutant induction and selection is outlined in Figure 1. Cells were mutagenized by a two- step procedure consisting of (l) the incorporation of 5- bromodeoxyuridine (BrdU) (National Biochemicals Corporation, Cleveland, Ohio), and subsequent exposure to black light, and (2) UV irradiation. 12 x 106 cells were plated in 40 35 .Hooououm cowuooaom ucmusz .H ousmflm 93:3 :9 no 202532303 0.. 93 2 2:3 8 m 0.3 m¢O>_>¢:w Sam! #5.. ZO_h<¢On¢002_ so“. can...» pmm» E F E o E a 93 c 953 a: 5 ca 2 «a _ I . .4 . I . or..." E: x N Amer. a: $5.. as :29 mo“. 4.. 0 >3 >3 muam: :93 69. so: :6: 02.5.... m2 55am: 53¢ .33 m~.2.wn>¢._. 2. w 36 plastic tissue culture dishes and allowed 24 hours to attach and achieve log phase growth at 38.5°C. Cells were grown for an additional 24 hours in the presence of 0.1 mM BrdU in the dark and were subsequently exposed to black light from a series of bulbs (General Electric F15T8-BL 15W), at a distance of 5.5 cm for one hour with medium remaining on the cells. Cells were washed with PBS and supplied with fresh medium. After approximately 10 days of growth, subculturing when necessary, 12 x 106 cells of the resulting population were plated and allowed 3.5 hours for attachment. Medium was removed and cells were exposed to 20 joules/m2 from a germicidal lamp (General Electric 25T8-25W) which was positioned to deliver a fluence of l joule/mZ/second, as measured by a Blak-Ray Ultraviolet Meter Model J-225 (Ultraviolet Products, Inc., San Gabriel, California). Mutant Selection and Isolation Mutagenized cells were trypsinized and replated at 2.5 x 106 cells/plate (8 plates) in medium containing 1% fetal calf serum. Twenty-four hours later the cells were exposed to 10 joules/m2 UV and supplied with medium con- taining 1% fetal calf serum plus 20 uCi/ml 3H-thymidine, 64.7 Ci/m mol (New England Nuclear, Boston, Massachusetts), for 6 hours. Cells were washed twice with PBS and then incubated in medium containing Hepes buffer, 1% FCS and 2x cold thymidine (4.12 x 10.-5 M) for 1 hour. Plates were 37 then moved to a Sherer automatically controlled cold room (4°C) for 10 days. Following radioactive thymidine killing the cells around the periphery of each plate were removed with a rubber policeman to eliminate survival due to a shadowing effect. HAT medium [5 ug/ml hypoxanthine (Sigma Chemical Company), 5 ug/ml thymidine (Sigma Chemical COmpany), and 3.2 uM amethopterin (National Biochemicals Corporation)] was added to each plate and cells were grown for 10 days at 34°C. Surviving clones were isolated and grown in dupli- cate in 24-well plates (Corning Glass Works, Corning, New York). UV Sensitivity One set of multiwell plates containing isolated colonies was reserved to provide cells for the establishment of individual cell lines for freezing, the unscheduled DNA synthesis assay, and additional work. The duplicate set of multiwell plates was used as a preliminary screen for UV sensitivity. With medium removed, cells in multiwell plates were exposed to 10 joules/m2 UV. Fresh medium was added and several days later individual wells were examined for qualitative evidence of sensitivity or resistance. Wells exhibiting abnormal response as compared to the parental line were considered to be of special interest, and the cell lines established from their duplicate wells were examined by more quantitative means. For each cell line 38 demonstrating preliminary sensitivity or resistance, UV sensitivity was determined by means of colony forming abil- ity after exposure to 5.0 and 20 joules/m2 UV. Survival was determined by averaging the percentage of survivors in quadruplicate plates for each dose of UV. Detailed sur- vival at many doses of UV was then determined for a few select lines in a similar manner. Unscheduled DNA Eynthesis (UDS) Excision-repair capacities of the various cell lines were estimated by means of the unscheduled DNA synthesis assay as develOped by Trosko and Yager (121). Preliminary measurements were made by establishing one monolayered 60 x 15 mm petri dish of cells for each cell line isolated through the employment of the mutant selection protocol. Monolayered cells were maintained on deficient medium lack- ing arginine and isoleucine (GIBCO, Grand Island, New York) and supplemented with 5% dialysed fetal calf serum (GIBCO, Grand Island, New York) for 24 hours. Medium was then changed to fresh deficient medium with 5% dialysed fetal calf serum for an additional 48 hours. Five mM hydroxyurea (Sigma Chemical Company) was added 1 hour prior to the col- lection of medium and exposure of the cells to 20 joules/m2 of UV irradiation. 3H-thymidine (40-60 Ci/m mol) was added to the collected medium to a concentration of 5 uCi/ml. Medium was returned to the cells for 3 hours of incorpora- tion. The parental cell line was assayed as a control, 39 using duplicate plates and groups with and without UV, to allow for more accurate estimations of normal values. At 3 hours all cells were collected in PBS, frozen, and later assayed via trichloroacetic acid (TCA) extraction of the DNA. Specific activities (DPM/ug DNA) were calcu- lated for each cell line. A few selected lines were anal- yzed in greater detail using the same techniques with the arginine/isoleucine deficient medium. Various doses of UV were examined with duplicate plates per dose. Sensitivity to Additional Mutagens Selected lines were examined for sensitivity to several mutagens, in addition to the aforementioned ultra- violet light. For x-ray sensitivity cells were trypsinized, suspended in medium in 25 cm2 plastic flasks, and irradiated with x-rays generated by a General Electric Maxitron 300 x-ray machine. The exposure rate was 184 R/min (250KV, ZOmA, with 3 mm Aluminum filtration). Cells were immedi- ately plated in 9 cm petri dishes and 7-10 days later, colonies were scored. Percent survival was again deter- mined by averaging quadruplicate plates per point. For examining sensitivity to N-methyl-N-nitro-N- nitrosoguanidine (MNNG) (Sigma Chemical Company), trypsin- ized cells were plated in 10 ml of medium and allowed 3.5 hours for attachment. MNNG was dissolved in acetone and immediately added to plates with acetone alone added to some plates as control. After 2.5 hours the plates were 40 washed with PBS and supplied with fresh medium. Seven to ten days later colonies were scored and percent survival determined as previously described. Similar experiments were conducted with N-acetoxy-2-acety1aminofluorene (NAc- AAF) using dimethylsulfoxide (DMSO) as a solvent and 24 hour exposure. NAc-AAF was a gift to Dr. James Trosko from Dr. James Miller. Cell Growth Rate For each cell line to be examined, approximately 1x105 cells were plated in a sufficient number of 9 cm tissue culture dishes and at various times thereafter, two plates were trypsinized for each cell line, and the cells were counted and the values averaged. Recovery to High Molecular Weight DNA Following UV—Irradiation Following UV-irradiation, daughter strand DNA is found to be of low molecular weight. This observation sug-~ gests either the occurrence of postreplication "gaps" adjacent to pyrimidine dimers (81), or the inhibition of normal DNA synthesis by such dimers (83, 84). With increasing time, the DNA returns to high molecular weight as a consequence of either the filling of gaps or resumed replication. Since the examination of this recovery to large molecular weight has been proposed as a measure of postreplication repair, it was of interest to examine the mutants reported here and evaluate their competence at 41 conducting this molecular function. UVr-23, UVS-40, and UVS-44 were examined with a parental control in two separate experiments. In the first experiment, cells were grown in 60 mm tissue culture dishes and labeled with 14 C-thymidine for 24 hours. All cells were rinsed with PBS, exposed to 7.5 joules/m2 UV. Fifteen minutes after irradiation, cells were exposed to 3H-thymidine (67 uCi/ml, so Ci/m mol) for 30 minutes, rinsed with PBS, and subsequently permitted 0, 45, or 90 minutes chase with cold thymidine. The experi- mental procedures were spaced so all groups were ready to collect at the same time. Cells were exposed to 1500 Rads of x-ray and collected. A 50 ml aliquot of each treatment group was lysed for 1 hour in 1.0 N NaOH and 0.01 M EDTA on top of a 5-20% alkaline sucrose (high salt) gradient in a 7/16 inch diameter x 2-3/8 inch polyallomer tube (Beckman Instruments, Inc., Palo Alto, Ca.). Sedimentation occurred at 30,000 revolutions/minute at 20°C for 130 minutes. Gradients were collected in 5 drop fractions from the bottom, and radioactive content of the fractions was deter- mined for both 3H and 14C. The second experiment was conducted in a similar manner with the following changes. Only UVS-44 and control were examined, and all cells were permitted a 45 minute chase time. Cells were exposed to UV doses of 0 or 7.5 joules/m2 or a split—dose procedure of 2.5 and 7.5 joules/ 42 m2, separated by 2 hours. The split-dose procedure has been demonstrated to enhance the recovery of DNA to high molec- ular weight, possibly by inducing a repair mechanism (82). Chromosome Analysis Cells were inoculated into 60 mm tissue culture. dishes (Falcon Plastics, Oxnard, Ca.) containing 5 ml of medium and a sterile glass cover slip. Twenty—four hours later, colcemid (GIBCO, Grand Island, New York) was added to a final concentration of 0.1 ug/ml for 3 hours. Cover slips were collected, treated with 0.7% sodium citrate for 20 minutes and fixed with methanol and acetic acid (3:1) for a few minutes. After air drying, cover slips were stained in Giemsa stain for 10 minutes. Giemsa stain con- sisted of 3 m1 Giemsa stock selection (Bio/Medical Special- ties, Santa Monica, Ca.) and 100 ml 0.025 M phosphate buffer (pH 6.8). Slips were then rinsed in water, air dried, soaked in xylene and mounted for examination. Establishment of Mutation Freqpencies A variety of resistant markers and mutation proto- cols were used throughout these experiments. The specific procedures employed for any given experiment are indicated with the data in the RESULTS portion of this dissertation. The details of the procedures are described here. Mutation induction was always achieved with UV from one of several germicidal lamps, positioned to deliver a 43 fluence of 1.0-1.4 joules/mZ/second. Trypsinized cells were plated in 9 cm tissue culture dishes in 10 m1 of medium at the density indicated for each experiment, and allowed 3.5 hours for attachment. Exposure consisted of removing both medium and dish cover for a sufficient time to achieve the total dose desired. In addition to those cells plated for mutation selection, an appropriate number of cells for each treatment group were plated to determine both plating efficiency and UV lethality, as previously described. The induction of ouabain resistance in Chinese ham- ster cells by UV has been quantitatively characterized (122). Ouabain resistance is rendered by mutations in the membrane-bound Na+/K+ ATPase (123). Mutation expression of ouabain resistance has been achieved by either igggigg or "replating" techniques. Both techniques were utilized for the experiments presented here. For lg E122 expres- sion, survivors of UV mutagenesis were permitted to develop into colonies before exposure to 1 mM ouabain (Sigma Chemical Company). Resistant clones were scored visually in 6-7 days. Expression time permitted before exposure to the selective agent was determined either in terms of cell division or time (e.g., days). Maximum expression of wild type Chinese hamster fibroblasts has previously been shown to occur at four cell divisions (122). Therefore, in some experiments, the visual identification of 16-ce11 colonies was used as evidence that maximal expression had occurred. 44 For the replating technique, mutagenized cells were permitted to grow continuously in log phase in the tissue culture dishes. When necessary, these cells were subcul- tured and transferred to plastic flasks to maintain suffici- ently low density and encourage log phase growth. At 3, 6, 9 and 12 days following mutagenesis, cells were replated in tissue culture dishes, allowed 3.5 hours for attachment and exposed to ouabain. Colonies were scOred visually when sufficiently large. A second locus utilized in these experiments was that of hypoxanthineguanine phosphoribosy1transferase (HGPRT). Inactivity at this locus has been demonstrated to be responsible for 6-thioguanine resistance (124). The induction of 6-thioguanine—resistant (6-TGr) mutants by both UV and x-rays has been reported (122), and 6-7 days has been determined to be sufficient time for maximum expression when utilizing a replating technique (122). Therefore, the UV-induced 6-TGr mutagenesis experiments discussed here employed a replating technique identical to that described for ouabain, with the substitution of 10 ug/ml of 6-TG (Sigma Company) as a selective agent. The forward mutation to diphtheria toxin resistance (DTr) has also been characterized as a UV-inducible mutation assay in Chinese hamster fibroblasts (125). Maximum mutant yield is achieved by employing a combined replating-1g glpg protocol. The DTr assay was used to examine UV-induced 45 mutagenesis in one experiment. The protocol is presented in Figure 2. Mutagenized cells were grown for various lengths of time and plated as per the replating technique. Before the addition of 0.1 Lf/ml (LF : floculating unit) of the diphtheria toxin (Connaught Laboratories, Toronto, Canada), however, the cells were permitted to undergo four rounds of replication. After two days, toxin was removed and fresh medium added. Following sufficient time for development, colonies were scored visually. Determination of Spontaneous Mutation Rates To determine the rate at which spontaneous mutations occur in the parental Chinese hamster cell line and one iso- lated mutant line, a modification of the technique initially developed by Newcombe (126) was used. As illustrated in Figure 3, the technique consists of the determination of mutation frequencies at various times and a concurrent monitoring of cell divisions. This permits the determina- tion of the change in frequency per number of cell divi- sions. Cells for both lines were grown in HAT medium (previously described) for 16 days, subculturing as neces- sary. After an additional 5 days growth in normal medium, cells were trypsinized and plated in 144 tissue culture dishes at 5 x 104 cells/dish. In addition to the dishes, 2 a total of 20 x 106 cells were distributed to four 150 cm flasks. After 3.5 hours, cells in dishes were exposed to 6 4 macaw mocmpmwmom waoe mwhospmfia mo hosedwoam map mcfisdmmo:.%mmm< QOHPHpDZ .m opsmflm Edwwo: Q QmOHm mama OHIR .~ meson em eoeee easoe oeafid\maaoo sea a a msaeaae Haoo aeoaaaeao Heme e _ mcwpwadom _ osdmooxm >3 HOOOpopQ mcflpmammm meassae Heme mason m.m _ hero NH asa.o.o.m wowhom gowmmosaxm 4 HOOOPONQ Cowmmoamxo one mammzmmmpdz 47 Mutation Rate Methodology N1 N2 N3 2 (F - F ) mutation rate = 3 a In 2 In N3 IN,I where: initial alter x divisions mutation frequency 0 B F N total cell number Figure 3. Mutation Rate Methodology. 48 10 ug/ml 6-TG. Flasks were subcultured when necessary by pooling and counting the total cells at each subculturing and seeding 20 x 106 cells into four new flasks. On the basis of cell counts, it was possible to determine the total cell growth that occurred between two mutation fre- quency samplings. Cultures were maintained for a total of 80 days, with a number of platings for frequency deter- minations to permit the calculation of several different mutation rate values for each cell line. The calculated mutation rates for the two lines were compared by means of a standard Student t-test. RES ULTS Selection Protocol The selection protocol was designed for the isola- tion of mutants defective in excision repair or hypersensi- tive to UV for alternative reasons. Protocol design, there- fore, focused on qualitative rather than quantitative char- acteristics. Past laboratory experience has demonstrated that 24 hour BrdU incorporation and 1 hour black light com- monly yields less than 1% survival and 20 joules/m2 UV yields less than 10% survival. Although measures were not employed to examine quantitatively, the lethality of these mutagenic treatments, visual examination confirmed that significant cell killing had occurred. The tritium suicide technique involving the incorporation of high specific activity tritiated thymidine after exposure to 10 joules/ m2 UV and subsequent accumulation of damage at low tempera- ture was also noted to produce significant cell killing. All culture dishes showed greater survival around the periphery than in the center of the dish. As culture dishes commonly produce a shadowing effect around the per- iphery during UV exposure, all the plates were scraped at the periphery with a sterile rubber policeman to eliminate 49 50 clones that may have survived as a result of this shadow- ing. Surviving clones were grown in HAT medium for 10 days before isolation to permit the elimination of cells that survived due to an inability to utilize exogenous thymidine. Of 2 x 107 mutagenized cells plated for selection, approximately 100 survived to produce colonies through the procedure. Seventy-two colonies were isolated and tested for preliminary evidence of UV-sensitivity or deficiency in unscheduled DNA synthesis. Initial Estimates of UV Sensitivity Exposure of a duplicate set of all isolated clones in multiwell plates permitted the qualitative identifica- tion of 9 UV-sensitive (No. 17, 27, 32, 33, 40, 44, 54, 58, 59) and 4 UV-resistant clones (23, 26, 61, 64). Preliminary analysis of repair synthesis for 57 clones is presented in Table 4. The results were used to identify lines 13, 17, 19, 32, 33, 41, 42, and 59 as poten- tially deficient in excision repair. Ten additional clones were analyzed at a later date permitting the identification of clones 4, 7, and 20 as potentially deficient. Of the 11 potentially repair deficient mutants, only four (No. 17, 32, 33, 59) were among the 9 lines identified as UV- sensitive in the qualitative prescreening. 51 Table 4.--Initial Crude Repair. Cell Line Sp. Act.* Cell Line Sp. Act. V79** 722.49 35 872.36 1 1132.16 36 1414.47 2 5555.84 37 918.65 5 879.37 39 790.39 6 975.39 40 674.82 9 737.94 41 289.04 10 930.55 42 299.34 11 646.66 43 765.48 12 702.75 44 605.72 13 415.64 45 714.76 14 600.65 46 626.44 15 562.58 47 488.39 17 424.51 48 522.45 18 995.84 49 937.34 19 362.34 52 953.15 21 1023.54 53 891.43 22 480.03 54 799.35 23 597.88 55 715.65 24 601.60 57 617.38 25 845.70 58 507.73 26 738.75 59 198.25 27 775.45 60 398.08 28 894.65 61 750.91 29 1036.39 62 866.56_ 30 819.52 63 501.40 31 839.59 65 638.58 32 240.78 66 774.26 33 325.61 67 527.08 34 674.26 68 897.23 *Sp. Act. = Specific Activity (DPM/ug DNA). **V79 background Sp. Act. = 156.24. 52 To further evaluate the 20 strains of potential interest, more quantitative UV survival experiments were conducted. The experiments illustrate that the strains exhibit a wide range of UV sensitivity, as exemplified by the data in Figure 4. Although all of the 20 variants appeared to be of further interest, it seemed most efficient to concentrate on a few cell lines for further characterization. There- fore, attention was focused on the two lines most sensitive to UV (UVS-40, UVS-44), the one line most resistant to UV (UVr-23) and the one line demonstrating the greatest deficiency in UDS (UVS-7). Radiation Sensitivity Detailed UV and x-ray survival curves for the four cell lines analyzed are presented in Figure 5. Because of its very slow growth characteristics, UVS-7 was often analyzed after the other cell lines. As shown in Figure 5A and 5C, UVS-40 and UVS-44 are sensitive to both UV and x-ray. UVr-23 is similar to wild type in x-ray sensitivity but more UV-resistant. UVs-7, which was not detected as UV-sensitive in the preliminary screening, is slightly, but consistently UV-sensitive as presented in Figure 5B. The x-ray sensitivity of UVS-7 (Figure 5D) is not at variance with the wild type. The UV survival curves have been used to estimate Do and N values for the four mutant lines and controls. 53 100 1 " s2=== \\ uv-23 uv-61 uv- Egfiiéoi 10 . uv-27 uv-Su uv-58 d >- 53 I: a» an * 1 . uv-hO 0.1 5, *fgbv uv DOSE (J/hz) Figure 4. Preliminary Range of UV Sensitivity. 0'» GO 6 PUKINTSUAMAL Figure 5. 54 1 1 l A L 2004000000000” X‘ RAY DOSE (RADS) X'MY m (RADS) UV and X-Ray Survival for Selected Clones. 55 Do is commonly defined as the increase in dose necessary to reduce survival by l/e (.37) on the straight-line por- tion of the survival curve. N is the multiple of 100% to which the straight-line portion would extrapolate at 0 UV exposure. The values are presented in Table 5. Table 5.-—Do Values for UV Exposure. Cell Lines Slope DO (ergs) N Control -8.8 x 10"3 49.1 3.34 UVr-23 -8.0 x 10’3 53.7 6.84 UVs-40 -9.6 x 10‘3 43.9 1.93 UVS-44 -9.8 x 10‘3 46.9 1.48 Control -16.1 x 10'3 26.7 43.64 UVS-7 -16.2 x 10‘3 26.7 33.80 The differences observed between the control lines for the two experiments are a consequence of varying UV sources, inaccurate monitoring of dose (a consequence of faulty monitoring equipment) and cell growth conditions. The interexperimental differences are inconsequential. The data suggest a minimum of intraexperimental variation in Do values. The N values change considerably from line to line, presumably a consequence of the size of the shoulder portion of the survival curve. Detailed analysis at low doses has not been conducted to precisely 56 evaluate the shoulder of the survival curve for the various lines. Sensitivity to Chemical Mutagens The mutants were also tested for their sensitivity to two chemical mutagens; NAc-AAF and MNNG. As shown in Figure 6, UVs—40 and UVS-44 are very sensitive to NAc-AAF, whereas the sensitivity of UVS-7 and UVr-23 to this chemical is similar to the wild type. UVS-40 and UVS—44 are also sensitive to MNNG (Figure 7). UVS-40 is more sensitive to the two chemical mutagens than UVS-44. Unscheduled DNA Synthesis (UDS) UV-induced UDS for the four mutant cell lines and the wild type were determined in two experiments, presented together in Figure 8. The results clearly indicate that UVS-7 is deficient in UDS. In contrast, UVr-23 is more proficient in UDS. The other two mutants, UVS-40 and UVS-44, which are sensitive to both radiation and chemical mutagens, are only slightly reduced in their ability to conduct UDS. Temperature Sensitivity Test Since the potential for temperature sensitivity had been incorporated into the selection protocol, UVS-40, UVS-44, and UVr-23 were examined for UV sensitivity and UVr-23 and UVS-7 for UDS at low temperature (34°C). The experiments were conducted in a manner similar to those previously described but in an incubator maintained at 57 IOO _\ T“-» o (kqu I uv O uv23 A uv40 " uv44 \, IO \\\\ é § I it b V) k. 2 lo ‘3 $1 at Q~ OJ Of» L 1 1 1 °'°°' 0.5 I L!) 2 NAc-AAF(»g/ml) Figure 6. N—acetoxy-Z-acetylaminofluorene Toxicity. 58 IOO ~ oControl Auv"40 Xu¢L44 D I— ‘J E S 0') I.0 - K 53 OJ — 00' 1 J l (18 l6 32 MNNG (pg/ml) Figure 7. N-methyl-N-nitro-N-nitrosoguanidine Toxicity. 59 4" Control 0‘. qu-7 ‘ uvR-za V qu-40 I qu-44 D RELATIVE ENHANCEMENT OF SPECIFIC ACTIVITY UV DOSE 20 (Joules/m2) Figure 8. UV-Induced Unscheduled DNA Synthesis. 60 34°C. The experiments were intended to provide crude data relevant to the temperature sensitive nature of the mutant lines. Table 6 presents the data of an experiment examining the percent survival of mutants and control after exposure to one dose of UV (l8 joules/m2) at low temperature. At low temperature UVS-40 and UVS-44 remained highly sensi- tive and UVr-23 was resistant to the lethal effects of UV. Table 6.--Low Temperature Effects on UV-Survival. Cell Line Control UVr-23 UV -40 UV -44 % survival 13.2 30.1 2.3 2.6 Figure 9 presents the unscheduled DNA synthesis results at low temperature. The observed alteration in phenotype (UVr-23 enhancement and UVs-7 deficiency) per- sists at low temperature. The alterations responsible for the phenotypes of all four mutants, therefore, appear not to be temperature sensitive in nature. Chromosome Examination The examination of chromosomes from the parental and four mutant lines produced a modal number of twenty chromosomes for all lines. Representative preparations are shown in Figure 10. The chromosomes have not been analyzed in greater detail. 61 6- CONTROL 0 uv'-7 . uv'-23 - 5%- * C >4— 0 o o- E > c o- 0 '0- C 0 a o i o o :;3- 0 0 0. > a. z (D o O — '0- ‘: o 2.. II— - 1 O 5 2C) Figure 9. u v (J/mzl Low Temperature Repair. 62 UV V79 qu-«Io UVS-44 Figure 10 . Chromosomes . 63 Recovery to High Molecular Weight DNA Following UV-Irradiation Figure 11 presents the data for the first experi- ment. With increasing chase time, most lines show a pro- gressive increase in distance sedimented indicating an increase in molecular weight of 3H-thymidine labeled DNA. The 3H label represents DNA synthesized after UV-irradiation (daughter DNA). Disagreement exists in the literature as to whether small weight DNA following UV irradiation is a consequence of postreplication "gaps" or alternatively, a delay in normal DNA synthesis. Progressive recovery either represents the filling of gaps (postreplication repair) or recovery from a delay in normal synthesis. UVS-44 is the only line examined that does not show a progressive increase in the size of the DNA with increasing chase time after 3 irradiation. The distance of H-TdR peaks sedimentation in these experiments is defined as: 3 (Total Fraction # — Peak H-TdR Fraction #) Total Fraction # It is a crude estimation of relative size and should not be considered an accurate estimate of molecular weight. The data for the second experiment are presented in Figure 12. It can be seen that for control cells, a 45 minute chase following either a single or split dose was sufficient to permit recovery of the DNA to unirradiated 64 0 control * uv-EB v uv- O 1'0" x uv-hh E4 :r: U H I!) 3 c: < I—‘l D S / 2:05 // O / // E / // i E // / 7 < 9 / // ” a / / /’ I / / / // // / / / I / // ’IV’ / / I / ‘/ x ---¥-— --——————- ------- / /’ / X ” O L , 45 9o CHASE (min) Figure 11. Relative Size of Irradiated DNA. 65 - no II o w IIIIIII ' cInIIIII . SPl|l+c|lIlSE ‘ y % Radggactivit III I: FractIOn Figure 12. Gradiant Sedimentation Profiles. 66 size. For UVS-44, neither condition permitted the DNA to recover to full size. Cell Growth Rate The growth curves for the parental line and four mutant lines are shown in Figure 13. All mutants grew at a rate slower than the parental line. The cell division times have been estimated at 16.0, 20.8, 21.5, 22.5, and 26.6 hours for the parental line, UVr-23, UVs-44, UVs-40, and UVS—7 respectively. UV-Induced Mutation Frequencies--UVs-7 Table 7 summarizes the data from three different experiments examining the UV-induced mutability of UVS-7 at the Na+/K+ ATPase and HGPRT loci. In the first experiment, cells were plated at l x 105 cells/plate for spontaneoUs frequency determination and 2 x 105 cells/plate in plates which were irradiated with 18 joules/m2 UV after 3.5 hours for cell attachment. Cells for spontaneous frequency deter- mination were allowed one day of lfl.§l£2 growth before selec- tion with 1 mM ouabain. UV-irradiated cells were selected after three days 12 gigp growth, a period of time previously demonstrated to be sufficient for maximal expression of wild type UV-induced ouabain-resistant mutants (97). Under these conditions, the UV-induced ouabain-resistant mutation frequency for UVS-7 was three times that of the parental line. 8 83388 Illlll 3. l I 30 20!— CELLS/PLATE x 105 O N O (D O l l l l l I I I Figure 13. 67 Parental V79 UVS-7 UV '-23 UV9-40 X I> D'¢3 O UV3-44 48 TIME IhourSI Mutant Growth Rates. 3L 613 Table 7.--UV-Induced Mutagenesis for UVs-7 and Its Parental V79 Cells. EXPRESSION TorAL # MurArIon UV 005E SURVIVAL TINE or CELLS FREOUEch ExPERINENr CELL LINE (J/HZ) 2 (DAYS) ASSAYED x10S IUETSURVIVORS V79 0 100 1 30.8 3.3 #1 uv5-7 0 100 1 23.0 1.3 00AR (A) V79 18 7.4 3 4.1 448.8 qu-7 18 5.6 3 2.4 1529.6’ V79 0 100 2 19.9 8.5 #2 UV5-7 0 100 4 19.1 2.6 00AR (A) V79 20 2.0 2.5 0.8 1093.3 uv5-7 (a) 20 1.1 s 0.3 29444.4* V79 0 100 6 9.9 9.1 UV5-7 0 100 6 10.3 10.3 V79 0 100 9 7.1 29.6 qu-7 0 100 9 7.8 10.3 V79 0 100 12 10.4 12.5 UV5-7 0 100 12 8.1 2.5 #3 V79 0 100 15 7.7 29.9 5-158 (c) UVs-7 0 100 15 12.4 7.3 v79 14 11.2 6 7.7 163.6 uv5-7 14 10.0 6 8.6 174.4 V79 14 11.2 9 5.1 309.8 qu-7 14 10.0 9 7.3 574.0+ V79 14 11.2 12 9.2 220.7 qu-7 14 10.0 12 8.0 411.3* V79 14 11.2 15 7.5 240.0 qu-7 14 10.0 15 11.6 424.1’ "0. OF CELLS USED PER PLArE (9cn) NAs A= 1x105 FOR SPONTANEOUS AND 2x105 FOR UV-INDUCED EXCERr FOR 8- 5x10". c- 5x10“ FOR ALL PLATES. * HurArIoN FREQUENCIES roR UV5-7 ARE SIGNIFICANTLY HIGHER THAN THOSE FOR V79 coNrRoL WITH EOUIVALENI DOSE or UV. P < 12. 69 Due to the slow growth of UVS—7, three days ifl.§l£2 does not result in colony size equal to the parental con- trol at the time of selection. This factor was taken into account in the second experiment, which also employed the $2 ElEB ouabain selection technique.. After exposure to a high dose of UV (20 joules/m2), five days growth permitted surviving UVs-7 cells to achieve a predominance of 16-ce11 colonies. The parental line was noted to achieve such colonies in 2.5 days. Employing such a variable expression protocol resulted in an induced mutation frequency for UVS-7 nearly 27 times that of the parental line. In the third experiment, the spontaneous and UV- induced 6-thioguanine resistance frequencies were examined with an 12 glgp protocol, using one moderate dose of UV (l4 joules/m2) and multiple expression times of 6, 9, 12, and 15 days. Plating 5 x 104 cells/plate has previously been shown to be of sufficiently low density to permit the recovery of virtually all resistant cells for the control line (127). The results indicate that for both the parental control (V79) and mutant (UVS-7) lines, maximal expression was not fully achieved by six days, but was by nine days. In addition, further expression did not result in an increase in the mutation frequency for UVs-7, suggesting that the expression period for both the control and mutant line are similar for 6-thioguanine resistance. Most importantly, with this moderate dose of UV, the mutant line demonstrated 70 a slight increase in UV-sensitivity and a nearly twofold increase in mutability. The mutability of UVS-7 was also examined at the diphtheria toxin (DT) resistance locus (elongation factor II). The experiment was conducted concurrently with the 6-thioguanine experiment presented in Table 7. The same spontaneous and UV-induced (14 joules/m2) populations were used to determine the frequency of DT resistance as were used to examine 6-TG resistance. The same expression times of 6, 9, 12, and 15 days were examined. The DT results are presented in Table 8 and summarized in Figure 14. The results demonstrate that both the parental control and mutant line share equivalent UV-induced maximal expression times of approximately nine days. Both lines demonstrate a sharp decline in the frequency of mutants after the maximal expression time, indicating a strong selective dis- advantage for DT resistant cells in the population. At the maximal expression period of nine days, the UV-induced muta- tion frequency for UVS-7 is seven times that of the wild type cells. UV-Induced Mutation Frequencies-~UVs-40 The UV-induction of ouabain resistance for UVS-40 was examined in two separate experiments. The first, pre- sented in Table 9, examines the frequency of ouabain resis- tance following 0, 5, 10, or 20 joules/m2 of UV. The $2 Situ expression technique was used, with the variations in 71 . ca x H was A50 m. mumflm Hum poms maamo mo HOQESZR m m.omo o.a~ ma o.oa as a- >2 o.ee a.eH ma N.HH as we> a.mme o.oa NH o.oa as a- >o ~.ea m.ma NH N.HH ea me> m.amaa e.ea a o.oa as a- >2 m.me~ H.oH a ~.HH ea wa> o.m~ma ~.ea o o.oa ea a- >o m.eoa e.mH o ~.HH as me> o.am e.e~ ma ooa o a- >3 H.HH m.mH ma ooa o me> a.m m.oa NH ace 0 a- >o m.m e.om as cos 0 ms> a.aea m.ma a sea o eun>o o.aa ~.ea a ooa o ae> m.oaa e.o~ o ooa o sun>o m.om m.aa o ooa o as> muo>fi>usm moa moa x newsmmd Am>mov Am. A E\h. wocmnqmum Homwwmo mo oeflam Hm>fl>usm O on >3 mafia HHOU cowumusz a o z Hmuoe :OHmmmH xm .Houucou Hmucmumm muH pom hIm>D How oucmumwmom saxoa manonundwa ooosocHI>DII.m manna 72 .Houucoo Hmucoumm mUH can hIm>b How oocwumfiwom CANOE mflwoaunmfin Am><9 w}: ZO_mmw¢uxm l 8 an SlOAanS 901 18d sIueInIu “a 1 SN— 1 8: _>: 0:. a~> 5:22. OI Ilo $3 257;: $1.14 I. 80— .15.. 3 5:3; .25.... 0.10 :15. 3 .2: 7;: I .ea enemas 73 Table 9.--UV-Induced Mutagenesis for UVs-40 and Its Parental V79 Cells. EXPRESSION TOTAL # OUAR UV DOSE SURVIVAL TIME OF CELLS HUTATION FREQUENCY CELL LINE (J/MZ) (2) (DAYS) ASSAVED x105 106 SURVIVORS V79 0 100 1 20.7 (a) 1.5 qu-40 o 100 1 11.7 (a) 7.7 v79 5 91.6 2 9.5 (A) 77.9 uv5-40 5 71.9 2 8.0 (a) 58.8 v79 10 65.7 2 6.1 (A) 75.4 v79 10 65.7 3 6.8 (A) 117.6 uvs-40 10 16.6 3 3.7 (c) 127.0 qu-4o 10 16.6 4 3.9 (c) 146.2 v79 20 14.0 2 5.8 (c) 89.7 v79 20 14.0 3 5.8 (c) 243.1 v79 20 14.0 4 2.9 (a) 410.3 uv5-40 20 1.1 3 1.3 (D) 346.2* ** qu-4o 20 1.1 4 1.3 (6) 584.61 ** uv5-40 20 1.1 5 1.1 (D) 1145.5** NO. or CELLS USED PER PLATE (9cm) WAS A- 5x10“. 6- 1x105. c- 2x105, 6- 8x105 1 MUTATION FREQUENCIES EOR UV5—40 THAT ARE SIGNIFICANTLY HIGHER IHAN THOSE FOR V79 CONTROL AT EQUIVALENT DOSE OF UV AND EQUIVALENT EXPRESSION TIME. P < 11 ++I‘IUTATION FREQUENCIES FOR UV5-40 THAT ARE SIGNIFICANTLY HIGHER THAN THOSE FOR V79 CONTROL AT EQUIVALENT DOSE OF UV BUT ONE DAY LEss EXPRESSION TIME FOR V79 CONTROL. P < 12. 74 expression time as indicated. As UVS-4O divides slower than the V79 control, equivalent or longer expression times were commonly examined for UVs-40 to permit the development of equivalent colony size before selection. The data indi- cate no difference in the resistance frequencies at a low UV dose of 5 joules/m2 and only a slight, but statistically insignificant, hypermutability of UVS-40 at a moderate UV dose of 10 joules/m2. At 20 joules/m2, the hypermutability of UVS-40 is quite evident. A second UV-induced ouabain resistant experiment was conducted, examining the frequency of resistant colonies at 2, 3, 4, and 5 days ;p gigp expression, A single UV dose of 20 joules/m2 was used. The data are presented in Table 10 and summarized in Figure 15. UVS-40 is clearly hypermutable compared to the V79 parental control with 20 joules/m2 of UV-irradiation. UV-Induced Mutation Frequencies-~UVr-23 Table 11 presents the data derived from two separate experiments examining the UV induction of ouabain resistant colonies for UVr-23 and the parental control line. Expres- sion for both experiments was achieved Via lg EEEE colony growth following the various UV doses indicated. The data suggest that UVr-23 may be hypomutable, as the frequency of ouabain-resistant mutants is less than that for the parental control at all UV doses. However, only at the highest dose (20 joules/m2) does that difference appear significant. 75 . OH x m.m u m .40H x m u U . OH x m u m . OH x H u a "mos .80 m. oumHm mom pom: mHHmo mo Hmoadz m m H.mmh ADV m.H m H.H om owl >D e.ee~ loo H.H m a.oa om ma> 5.0mm ADV N.H v H.H om o¢Im>D m.NHN Aflv v.N v m.OH om mb> m.emm 1o. m.H m H.H om oeun>o H.NVH Amy ©.v m m.OH om mh> m.amm 1o. m.H a H.H om oeun>o N.Hm Amv m.v N m.OH ON mh> H.mN Afiv m.mH m.N OCH 0 ovlm>D v.H Afiv H.NN m.N OOH o mh> muo>fl>usm ooH OH x omwmmm< Amwmov m nHHoo no mass is. .ms\o. moan HHOO wucmsqmnm Honesz Hmuoe conmoumxm HO>H>HDm O on >D coHumusz . .Houucoo Hmucmumm muH pom ov1m>D MOM mHmmcmmmusz UOODUsHI>D mo conmoummeI.0H OHQOB 76 .Houuooo Hmucoumm muH pom oqu>D How mHmOcommunz omosocHI>D mo conmOmem Am>: 4 II 4 .mH musmflm uowuns 9m 10d sIueInun ,vno .moH x m u o .mmH x H u o 77 .vOH x O u m .OOH x m u 4 "mos .80 m. OumHm you new: mHHwo mo Hon 2 m.mmh A.mH ON mmIH>D ~.OOO~ ADV 0.0 m O.H ON mn> v.Om Adv O.m m.m O.vh OH mmIH>D €30 O.~m Am. O.m m.m 0.0m OH O>> NH O.N AD. m.om H OOH O mqu>D H.v ADO m.mH H OOH O mh> v.mmm Am. O.m m m.mm OH mmIH>D 0.0mm HOV O.H m m.n OH mn> moo m.O AGO «.mH mm.H OOH O mqu>D H¢ v.5H HOV O.HH m~.H OOH O Oh> mHo>H>uom OOH mOH x pommmmd Am>oav Am. A E\OO hocmsvmum ummwwou WOO :onMWwwx Hm>H>H5m Ommo >9 OGHH HHOU pameHummxm GOHDOUOE o 2 H u 9 . m .conmoudxm DuHm mMIImHHmU mh> Hmucmumm muH pom mmIH>D MOM mHmmcmmmusz UOOSOcHI>DII.HH OHQOB 78 It was suggested for both UVS-7 and UVS-40 that slower division time might necessitate longer expression periods than the control cells to achieve a maximal value. This is particularly important when utilizing 13 E222 selec- tion techniques, where one hopes to achieve equivalent colony size for selection. This hypothesis, however, might be used to argue against the hypomutability of UVr-23, which also grows slower than the parental control, and may not have fully expressed at the time of selection. Therefore, a third experiment was conducted, examining the UV- induction of both ouabain-resistant and 6-thioguanine- resistant colonies for UVr-23 and the parental control using the replating technique for expression. One dose of UV (16.8 joules/m2) and multiple expression times of 3, 6, 9, and 12 days were used. The results are presented in Table 12 and the ouabain and 6-thioguanine portions summarized in Figure 16. The previous tendency for UVr-23 to appear hypomutable was confirmed. For the expression periods examined, no delay in ouabain or 6—thioguanine expression was noted for UVr-23 cells. A sharp reduction in the frequency of UVS-23 ouabain-. resistant cells was noted between the third and sixth days of replating. This observation suggests a great selective disadvantage for UVr-23 ouabain-resistant cells. These findings coincide with the qualitative observation that both 6-thioguanine- and ouabain-resistant clones derived 79 O.mmv 0.5 O.HN 0.0H NH O.HN m.OH MNIH>D N.NOO O.v O.mH 0.0H NH O.vH 0.0H mb> H.Omv O.h O.HH 0.0H m O.HN OSOH mNIH>D H.5mm N.v 0.0H O.m m m.vH 0.0H mh> O.N¢v N.O O.mm O.HH O O.HN m.OH MNIu>D H.mmm N.m H.5v O.HH O m.vH 0.0H 05> H.ONN O.m «.0v m.OH m O.HN 0.0H MNIM>D m.mNN o.n n.nv m.OH m O.VH m.OH mb> O.mm O.h O 0.0H NH OOH O MNIH>D 0.0VH m.n 0.0 0.0H NH OOH O mw> 0.00 O.h 0.0 v.5H m OOH O mNIu>D m.HNH h.m O O.MH O OOH O mh> m.MO O.m m.o O.mH O OOH o MNIM>D H.OvH O.¢ o 0.0H O OOH o m>> v.Om m.w O O.mH m OOH O MNIH>D N.vh 0.0 0.0 H.OH m OOH O mn> OCHGODOOHneIO :Hmnmso muo>H>Hsm OOH OOH x woammmd muO>H>Hsm OOH OOH x Omummmm Hmmmov Hwy H E\OV aocmsvmum MOMWMMUHMMOB mucosvoum NOMWMMOHMMOE coawmwwmxm HO>H>Hsm omma >5 OCHH HHOU :oHumuaz coHumusz . .soamnmndxm oouoHdom EDA: Hosusoo Houseman nnH one mmIA>O no SDHHHDBDOS omososHI>OII.~H OHnea 80 A (OUABAIN) 0:) 0 Control 3 50 _ f. 3 . 0 UV 23 p. z 0 Q "3 40- O W o 3 5?: ; x 30— o z W D 8 c 20— IL 2 9 2 I- 10*— 3 2 l l l A] o 3 6 9 12 EXPRESSION TIME (DAYS) 3 (6- THIOGUANINE) 600 — 500 '— 400 *— 300 - 200 I— 100)— MUTATION FREQUENCY (INDUCED-SPONTANEOUS) x 105 l l 6 9 12 EXPRESSION TIME (DAYS) (ADI-— b Figure 16. Expression of Ouabain and 6-thioguanine Resistance for UVS-7 and Parental Control Cultures. 81 from UVr-23 cells appeared less healthy than their parental equivalents, even though UVr-23 cells generally appeared as healthy as the parental cells when grown as clones or in mass culture. UV-Induced Mutation Frequencies--UVs-44 The UV-induced mutability of UVS-44 was examined in three experiments, the results of which are presented in Table 13. With a single UV dose of 18.2 joules/m2, and replating expression times of 8 and 13 days, the 6- thioguanine-resistant mutation frequency for UVs-44 was found to be comparable to that for the parental control cells. The two ouabain experiments were conducted utiliz- ing in gitg expression and 18 joules/m2 of UV. In both cases, UVS—44 was provided a single in sign expression time of three days. No significant difference was noted between the mutability of UVS-44 or parental control cells. It was, however, noted that UVS-44 ouabain-resistant colonies exhibit poor growth and colony formation. Retesting of Induced Mutants UV-induced ouabain-resistant mutants from UVs-7 were isolated (from Table 7, Experiment #2) and reselected in 1 mM ouabain. Of thirty clones retested all were con- firmed as ouabain-resistant mutants. Following the diphtheria toxin experiment (Figure 14), UV-induced DTr mutants of UVS-7 were isolated and tested for sensitivity 82 . OH x m was «EU m. mumHQ you poms mHHmo mo HmnE:z« O O.OOO O.O OH O.H ~.OH OO-O>2 O.NOO O.O OH O.O ~.OH OO> 0.000 O.O O O.H ~.OH OOIO>2 O.OOO ~.O O 0.0 m.OH OO> usuO O ~.O OH OOH O Ovum>2 OO O.~H O.O OH OOH O OO> O O.O O OOH O OO- >2 0.0~ «.mH O OOH O MO> 0.0HO m.O m O.~ OH «O. >2 0.0HO O.~ O H.O OH MO> <22 O.H N.O O.H OOH O OO. >2 NO O.O O.OH O.H OOH O MO> O.OO~ O.m O m.m OH OO- >2 0.00~ O.H O ~.O OH NO> 420 0.0 O.HH ON.H OOH O OO- >2 HO 0.0H O.HH O~.H OOH O NO>‘ muo>fi>usm mod OH x cm>Mmm< Amamav A V A E he hocmsvmnm m mHHmU mo mafia Hm>HMH5m mmmo\>b mafia Hamu ucmsfluomxm coaumusz «Honesz Hmuoa :onmmumxm . .Honucoo Hmucmumm moH paw vv|m>D mo mwmwcwmmusz pmuschn>Dll.MH manna 83 to both diphtheria toxin and pseudomonas exotoxin. Of twenty-four clones retested all were found to be resistant to both toxins. The results lend support to the frequencies reported and to the hypermutability of UVS-7. Spontaneous Mutation Rates UVS-7 demonstrated UV-induced hypermutability at three loci, a slightly increased sensitivity to UV, and a greatly reduced capacity to conduct unscheduled DNA syn- thesis when stimulated by UV. This mutant cell line, how- ever, shows no increased sensitivity to x-ray or NAc-AAF. As all abnormal phenotypes appeared to occur in response to UV, it was of particular interest to examine the spontaneous mutability of UVS-7 relative to the parental control. As previously described, to evaluate the spontaneous mutation rate, it is necessary to determine the change in mutation frequency relative to cell growth. Populations of UVs-7 and parental control cells which had been preselected in HAT medium were maintained for a period of 35 days with frequent samplings of mutation frequencies. Figure 17 pre- sents the results obtained with these frequency determina- tions. Both cell lines showed a consistent increase in the frequency of 6-thioguanine-resistant cells. This increase was faster in the control line than the UVS-7 mutant, a result anticipated since UVS-7 grows slower and thereby accumulates new mutants less quickly. Frequency determina- tions were also made at later times as the two cultures 84 48 '- Cell Line 810 e o = V75 -ITE65 x = UV3-7 1.059 x d “L— o 32 -‘ x ~o I c: H N 24 .- O E E; x a. 2: S 9. <1: E3 16_ x r 8 I. o' '7 14 21 28 33'— DAYS Figure 17. Spontaneous Mutation Frequencies in UVS-7 and Parental Control Cultures. 85 were maintained for a total period of eighty days. At various intervals throughout this eighty day period, the increase in population size was monitored between fre- quency platings to permit the calculations of spontaneous mutation rates. The data are presented in Table 14. A standard Student t-test indicates that no significant dif- ference exists between the average spontaneous mutation rate for UVS-7 and that for the parental control. Stability of Mutants The four mutant cell lines that have been most extensively studied throughout this research appear to be quite stable. Each line was maintained continuously in culture for a period of time in excess of thirty days and in the two and a half years since their initial isolation, no reversion of any altered phenotype has been observed. .AH0.0 A me ucmummmwp mHucmoHMHcmHm uo: mum mammz« 86 O.OH x OOO.~ u Oum>2m. O..OH x Omm.~ u OO>m« OH x m.m O0.00 OH x O.H Hm.mO OO OOO.~ Om OH0.0 NO x O I 0 O ONOH N n on mv ONOH x O m mm mv mo mHOH x m.¢ OO.~¢ u: I: mm OHH.O mHOH x v.m OO.hN NHOH x H.> >O.H¢ mm mOH.N Hmv.H NHOH x m.H Om.Hm mOH x v.m HH.vm Hm Omm.m Omm.m OHOH x v.~ mm.HH N.OH x O.~ Om.mm OH m>>.O OOH x O.m OO.m OHOH x O.H mm.mH O OOO.N mmH.~ N.OH x O.N >O.N hOH x O.~ mm.~ O OIOH x coHumHsmom mHHoU O OIOH x coHumHsmom mHHmU O cowumms cw MmeU aocmsvmum cowumms CH MHMmU mocodvmum pmumHm .u u 2 H u a :oHumusz .2 u 2 H u a :oHumusz and >Im>D mh> .mwumm :oHumusz msomamucommll.vH mHQMB DISCUSSION The Isolation Protocol and Its Success A new isolation protocol (Figure 1) was designed and implemented to aid the selection of mammalian cell mutants with defects relevant to the mechanisms of DNA repair. The first phase of this protocol specifically focused on the generation of new mutants. It has long been recognized that most new mutations are recessive (128). On the assumption that wild type cells in culture are homozygous for normal alleles at repair related loci, the likelihood of generating a homozygous recessive mutant with a point mutagen such as UV, would be exceedingly small. The generation of a hemizygous state through the deletion of one of the homologues, and subsequent point mutagenesis of the remaining homologue should provide an alternative theoretical approach and an adequate means for the genera- tion of temperature sensitive mutants. In the studies reported here, an attempt to generate hemizygosity involved the incorporation of BrdU for approximately two rounds of replication followed by exposure to black light. Such treatment has previously been shown to induce strand breaks (128) and is also capable of producing temperature sensi- tive mutants (130). Subsequent exposure to UV, primarily 87 88 resulting in the formation of pyrimidine dimers, could result in the formation of a point mutation in the hemi- zygous locus. This induction technique should permit the generation of either complete deficiencies or partial reductions in repair related functions. The two-step mutation induction is relatively harsh, and may result in the generation of mutants with alterations at a variety of loci in addition to those related to repair. From the chromosomal observations reported, it does not appear that the induction or selec- tion procedures encouraged the isolation of mutants with altered ploidy. The induction procedure appears to have been affec- tive in generating a variety of mutant phenotypes. Although unmutagenized populations have not been tested in the selec- tion portion of the isolation protocol, it is unlikely that such a variety of mutants would readily exist in the general population. Isomura and colleagues attempted to isolate UV-sensitive mutants from populations of unmutagenized HeLa 83 cells without success (114). Furthermore, the data pre- sented here and the personal experience of this researcher indicate that the mutants exhibit poorer plating effici- encies, slower growth characteristics, and a general ina— bility to effectively tolerate medium deficiencies and depletions as compared to wild type cells. Such mutants are likely to experience a strong selective disadvantage 89 in the general population. This hypothesis is testable and may be of considerable importance in future con- siderations of somatic cell population genetics. The selection portion of the isolation protocol is a modification of the tritium suicide technique previously reported to be an effective selection procedure in Chinese hamster ovary cells (131). Modifications included the growth of cells in reduced levels of fetal calf serum (1%) to accumulate cells in non-S phase, thereby avoiding 3H- TdR incorporation due to normal DNA synthesis. The 10 joules/m2 dose of UV just prior to 3H-TdR exposure was designed to encourage the uptake of tritium by excision repair proficient cells. Following the accumulation of tritium damage at low temperature, cells which survived as a consequence of culture dish shadowing were eliminated by scrapping and those which survived because of an ina- bility to utilize exogenous thymidine (thymidine kinase deficient) were eliminated by growth in HAT medium. The selection scheme is also harsh. Although the lethal effects of selection were not quantitated, signifi- cant killing was observed. Various treatments, and in particular the 10 joule/m2 dose of UV, may have selectively eliminated desirable mutants, including those highly sensi- tive to UV. For this reason, Kuroki and Miyashita (103) have criticized the use of harsh selection and have focused on replica plating as a means of mutant identification. 9O Replica plating, however, involves no means of enrichment and thereby proves tedious, particularly if attention is focused on repair deficiency, which cannot be easily iden- tified. It was, therefore, beneficial to employ some form of selection protocol with the risk of eliminating some mutants. It can be noted that uvs-7 would be difficult to detect via replica plating, except by virtue of its slow growth. Since 20 of the 78 isolated clones appeared to have altered UV sensitivity or DNA repair, the isolation protocol appears to have functioned as originally expected. This has been further verified by the detailed characterization of a few selected isolated mutants, and a second attempt at the isolation protocol, which also produced a number of clones qualitatively identified as UV sensitive. Although greater consideration may have been focused on improvements in the isolation technique, it seemed most beneficial to concentrate attention on the characterization of the spe- cific isolated cell lines. A great many loci influence the quality and quan- tity of DNA repair, as has been extensively demonstrated in Escherichia coli. It was, therefore, not surprising to find such extensive diversity among the isolated mammalian mutants. This diversity is illustrated in Figure 4 and Table 4. The four mutants most extensively characterized, UVS-7, UVS-40, UVS-44, and UVr-23, illustrate the extreme 91 examples of this variation. Although the observed diversity was not surprising, the recovery of large numbers of clones, exemplified by UVr-23, with resistance to UV and/or enhance- ment of unscheduled DNA synthesis was unexpected. Such clones may have survived as a consequence of their resis- tance to UV. The isolation procedure utilizes several expo- sures to UV. These exposures may select for resistant cells and enrich their proportion in the general population. This would increase the likelihood that several colonies surviving the tritium selection would actually be resistant to UV, with normal or enhanced UDS. UVs-7 is precisely the type of mutant that the iso- lation protocol was intended to select. Greatly reduced UDS would facilitate survival through the lethal growth selection. As UVS-7 is only slightly sensitive to UV, the 10 joule/m2 exposure prior to 3H-TdR incorporation would not have been excessively lethal. Had UVS-7 been highly sensitive to UV, comparable to complementation group D XP cells (52), a 10 joule/m2 dose would have killed the mutants. UVS-4O and UVS-44 are moderately sensitive to a variety of mutagens and only slightly depressed in their ability to conduct UDS. The selection of UV sensitive mutants with relatively normal UDS was expected, as the laboratories of both Isomura (114) and Randtke (113) have 92 previously used UV in conjunction with letal growth tech- niques to select for cell lines which were UV sensitive. All four of the mutants examined in detail exhibit varying degrees of slow cell division. This factor may also have encouraged their selection, as residual incor- poration through normal synthesis would be lower in such lines. Each of the mutants, however, expresses character- istics that suggest selection was enhanced by features less trivial in nature. As the isolated mutants were found to be of signifi- cant interest and worthy of detailed studies, the isolation procedure was not characterized in more complete detail. Future considerations for the use of such a procedure might focus on better quantitation and on the isolation of tempera- ture sensitive mutants. Comparative Evaluation of UV Sensitivity and Unscheduled DNA Synthesis The data for UVS-7 and UVr-23 correlate with what one would expect of mutants with altered nucleotide excision repair. UVr-23 exhibits enhanced UDS and corresponding resistance to UV. UVS-7 is deficient in its ability to conduct UDS and is sensitive to UV. The degree of UVS-7 sensitivity is not as great as that observed for the XP complementation group D, which exhibits approximately equivalent reduction in UDS. One must consider, however, that while Chinese hamster and normal human fibroblasts are 93 roughly equisensitive to UV exposure, the human cells possess an ability to conduct UV-induced UDS many times that of the hamster cells. A reduction in UDS in hamster cells might, therefore, contribute less to UV sensitivity than in human cells. The UV sensitivity of UVs-4O and UVS-44 was greater than that for UVS-7, however, their UDS was only slightly reduced. The data suggest that the basic defect in both of these cell lines is associated with a mechanism other than nucleotide excision repair. Similar UV sensitivity without a corresponding reduction in UDS has been observed in the cells from patients with many of the radiation sensitive syndromes listed in Table 2. The UV survival curves for all the new mutants were used to generate DO and N values. As the data indicate, only slight differences were observed between the DO values for the various mutants and the parental control. The major differences in UV survival appeared to be a conse- quence of the size of the shoulder portion of the curve. A theoretical explanation for this observation is as yet not ascertained. If the size of the shoulder reflects a measure of excision repair, this would be consistent with the apparent shoulder extension for UVr-23 and reduction for UVS-7. UVS-4O and UVS-44, however, have virtually no shoulder, and yet have near normal excision repair. Studies of the various complementation groups of XP 94 demonstrate a lack of correlation between DO and excision repair. It appears that both the shoulder and straight- line portions of UV survival curves are influenced by factors in addition to excision repair. The Damage of UV Versus That of N-acetoxy-2-acetylaminoflugrene The controversy in the literature regarding the similarities and differences in the repair of damage pro- duced by UV and NAc-AAF was eluded to in the LITERATURE REVIEW. The present data suggest that while the mechanisms of repairing damage caused by these two mutagens show simil- arities, specific differences must also exist. UVS-4O and UVS—44 were found to be sensitive to both mutagens, a result previously demonstrated for XP fibroblasts (52). Though the defect in XP cells is specific to nucleotide excision repair, the defects of UVS-40 and UVS—44 appear to be of a more general nature, as the mutants also exhibit sensitivity to the mutagens MNNG and x~ray. The latter are believed to produce damage repaired by mechanisms other than nucleotide excision repair. The results observed for UVs-7 and UVr-23 emphasize differences that exist between the mechanisms of repair for UV and NAc-AAF damage. UVS-7 is slightly sensi- tive to UV, but consistently shows no sensitivity to NAc- AAF. UVr-23 is resistant to UV, but exhibits no resistance to NAc-AAF, as compared to control cells. The results pre- sented have focused solely on survival. Additional 95 experiments could be considered examining NAc-AAF induced UDS or mutagenesis for UVS-7 and UVr-23. In addition, modifications in the isolation procedure may lead to the selection of mutants altered in their response to NAc-AAF, but not to UV. UV-Induced Mutagenesis The UV-induced hypermutability of UVs-7 was clearly demonstrated at three loci, which have previously been quantitatively characterized in Chinese hamster fibroblasts. These results are consistent with the observations that UVS-7 is deficient in UDS and sensitive to UV. Similar correlations have been observed with the classical XP fibro- blasts (52), which inferred that excision-repair is an error- free process. Deficiencies in excision-repair could lead to the saturation of error-free processes at lower doses of UV, resulting in increased UV sensitivity and hypermutability through error-prone mechanisms or error-prone physiological states. The biological realities are clearly more compli- cated than this simplistic explanation, as evidenced by the previously discussed lack of correlation between UV sensi— tivity and UDS among the various XP complementation groups. The degree of hypermutability for UVS-7 varies from locus to locus. The most dramatic effect occurs at the ouabain—resistant locus, where expression of four cell divisions for both control and mutant cells (Table 7, I Experiment #2) results in a UVS-7 induced frequency nearly 96 27 times control values. At the diphtheria toxin-resistant locus, UVs-7 mutability is as much as seven times control values, while the inducibility of 6-thioguanine resistance is only two- to three-fold higher than the control. An important consideration in evaluating hyper- mutability is the type of mutagenic event that can be detected at a given locus. UV-induced damage results pri- marily in the production of pyrimidine dimers which yield a predominance of point mutations. As both ouabain (Na+/ K+ ATPase) and diphtheria toxin (elongation factor-2) resistance require functional products for cell survival, resistance is thought to be rendered through minor base changes, such as point mutations. The observation in ham- ster cells that ouabain resistance is inducible by UV but not x-ray has been taken as confirmation that the induction of ouabain resistance is dependent on point mutagenesis. The induction of diphtheria toxin resistance in hamster cells is possible with both UV and x-ray, suggesting that small deletions may result in a functional product with resistance. 6-thioguanine resistance, however, requires the complete loss of HGPRT activity (124). A variety of mutational changes have been identified in 6-thioguanine resistant clones (124), including point mutations. Although resistance at all three loci can be induced by UV, factors including the size of the gene, availability of damage to repair enzymes, and expression 97 of mutated genes may influence the degree of hypermutability observed for UVS-7. UVS-4O was examined for UV-induced resistance to ouabain in two experiments (Table 9 and Table 10). The first experiment examined the relationship between UV dose and mutability. Hypermutability of UVS-4O was clear only at high doses. Data at a moderate dose of UV (10 joules/ m2), stimulates some interesting questions regarding the hypermutability. When UVS-40 and the parental control were permitted equivalent expression in units of time (three days), the UVs-40 and parental control mutation frequencies were 127.0 and 117.6, respectively. The difference was clearly insignificant. When mutation frequencies were examined with expression in units of equal cell divisions, for which UVS-40 requires four days to achieve the same colony size as three days growth for the parental control, the differences appeared more significant with observed frequencies of 146.2 and 117.6 for UVS-4O and the parental control, respectively. The data in the second experiment indicate that both the mutant and control mutation fre- quencies can be influenced by increasing in gitg expression. UVS-40 ouabain-resistant recovery appears to be affected to a slightly greater extent. Ouabain expression will be con- sidered in greater detail later in this dissertation. Both of the experiments illustrate the hypermutability of UVS-40 98 at higher doses, but differences at moderate doses are minimal. The experiments for UVs-4O were not designed to permit the evaluation of mutation frequencies per unit survival. From the data in Table 9 and Table 10, one cannot deduce if control and mutant cell lines share approximately equivalent mutability at equitoxic doses of UV. Such a relationship had not been demonstrated for XP variant cells (60). This obser- vation was interpreted to infer that repair in the XP variant is more error-prone than that in control cells. Such error- prone repair may be a consequence of greater dependence on normal error-prone tolerance function, alterations in enzymes which are normally error-free, or modified physiological states generating error-prone conditions for error-prone enzymes. The UV-induced mutability of UVr-23 was examined with both ouabain and 6-thioguanine resistance. Ouabain experiments employed both i3 sitg and replating expression techniques. The data for ouabain and 6-thioguanine suggest a slight hypomutability for UVr-23. Lower mutation fre- quencies were noted for UVr-23 in all three experiments reported. The frequency differences observed between con- trol and UVr-23 are not large for any single comparison, but the consistency of observed hypomutability throughout the experiments supports the concept that UVr423 is hypo- mutable. 99 Both ouabain and 6-thioguanine resistant clones derived from UVr-23 were found to be unusually sensitive to modifications in growth conditions. If these clones are at a greater selective disadvantage in the population, the apparent hypomutability observed may have been an arti- fact. This possibility is supported by a number of reports in the literature, demonstrating the dependence of the recovery of ouabain-resistant mutants on factors which influ- ence membranes and/or Na+/K+ ATPase, including caffeine (132), 12-o-tetradecanoylphorbol-13-acetate (133), P,P- diclorodiphenyl-tricloroethane (134), and adenocine 3',5'- cyclic phosphoric acid (cAMP) (135). High levels of cAMP have been associated with both slow growth and a decrease in ouabain-resistant mutant recovery (136). The slow growing UVr-23 may exhibit enhanced cAMP levels and thereby be at a selective disadvantage to express ouabain resis- tance. Therefore, although the data suggest hypomutability, such a phenomenon appears difficult to demonstrate, particu- larly when the magnitude is small. The mutability of UVS-44 was examined and UV-induced frequencies were found not to be at variance with control values. UVs-44 was found sensitive to a variety of mutagens, exhibited near normal levels of UDS, and demonstrated a reduced ability to chase low molecular weight DNA into high molecular weight DNA following UV irradiation. The molecular observations for UVs-44 suggest the mutant possesses a 100 defect in ligation or suffers from an extended delay in normal synthesis following UV exposure. In either case, the alteration appears to influence survival, but not mutagenesis following UV damage. In addition to the specific details relevant to the individual mutants, the induced mutagenesis studies have provided some important details concerning the utility of the various mutation assays employed. The importance of the type of mutagenic event (base change, deletion, etc.) has already been eluded to. In addition, the studies reported here confirm earlier speculations regarding muta- tion expression. Experiments examining 6-thioguanine resis- tance indicate that all cell lines achieve maximal expres- sion of UV-induced resistance at approximately seven days. As the cell lines grow at different rates, it can be deduced that expression is not dependent on cell division. This finding confirms earlier observations suggesting independence of 6-thioguanine expression on cell division (136). The independence of this assay on divisions is particularly attractive for comparative studies examining cell lines or experimental treatments that exhibit variations in division time. Studies reported here examining the induction of ouabain resistance, particularly those with UVS-7, indicate a dependence of expression on cell division. As Na+/K+ ATPase is a membrane-bound function, the elimination of 101 sensitive molecules could require dilution through the pro- duction of new membranes during cell division. The data for UVS-4O also support the dependence of ouabain-resistant expression on cell division. The increase in mutation frequency for the slower growing UVS-40 cells over four days ip gigg expression, in conjunction with a less dramatic increase in parental control frequencies over the same period, suggests resistance for UVS-40 does not express as quickly as control cells. The observed increase in control may be a consequence of new spontaneous mutations and colony selection factors previously described for diphtheria toxin resistance in hamster cells. In regard to the diphtheria toxin resistance assay, equivalent maximal expression occurred for both mutant and control lines at approximately nine days. As UVS-7 would have experienced considerably less cell divisions than the control cells in equivalent periods of time, expression appears not to be dependent on cell division. Spontaneous Mutation Rate Modifications of both the fluctuation analysis of Luria and Delbruck (137) and the replating technique of Newcombe (126) have been used to determine spontaneous muta- tion rates in mammalian cells. Most of these studies have been conducted to provide inferential evidence that an observed phenotypic change is a true mutation (i.e., demon- 6 9 stration of a mutation rate in the range of 10- to 10- ), 102 with a de-emphasis on significance. The use of less plates and simple estimations for calculations is common. Com- parative spontaneous mutagenesis, however, requires greater emphasis on significance and consideration of all aspects which effect the calculated rate. Three such aspects which have commonly been ignored in the past are metabolic cooperation, expression time, and selective advantage. The first two of these aspects may lead to a gross underestimation of mutation rate when the fluctuation anal- ysis is used. While metabolic cooperation can be compen- sated for with low cell density at selection, expression time is a major complication that cannot be eliminated from fluctuation type experiments. Starting populations are far too small to expect the existence of occurred, but unex- pressed mutants, which might compensate for those which are not yet expressed at the time of selection. With a modified Newcombe technique one examines the change in mutation frequencies over time, keeping track of the increase in the population. Through the generation of theoretical data, it has been determined that this relation- ship may be expressed as: azi‘fifi’. 1O ln(N2/Nl) where a is the mutation rate, F1 and F2 are mutation fre- quencies at different times, and N1 and N2 are the total 103 number of cells in the population at the time of‘Fl and F2 determination, respectively (Stephen Warren, personal com- munication). Since the technique permits a large initial population and division over long periods of time, expres- sion time is less of a problem than it is with the fluctu- ation analysis. As with the fluctuation test, appropriate compensation can avoid problems with metabolic cooperation. A major drawback of the Newcombe technique comes through the examination of a locus at which selective disadvantage exists for the phenotype being selected. DTr and OUAr mutants, for example, appear to be at a selective disadvan- tage as compared to wild type cells. Resistant cells would continuously be eliminated from the population, prohibiting an adequate estimation of mutation rate. As there is no apparent selective disadvantage to 6-thioguanine resistant cells in the population, this locus was selected to examine the mutation rate in UVs-7 and con- trol cells using a modified Newcombe technique. The tech— nique proved easy to employ and permitted the determination of several rate values for each cell line. No significant difference was detected between the mean values of 2.228 x -6 6 10 and 2.596 x 10- mutations/cell/division. The data once again confirm the UV-specific nature of UVS-7's defect. S UMMARY An immense contribution has been made to the cur- rent understanding of prokaryotic repair function through the isolation and characterization of relevant bacterial mutants. Despite the success of these efforts and similar efforts in lower eukaryotes, little attention has been focused on attempts to isolate new mutants in mammalian sys- tems. The present study reports the use of a new protocol to isolate mammalian cell mutants with alterations in DNA repair and/or UV sensitivity. The employment of this proto- col was successful in generating a variety of Chinese ham- ster fibroblast clones of potential interest. These clones exhibited phenotypes including UV sensitivity, with or with— out a reduction in the capacity to conduct unscheduled DNA synthesis, and UV resistance with enhanced unscheduled DNA synthesis. Four specific clones were selected for detailed studies, including an examination of UV-induced mutability. One clone was found to represent a new class of mutants, exhibiting reduced unscheduled DNA synthesis and UV- induced hypermutability similar to classical xeroderma pigmentosum cells, but only a slight sensitivity to UV. The spontaneous mutation rate for this clone was not found 104 105 to be significantly different from control values. The defect appears to be specific for UV—induced damage. Two additional clones have demonstrated moderate sensitivity to a variety of mutagens and only slightly reduced unscheduled DNA synthesis. One of these cell lines proved hypermutable to UV, while the other, a cell line which also exhibited poor "postreplication repair," showed no alteration in mutability. The fourth clone characterized in detail exhibited a resistance specific for UV, conducted enhanced unscheduled DNA synthesis and appeared slightly hypomutable. The characterized clones demonstrate the wide vari— ety of mutants that may be isolated through the implementa- tion of the isolation protocol. Their relevance to the study of DNA repair and mutagenesis has been clearly shown. 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