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CHARACTERIZATION OF ULTRAVIOLET LIGHT-INDUCED DIPHTHERIA TOXIN-RESISTANT MUTATIONS IN NORMAL AND XERODERMA PIGMENTOSUM HUMAN FIBROBLASTS BY Thomas Warren Glover A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Interdepartmental Program ABSTRACT CHARACTERIZATION OF ULTRAVIOLET LIGHT-INDUCED DIPHTHERIA TOXIN-RESISTANT MUTATIONS IN NORMAL AND XERODERMA PIGMENTOSUM HUMAN FIBROBLASTS BY Thomas Warren Glover Quantitative mutagenesis studies in human cells have been severely limited by the lack of reliable genetic markers. Experiments were therefore performed to develop and characterize a better quantitative mutation assay for human cells. The UV-induction of diphtheria toxin resistant (DTr) mutations in normal and excision repair defective xeroderma pigmentosum (XP) fibroblasts has been quantita- tively characterized. A concentration of diphtheria toxin to use in the selection of resistant mutants was determined whereby DTr cells are cross-resistant to Pseudomonas aeurginosa exotoxin A, indicating mutants have altered elongation factor-2 (BF-2) which is not susceptible to ADP-ribosylation by either toxin. Recovery of mutants was not influenced by the presence of wild-type cell densities of 1-8 x 105 per 9 cm plate, indicating no metabolic co- Operation, or cell density effect, exists in the selection Thomas Warren Glover of mutants. Expression periods for UV-induced DTr mutants differed with the dose of mutagen treatment and cell strain used. A relatively long (10-15 days after UV treatment) expression period was required for the maximum recovery of DTr mutants. Maximum recovery was followed by a decrease in mutation frequency on subsequent days evaluated. A linear dose-response was observed for UV-induced mutations in both normal and XP fibroblasts. Results of this study indicate that XP fibroblasts have higher UV-induced mutation frequencies per unit UV— dose but similar frequencies per unit survival compared to normal cells as measured using a new genetic marker for quantitative mutagenesis. Furthermore, these results support a prediction of the mutation theory of cancer, namely, that cells from individuals with certain human syndromes that predispose the individual to cancer will have higher induced mutation frequencies than cells from non-susceptible individuals. This newly characterized genetic marker should be useful in quantitative mutagenesis studies in human cells. To Sue, my parents, and family for love and support. To J.E.T. and C.C. for "the way". ii ACKNOWLEDGMENTS For their generous support, guidance, and friend- ship during this project and throughout my graduate training, I am deeply indebted to Dr. James E. Trosko and Dr. Chia-cheng Chang. I am extremely grateful to my major professor, James Trosko, for, among many other rewards, generating an atmosphere stimulating to thought and study of the nature of things, and for creating excellent working conditions in which to express my ideas. My profound appreciation goes to C.C. Chang who both directly and by example provided me with the special technical and mental training needed to perform this and future research. Moreover, the idea for the present study and the encouragement to pursue it were generated in discussions with him. Any rewarding results may thus be attributed to his unselfish support. I am thankful to have eXperienced and learned from the interaction between these two scientists which may be best described by the following quotation attributed to Werner Heisenberg, as seen in The Tao of Physics, "It is probably true quite generally that in the history of human thinking the most fruitful developments frequently take place iii at those points where two different lines of thought meet. These lines may have their roots in quite different parts of human culture, in different times of different cultural environments or different religious traditions: hence if they actually meet, that is if they are at least so much related to each other that a real interaction can take place, then one may hope that new and interesting developments may follow." I also thank the other members of my graduate committee, Dr. James Higgins and Dr. Jay Goodman, for their helpful comments and training. Also, I wish to express my gratitude to the faculty of the Genetics Program who have given of their time and efforts in my training. It is not possible to mention all those colleagues and friends who have helped in this and other projects, and whose friendship has made my graduate years enjoyable. However, I owe special thanks to Roger Schultz, Larry Yotti, Stephen Warren, Betty Dawson, Judy Funston, Carol Hunt, Marc Castellazi, and, last but not least, Phil Liu for his most appreciated aid at the intense end. In addition, to all the people who helped in the typing and editing of this dissertation - "mahalo". iv TABLE OF CONTENTS Page LIST OF TABLES 00.0.0000...0.......OOOOOOOOOOOOOOOOOO Vii LIST OF FIGURE I O O O O O O O O O O O O O O O O O O O O ..... O O O O O O O O O O O O Viii LIST OF ABBREVIATIONS ............................... ix INTRODUCTION ........................................ 1 LITERATURE REVIEW 0 O O O O O O O O O O C O O O O O O O O C C I O O 0000000000 4 Mutagenesis in Cultured Mammalian Cells ........ 4 Deve10pment of Selective Systems .......... 4 Human cells C I O O O I O O O ...... O O O O O ........... 8 In Xeroderma Pigmentosum: Relationship to Cancer 0 O O O O C O O O O O O O O O O O O O O O O O ........ 13 Diphtheria TOXin O O O O O O O O O O O O O O O O O O 0000000000000 15 Isolation of Diphtheria Toxin Resistant Variants ..... ................. 23 MATERIALS AND METHODS ............................... 29 Cell Strains ................................... 29 Culture Medium ................................. 29 Culture Vessels and Incubation Conditions ...... 30 Cell and Colony Counts ......................... 31 TOXinS ......OOOOOOOOCOOOOOO OOOOOOOOOOOOOOO .0... 3]- Plating Efficiencies and Cell Survival Determinations ........... ......... .. 32 Establishment of Selective Conditions .......... 32 Cytotoxicity of Diphtheria Toxin .......... 32 Cytotoxicity of Pseudomonas Exotoxin A .... 33 Cross Resistance of DTr Colonies to Pseudomonas Exotoxin A ........ .......... 33 Effect of Cell Density on Recovery of DTr Mutants .......................... 35 Expression of UV-Induced DTr Mutants ...... 36 Induction of DTr Mutations in XP Fibroblasts ... 38 Page Cytotoxicity of Diphtheria Toxin ........ 4O Cytotoxicity of Pseudomonas Exotoxin A .. 40 Cross Resistance of Isolated DTr Colonies to ET-A ...................... 42 Effect of Cell Density on Recovery of UV-Induced DTr Mutants ................ 44 Expression of UV-Induced DTr Mutants .... 45 Dose Response of UV-Induced DTr Mutants ...... 48 DTr Mutagenesis in XP Fibroblasts ............ 53 Expression of UV-Induced DTr Mutants .... 53 Dose Response of UV- Induced DTr Mutants . 58 DTr Mutation Frequencies as a Function of Cytotoxicity of UV—Light ................... 58 DISCUSSION 0............O.......O.........OOOOOOOOO 62 Characterization of the DTr Mutation Assay: Conditions for Selection of DTr Mutants .... 62 Effect of Cell Density .................. 66 Expression of UV-Induced Mutants ........ 67 Mutagenesis Dose-Response .................. 71 Comparison of Dose-Response in Normal and XP Fibroblasts .................... 71 The Genetic Nature of DTr Mutants .......... 72 SUMMARY .................... .......... ............. 74 LIST OF REFERENCES ...... ....... ................... 76 Vi LIST OF TABLES Induced mutations in human cells EVitro 00............OOOOOOOOOOOO ...... O. Cytotoxicity of Pseudomonas Exotoxin A .... Cross resistance of isolated DTr COlonieS to ET-A . . . . . . . . . . . . . . . . . . . ..... . . Effect of cell density on recovery Of DTrmutantS ......OOOOOOOOOOOOO......... Expression of UV-induced DTr mutants ...... Expression of UV—induced DTr mutants in normal and XP fibroblasts vii Page 42 43 46 50 56 Figure l. 10. ll. 12. LIST OF FIGURES Mechanism of diphtheria toxin action in intact cells ..........OOOOOOOIOOOOOOIOOO Protocol for quantitative selection of UV-light induced DTr mutations in human fibroblasts .................. ..... .. Cytotoxicity of diphtheria toxin in human fibroblasts ................. ........ Effect of cell density on recovery of DTr mutants ...O......OOOOOOOOOOOOIOOOOO... Survival of UV-irradiated normal human fibroblasts used in expression time experiment 00............OOOOOOOOOOOOOOCOOO Expression of UV-induced DTr mutants ...... Expression of UV-induced DTr mutants ...... Dose-response curve of UV-induced DTrmutantS ...00............OOCOOOOOOOOOOO Survival of UV-irradiated normal and XP fibroblasts used in determining expression time of DTr mutants ............ Expression of UV-induced DTr mutations in normal and XP fibroblasts .............. Dose-response curves of UV-induced DTr mutants in normal and XP fibroblasts ...... DTr mutation frequencies as a function of the cytotoxicity of UV—light ........... viii Page 21 37 41 47 49 51 52 54 55 57 59 61 EF-2 EMS ET-A GTP HGPRT Lf MNNG mRNA N-Aco—AAF NAD+ PBS LIST OF ABBREVIATIONS adenosine diphosphate bromodeoxyuridine 8-azaguanine 8-azaguanine resistant deoxyribonucleic acid diphtheria toxin diphtheria toxin resistant elongation factor-2 ethylmethane sulfonate Pseudomonas aeurginosa exotoxin A guanosine triphosphate hypoxanthine-guanine phOSphoribosyltransferase joule floculating unit minimum lethal dose N-methyl N'-nitro-N-nitrosoguanidine messenger RNA N-acetoxy-2—acetylaminofluorene nicotinamide-adenine—dinucleotide phosphate buffered saline ribonucleic acid 6-thioguanine ix 6-thioguanine resistant transfer RNA ultraviolet light xeroderma pigmentosum INTRODUCTION In View of the findings that most carcinogens and other environmental agents are mutagenic as tested in appropriate prokaryotic mutation test systems, the need for a reliable system for the quantitative detection of somatic mutations in human cells has been increasingly emphasized. It has long been realized that such a system, in addition to serving potentially as a method for monitoring genetic risk to man, would provide fundamental knowledge on the mechanisms of mutagenesis and on the genetics of heredity variation in human somatic cells. Quantitative mutagenesis studies in human cells have, however, been severely limited by the availability of reliable genetic markers. Of the relatively few such studies of this kind, most have employed the use of the HGPRT mutation assay. Because of problems inherent in this mutation system such as a cell density effect, effect of drug concentration, influence of serum components, and the possibility of an epigenetic change resulting in the altered phenotype, the purpose of the first phase of this study was to develop and characterize a better mutation assay system for human cells. Based on previous findings that mutants with altered elongation factor-2 (EF-2) could be singularly l isolated in Chinese hamster ovary (CHO-K1) cells by selection with high concentrations of diphtheria toxin (55), a series of studies was performed to characterize conditions for the selection and induction of similar mutants in normal human diploid fibroblasts in yitgg. In the second phase of this project, the newly characterized DTr mutation assay was applied in a compar- ative mutagenesis study with normal and excision repair defective xeroderma pigmentosum (XP) fibroblasts. Since the original observation that cells from classical type xeroderma pigmentosum were deficient in the ability to repair UV-induced DNA damage (71-73) when compared to fibroblasts from normal individuals, it was hypothesized that the clinical symptoms of this cancer— prone syndrome might be a result of their hypermutability due to a reduced ability to perform error-free repair of DNA damage. Maher gt El- using resistance to 8-azaguanine (8-AG) as a genetic marker, demonstrated that fibroblasts of several XP strains, when compared to normal cells, had increased cytotoxicity and mutation frequencies induced by UV-light or a number of chemical carcinogens (47). The question remained as to whether the higher induced mutation frequencies at the HGPRT locus would hold for other genetic loci. The purpose of these compar- ative mutagenesis studies was to test this hypothesis using diphtheria toxin resistance (DTr) as a genetic 2 marker. Additionally, it was felt that this study would provide valuable information on the sensitivity of the DTr mutation assay in human cells. LITERATURE REVIEW Mutagenesis in Cultured Mammalian Cells Development of Selective Systems The term "mutation" was coined by Hugh de Vries in 1901 for sudden hereditary changes seen in Oenothera lamarckinana (1). It was not until 20 years later that H.J. Muller, while working towards proving the chromosome theory of inheritance, stressed the link between mutations and the nature of the gene (2). As techniques to measure mutations were develOped, the first studies to successfully measure induced muta- tions were performed by H.J. Muller in DrOSOphilla (3) and L.J. Stadler in maize (4), both using x-rays. These and other early studies Opened many questions concerning the nature of the genetic material and the changes that occur during the mutation process (5,6). Micro-organisms were introduced in the late 1940's as subjects for quantitative mutagenesis in studies of drug resistance (7). The methodologies developed for use with micro-organisms inspired the development of systems for studying mutagenesis in mammalian cell culture. Because of the long generation time for mammals and the difficulties of whole animal experimentation, it was hOped that studies in mammalian cell culture would progress as rapidly as did genetic studies with micro-organisms (8). Once the observation was made that a single mammalian cell could routinely be grown to yield a colony of cells (9), efforts were directed to finding a genetic marker suitable for use in detecting mutations. The largest number of genetic markers successfully adapted to cell culture were initiated from animals having known hereditary variation (8). The genetics of the markers was thus clear; however, attempts to develop selective systems based on the hereditary variation in these cell lines were largely unsuccessful. The development of systems for the selection of drug—resistant variants from normal cell populations proved more successful. The selection of cultured mouse or human cells resistant to purine analogs or aminopterin were reported by Lieberman and Ove (10), Szybalski and Smith (11), and Littlefield (12); and of mouse cells resistant to bromodeoxyuridine (BUdR) by Kit gt 31. (13). Initial attempts to induce drug-resistant mutations in mammalian cells in zitrg with a variety of agents known to be mutagenic in micro-organisms were largely unsuccess- ful. Szybalski (14) reported that with the possible exception of x-rays, mutagens tested actually depressed the frequency of recovery of purine analog resistant mutants, and revertants, in human D98 fibroblasts. The mutagens used included ultraviolet irradiation, N-acetoxy- acetylaminofluorine (N-aco-AAF), nitrogen mustard, and other chemical mutagens. A number of possible explana— tions were proposed for these early results including the existence of a difference in the mechanisms of mutagenesis between micro-organisms and mammalian cells (14), and unforeseen technical problems associated with the mutation assays used (8). Due to these results, the genetic basis of the purine analog resistant marker remained unclear and was questioned by some investigators (8,15). The effects of mutagenic agents in mammalian cells were thus limited to studies of chromosomal changes in cultured mammalian cells (16,17), and dominant lethals and varigated-type position effects in whole animals (18,19). The first successful demonstrations of chemical induction of mutations in cultured mammalian cells were reported independently by three laboratories in 1968. Kao and Puck (20) employed a qualitative method to isolate nutritional mutants from Chinese hamster cells. Chu and Malling (21) and Shipiro 33 31. (22), also using Chinese hamster cells, selected for mutants resistant to purine analogs. Chu and Malling additionally measured mutations from L—glutamine auxotrophy to prototrophy. Chu and Malling importantly demonstrated that for selection of 8-azaguanine resistant (8-AGr) mutants, factors such as cell density, expression time, and concentration of selective agent were found to have a profound influence on the mutation frequency. Since these initial experiments were performed, extensive research has continued toward the development of a reliable in vitro mutation assay for mammalian cells. A large number of studies have shown that the frequency of mutations at a number of loci can be increased with various chemical or physical agents. Most of these studies have employed the use of permanent cell lines established from Chinese hamster ovary (CHO) (20,23-27), Chinese hamster lung (V79) (28-32), or mouse lymphoma (L5178Y) (33-35). Genetic markers used have included mutations to auxotrophy, or reversion (20,27,30,32), drug resistance (23-26,28,29,31,33-35), temperature sensitivity (32), and alterations in cellular products (36). By far, most investigations have used resistance to the purine analogs 6-TG or 8-AG, discussed in the following section. Recent reviews have dealt with the subject of selective systems characterized for use with, mainly, permanent rodent cell lines (37,38). Human Cells It is generally agreed by a number of investi- gators that for use in assessing the mutagenic risks of environmental agents for humans in 1129 and to accurately provide information about the basic mechanisms of muta- genesis in human cells, an in yitrg_mutation assay using human diploid fibroblasts is preferred over similar assays using transformed rodent cells (39—42). However, the development of such an assay has been hindered by a lack of suitable genetic markers and by technical problems including a finite life span of cells, relatively low plating efficiencies, and others described below. From the time of the unsuccessful attempts to induce mutations in human fibroblasts by Szybalski (14), little apparent progress was reported in quantitative mutagenesis in human cells until Demars and Held (43) characterized spontaneous 8-AGr mutants in diploid fibroblasts and Albertini and Demars (44) first reported induction of mutations in these cells using x-rays to induce 8-AGr mutations. Sato gt al. (45) concurrently reported induction of 6-TGr mutants with EMS in an established human lymphoblastoid cell line. In these and in subsequent studies a number of mutagens have been used to quantitatively induce HGPRT‘ mutants in human cells in vitro, and a number of attempts have been made to .coflmmsomflc u0w uxou moma macawum> mm 0222 .m2m In: mummHnounflm Hmum>om owumuonmouuomam nodumNfikus mew» Imho + mcwcoflnumfi mm 0222 .mzm In: mummHnounfiu mmwc0wcumum>o no mcownumumao COHuMNwku: He mamuix tin mummHnounfim in: mmouosuu wmwcmmouv>nmc mcflcwmum HMO“ hm mzm --- mommanounau mumcamozamummoosam -smnooumhs ammo AcocoHummsv no czocxcs manna cauocmuv "muosuo omHimuH mluouomw :onu canonuzmfic mm.mm.ov ozzz .mzm .>D «ucmcHEocioo mummHnounflm cowumocon ou mocmumwmmu aauflcmecue vm mzm ucmcHEocuoo mummHnounflw HH wmmumfixaom «zm ou wocmumflmwu camnmso mm.mv 022: .>D .mzm acmcHEocnoo mummHQOHQHw mmmm9D .mxmuux w>wmmmowu mumMHnounfiw Emmom oocmumwmwu >ufi>fimmwomm Um>ao>cfi hanmESmmum omxuoconm com: \mocmcHEoo cofiuocsm no msooH unawum> moocwumwmm mcmmmusz cmEdmwum wmxs HHoU Eoummm cofiuwuzz .ouuw> mm mHHmo ages: :a mc0wumude coonocH .H «Hams 10 utilize other genetic markers for induced mutation studies (Table 1). As is evident from Table l, the HGPRT’ mutation assay has, as with other cell types, been by far utilized the most with human cells. This choice is based mainly on the sex-linked nature of the HGPRT locus resulting in a high expression of the recessive mutant phenotype (43) and the fact that a biochemical basis for resistance has been comprehensively characterized (60,61). Extensive use of this mutation system, however, has promoted the publica- tion of a number of papers dealing with technical problems (38,51,62,63) or questioning the genetic origin of the resistant phenotype (64-66). The technical problems include accounting for the variables of cell density, expression time, and concentration of selective agent as demonstrated by Chu and Malling in 1968 (21). The influence of cell density on the recovery of mutants is a result of molecular exchange of nucleotides between resistant and sensitive cells. It is assumed that cell- to-cell contact is required (67). Thus, to insure optimal recovery of mutants, a limited number of cells can be seeded per given area of culture vessel in mutagenesis experiments. For human fibroblasts, this number has been fixed by various investigators to be equal to or less than 4 x 10” cells per 60 mm culture dish (39,46,47). This factor may seriously limit the maximum size of mutation 11 experiments using the HGPRT system. The occurrence of an expression period, or pheno- typic lag, before maximum recovery of induced HGPRT mutants is required for loss, by dilution or degradation, of most and perhaps all pre-existing HGPRT molecules and corres- ponding mRNA (51). Although this phenomenon is seen in all cells studied, the pattern of expression time curves may differ for different cell types. It is believed that for Chinese hamster V79 (30,63) and CH0 (23,24) cells, once maximum expression of mutants is attained, recovery is constant for many cell generations thereafter. In a limited number of studies with human fibroblasts, however, reports are conflicting regarding the pattern of expression time curves, with some reports of a rapid decline in muta- tion frequency following maximum recovery (39,46), and others of constant recovery (41). Cells were replated at relatively low cell numbers prior to selection in each of the investigations cited, thus eliminating cell density effects as a major factor in explaining this discrepancy. The practical implications of these and other findings have led to the prOposal by Jacobs and DeMars that empiri- cal determination of optimal expression time is necessary for every cell strain and mutagen used (39). Other technical variables which may influence results with the HGPRT selective system are the concen- tration of drug (43,44,63,65), the existence of an unknown 12 serum factor which degrades purine analogs, especially 8-AG (69), and competition of serum purines with purine analogs (69). There is much evidence to support the genetic origin of a1 least some HGPRT" variants, both from enzyme characterization and comparison to fibroblast strains lacking HGPRT activity derived from patients with Lesch Nyhan syndrome (43,44,60,61). However, the influence of epigenetic change resulting in the HGPRT" phenotype and thus affecting results from mutation experiments has been argued (37,64-66). These arguments have been based on, among other findings, the instability and high reversion rates of a number of isolated mutants, failure to show expected decreases in mutation rates with increasing ploidy levels, and the adaptive growth of some HGPRT+ cells in increasing concentrations of 8-AG. Of the other genetic markers utilized for induced mutation studies in human cells (Table 1) mutations to ouabain resistance and a-amanitin resistance, while presumably co-dominant, are recovered at very low frequen- cies. Mutation frequencies in EMS treated fibroblasts have been reported to be 5 x 10'8 for a-amanitin resistance (54) and 20 x 10‘6 for ouabain resistance (42). Use of these markers in mutation studies therefore necessitates large numbers of cells for accurate quantitative studies. This factor has prohibited further characterization of 13 these mutation systems in diploid human cell strains due to the relatively low plating efficiencies, long generation time, and finite life span of such cells. The mutation system utilizing resistance to diphtheria toxin is reviewed in detail in the following sections of this manuscript. The other mutation systems listed in Table 1 lack from incomplete characterization or a questionable genetic basis of the variant phenotype (38). Their usefulness for genetic studies, therefore, remains uncertain. In Xeroderma Pigmentosum: Relationship to Cancer Xeroderma pigmentosum (XP) is a rare autosomal recessive genetic disease predisposing affected individuals to sunlight-induced skin damage, pigmentation changes, and multiple skin carcinomas on areas exposed to the sun, and in some forms of the disease, to developmental and neuro- logical disorders (69,70). Cleaver (71), Setlow gt ii- (72) and Cleaver and Trosko (73) found that cultured cells from many classical forms of XP were deficient in the UV- endonuclease function necessary for the excision of UV- induced pyrimidine dimers in DNA. A number of subsequent studies have further characterized the disease on the molecular levels and cellular levels. The disease is genetically heterogenous with at least six distinct forms known: five excision repair l4 deficient forms which have been classified into complemen- tation groups A-E by cell hybridization studies (74) and a variant form with a normal rate of excision repair synthe- sis but abnormal post replication repair necessary for converting low molecular weight DNA, newly synthesized after DNA damage, into high molecular weight DNA (75). Cells within each complementation group A-E have similar rates of UV-induced unscheduled DNA synthesis (excision repair) and each group has a characteristic rate (74). All XP cells are more sensitive than normal cells to cell killing by UV-irradiation or a number of chemical mutagens as measured by colony forming ability in zitrg (47,76,77). Andrews 33 31. (77) have shown that the clinical manifestion of neurological abnormalities, seen in complementation groups A and D, is correlated with the greatest sensitivity to UV-induced cell killing. The effect of the DNA repair deficiencies seen in XP cells on induced mutation frequencies in 11259 has been investigated in a series of experiments by Maher 2E.§l- (47) using resistance to 8-azaguanine as a genetic marker. Fibroblast strains from complementation groups A, C, and XP variant were shown to have, compared to normal cells, increased frequencies of mutations induced by UV-irradia- tion (in groups A, C, and XP variant), N-aco-AAF, and the "K-region" epoxides of several carcinogenic polycyclic hydrocarbons (in groups C and XP variant). However, when 15 the frequency of mutations induced by UV-irradiation in excision repair defective group A and C cells was compared to normal cells as a function of cytotoxicity, all cell strains were found to be equal. XP variant cells had a somewhat higher induced mutation frequency per unit UV— dose. These studies with XP cells have been important in the study of DNA repair functions in human cells and their relationship to the mechanisms of mutagenesis (47,70,78,79) and as recently emphasized by Trosko gt gt. (79) have made what is, "Probably the most significant contribution to the mutation theory of cancer . . .". Originally postu- lated by Boveri in 1914 (80) and recently thoroughly reviewed (81), the mutation theory of cancer has as one of its predictions that humans having certain syndromes that predispose the individual to cancer will have higher mutation frequencies than non-susceptible individuals. The mutation studies of Maher gt gt. (47) and those reported in a preliminary report of this work (56) using cells from cancer prone individuals with the XP syndrome support this hypothesis. Diphtheria Toxin Because of the role of diphtheria toxin in the pathogenesis of disease, and the fact that the molecular 16 biology of diphtheria toxin has provided a model for the mode of action of other bacterial and plant toxins, a number of reviews have appeared. Collier (82) and Pappenheimer (83,84) have thoroughly reviewed the litera- ture through 1976 on the structure, mode of action and control of expression of diphtheria toxin. References not included herein on general features of diphtheria toxin appear in these recent reviews. Diphtheria toxin is synthesized by strains of Cornebacterium diphtheriae lysogenic for cornephage B carrying the tox+ structural gene. The expression of the tox+ structural gene is regulated by the bacterial host (84,85). Diphtheria toxin is highly toxic for many species, including man, guinea pigs, rabbits, and many fowl; however, rats and mice are notably resistant. About 50- 100 ng/kg is lethal in sensitive species but doses 3 orders of magnitude greater per unit body weight are required for lethality in rats and mice (82). The sensitivities of cultured cells from various animals, including humans, correspond approximately to those of the parent animal (84). Cultures from various organs within a given animal are similar in sensitivity indicating no apparent tissue specificity (84). In 1959, Strauss and Hendee (86), using cultured HeLa cells, first reported that diphtheria toxin inhibited 17 protein synthesis. These results have been confirmed in other systems using intact cells and cell free systems (87—107). NAD+ was found to be an essential co-factor for protein synthesis inhibition (87). Collier (88) and Goor and Pappenheimer (89) determined, in 1967, that diphtheria toxin specifically inactivated elongation factor-2 (BF-2; aminoacyl trans- ferase II) in cell free extracts from mammalian cells. Elongation factor-2 participates in the translocation of peptidyl tRNA from the acceptor site to the donor site on ribosomes, and movement of mRNA by one nucleotide triplet (condon) after each peptide bond is formed. Guanosine triphosphate (GTP) is hydrolyzed in the reaction. The exact mechanism of diphtheria toxin catalyzed inactivation of EF—Z was elucidated by Honjo and co-workers (90) and Gill gt gt. (91). The reaction was determined to occur by toxin catalyzed attachment of the adenosine diphosphate ribose (ADPR) portion of NAD+ to EF-2, as follows: EF-2 + NAD+ \.-_—“_ ADPR-EF-Z (inactive) + nicotinamide + H+ At physiological conditions of pH and concentrations of NAD+ and EF-Z, inactivation of EF-2 is virtually complete at equilibrium (90). Evidence exists that the interaction between toxin and NAD+ involves non-covalent forces, and a ternary intermediate consisting of toxin, NAD+, and EF-2 18 has been proposed (92). The ADPR moiety is covalently bound to EF-Z (90,92). The reaction is very specific for eukaryotic EF-2. No protein acceptor other than EF-2 has been found in mammalian tissue extracts (82,84,90). All eukaryotic EF-2 studied, including that from rats and mice, but not prokaryotic or mitochondrial EF-G, serves as substrate (82,84,110). There exists evidence that EF-Z contains only a single attachment site for ADPR; however, the nature of this site remains uncertain despite isolation and amino acid sequencing of different 11+C-ADPR binding tryptic peptides of EF-2 by three laboratories (93-95). The exact nature of the inhibition of the trans- location reaction by ADP ribosylated-EF-2 remains uncertain. ADPR-EF—Z is not inhibited from forming a complex with GTP, the first step in the translocation reaction (95,96). The ADPR-EF-2:GTP complex binds to ribosomes but may have decreased rates of association and dissociation (95,96). Interaction with other RNA species (mRNA, tRNA) appears to be blocked by the ADPR group (97). Diphtheria toxin is secreted from diphtheria bacilli as a single polypeptide chain with a molecular weight estimated at 62,000 to 63,000 daltons and containing two disulfide bridges (82-84). While toxic to animals and cells cultured from them, this form has been 19 shown to be enzymatically inactive in cell free assay systems. Collier (98,99) and Gill (100,101) and their co-workers determined that the ADP-ribosylation reaction is catalyzed by a proteolytic fragment of intact toxin. The toxin molecule is thus a proenzyme. Gill and Dinus (100) showed that intact toxin polypeptides are normally partially digested by proteolytic enzymes in bacterial culture medium before isolation. A species called "nicked toxin" containing two fragments - A (molecular weight 24,000), and B (molecular weight 38,000) - linked by a single disulfide bond, was found to co-purify with intact toxin. All the enzymatic activity of diphtheria toxin when tested in cell free systems 32 ttttg was found in Fragment A. Gill and Pappenheimer (101) and Collier and Kandal (99) showed that both a peptide bond and a disulfide bond normally connecting Fragment A to the rest of the molecule must be broken to activate Fragment A and to produce enzymatic activity £2,21EEQ- Intact toxin and Fragment B had no enzymatic activity in the cell free system used. It was shown that Fragments A and B could be separated by mild diges- tion with trypsin and reduction with thiols tg_ztttg. In intact cells from sensitive species, however, both fragments were found to be required for intoxication by low doses of toxin (102,103). Isolated Fragment A was found to be less than 0.01% as toxic for sensitive human 20 cells in culture (102) or in guinea pigs (82). No appreciable difference was found between "nicked" and intact toxin preparations in toxicity towards guinea pigs or HeLa cells (55,104-107). Moehring and Moehring (102) have shown that in naturally "resistant" cells such as mouse L929 or 3T3 cells and permeability class toxin resistant human cells (to be reviewed), from 105 to 106 times as much whole or partially nicked toxin was required as for sensitive KB—S or HeLa cells to reduce their rate of protein synthesis by 50% after 24 hours of exposure. However, protein synthesis was inhibited to the same extent in all cells studied by purified Fragment A. On a molar basis, the toxicity of Fragment A was equivalent to that of whole toxin on resistant cell lines. These, and a large number of other studies, have provided evidence for a model of diphtheria toxin action in whole cells first proposed by Gill and Pappenheimer (101). In a two step mechanism, "nicked" or intact toxin molecules are bound by the B fragment to specific cell receptors. Once bound, Fragment A is released into the cytoplasm where it catalyzes ADP—ribosylation of elonga— tion factor-2 (Figure 1). Data obtained from a number of sources support the hypothesis that specific receptors exist for diphtheria toxin on the plasma membrane of sensitive cells and that 21 Toxin McileculesN ~ Toxin / R ecipfors nic~ tina ide ‘DPR-EF-Z l NA u"! inactive) Figure l. Mechanism of diphtheria toxin action in intact cells (108). 22 Fragment B interacts with the receptors (82-84, 103,109). Such receptors are absent from naturally "resistant" cells (109,110). The exact number of receptors per sensitive cell has not been clearly determined but estimates of 50 (83) and 4,000 (103) have been proposed. The receptors interacting with diphtheria toxin are different from those interacting with other toxins (84) including Pseudomonas exotoxin (55,111-114) (to be reviewed). Cregan gt gt. (108), using mouse-human hybrids, have mapped what may be the receptor gene to chromosome #5 in human cells. Sensitivity acted as a dominant in these hybrids. After toxin binds to the cell membrane by its B fragment, it is not clear how Fragment A reaches the cytoplasm in active form in sensitive cells. Various models exist (82,101,103), all of which assume nicking of intact toxin molecules by cellular proteases, reduction of the disulfide bond connecting the A and B fragments, and release of Fragment A into the cytoplasm by active transport via pinocytotic or phagocytotic vessicles or passively via the hydrophobic B fragment acting as a carrier through the membrane. It is quite certain that the B fragment remains behind on the cell membrane (103, 109). The mechanism by which enzymatically active diphtheria toxin or its Fragment A reaches the cytoplasm in naturally resistant rat or mouse cells or in 23 "permeability" class resistant variants (to be reviewed) from sensitive cell lines is not clear but is believed to occur via normal endocytosis (82-84,103,110). Moehring and Moehring (110) showed that the sensitivity of mouse L cells to diphtheria toxin could be increased when poly-L- ornithine, which stimulates macromolecular uptake, was added to the cultured cells and that an increased sensitiv- ity to toxin was correlated with an increase in the number of pinocytotic vessicles in the cells. Similar results were obtained with permeability variants of sensitive cell lines using DEAE-dextran as a stimulator of endocytosis (55). Elongation factor-2 of these "resistant" cells is just as sensitive to inactivation by diphtheria toxin in cell free systems as sensitive cells, and the endocytosis mechanism has been proposed to explain the fact that resistance can be overcome in these cell lines by using high toxin concentrations (55,106,110). Gill and Pappenheimer (83) have prOposed that only a few molecules of Fragment A and perhaps only a single molecule need reach the cytoplasm in order to kill a eukaryotic cell. Boquet and Pappenheimer (103) have shown measuring 1L‘C-insulin uptake that HeLa cells in suSpension culture take up 1.2% of their cell volume per hour at 30°C, by endocytosis. Isolation of Diphtheria Toxin-Resistant Variants Moehring and Moehring in one of a series of 24 studies of the response of cultured mammalian cells to diphtheria toxin, isolated populations of human epidermoid carcinoma (KB) cells resistant to approximately 2,000 times more diphtheria toxin than the parental cells (104). Resistant cells were selected by sequential growth in increasing concentrations of toxin. Resistant cells were no more resistant than parental cells when toxin catalyzed inhibition of protein synthesis was measured in cell free extracts, and thus mimicked mouse L cells. Resistant cells were cross resistant to various RNA viruses (105). It was postulated that these resistant cell populations lacked specific membrane associated functions necessary for binding of diphtheria toxin or the activation or transport of toxin and RNA viruses. In subsequent studies (106), additional toxin resistant cell pOpulations were isolated and characterized. Resistant human KB and Chinese hamster ovary (CHO-K1) cells were compared to naturally resistant mouse L and rat All cells and to sensitive parental cells in terms of percent inhibition of protein synthesis by diphtheria toxin in whole cells and cell free extracts. All resistant cell lines were at least 105 times more resistant than sensitive cells and all were approximately equal in resistance. Resistance could be overcome by using high concentrations of toxin and, when done, resulted in survi- val curves with identical $10pes as sensitive cells. The 25 sensitivities of cell free extracts of two resistant KB cell lines studied was identical to sensitive cells and L cells studied previously (104,107,110). No difference was found in the toxicity of resistant KB and L cells to two different preparations of toxin containing different prOportions of nicked and intact toxin, indicating that inability to inactivate toxin, perhaps through preteolysis, was not the basis for resistance. It was concluded that these resistant variants, as with others previously studied, shared with naturally resistant cells the inability to bind toxin or transport it through the cell membrane, and were designated "permeability variants" (55). In subsequent studies, Moehring and Moehring (55) isolated a second class of diphtheria toxin resistant variants, designated "translational variants", from Chinese hamster ovary (CHO—K1) cells. These variants differed from previously isolated permeability variants in three ways: First, they were much more resistant to diphtheria toxin than permeability variants. Protein synthesis in whole cells was not inhibited, to a significant extent, by 24 hour exposure to toxin concentrations that almost com- pletely inhibited protein synthesis in permeability variants. Second, cell free extracts from translational variants were fully resistant to inhibition of protein synthesis by toxin. Furthermore, EF-2 extracted from translational variants was not susceptible to 26 ADP-ribosylation by diphtheria toxin as measured by 1L’C-NAD incorporated into 1"‘C-ADPR-EF-Z. Under the conditions of the assay, there was a linear dose-response following the addition of increasing amounts of EF-2 prepared from sensitive wild type CHO cells and perme- ability variants. Third, as opposed to wild type or permeability variants, translational variants were resistant to protein synthesis inhibition by Pseudomonas aeurginosa exotoxin A as measured in whole cells, and ADP-ribosylation of EF—2 by Pseudomonas exotoxin. Pseudomonas aeurginosa exotoxin A had been previously shown by Iglewski and Kabat to have the same enzymatic activity as diphtheria toxin (111). Both toxins catalyze ADP-ribosylation of EF-2 in eukaryotic cells. Results from competition experiments using the two toxins indicate that the same tryptic peptide and perhaps the same amino acid of EF-Z is ADP-ribosylated in an identical fashion (111,112). The two bacterial toxins, however, have different cellular and species sensitivities and different modes of entry into sensitive cells (55,111-114). Moehring and Moehring (55) were able to increase the frequency of occurrence of translational variants in CHO-Kl cell pOpulations by treatment with ethyl methane sulfonate (EMS). The authors speculated on the potential use of the diphtheria toxin resistant genetic marker in studies of mutagenesis in somatic cells. 27 Gupta and Siminovitch (115) have recently reported the isolation and characterization of what they believe to be a second class of translational mutants in CHO cells. As opposed to the translational mutants described by Moehring and Moehring (55), in which extracted EF-2 is fully resistant to toxin catalyzed ADP—ribosylation, in these mutants about 50% of EF-Z is still susceptible to ADP-ribosylation and about 50% is resistant. It was suggested that this represented the products of one mutated allele and one wild-type allele in functionally diploid CHO cells, whereas, the CHO-K1 cells used by Moehring and Moehring may be functionally haploid at this locus. The mutants described by Gupta and Siminovitch were stable and able to grow in high concentrations of diphtheria toxin indicating that resistance acted as a dominant in these cells. However, in resistant x sensitive hybrids in CHO cells, resistance acted as a recessive as measured by cell survival. It was hypothe- sized that this apparently conflicting behavior is due to a gene dosage effect. In addition, it was determined that the diphtheria toxin resistant marker is not linked to the emitine resistant (Emtr) locus or to the X- chromosome. Attempts to characterize the diphtheria toxin resistant mutation system for use as a mutation assay in human cells have recently been reported by Gupta and 28 Siminovitch (40) and in a preliminary report of this work (56). Gupta and Siminovitch selected for resistance to high concentrations of diphtheria toxin in human diploid fibroblasts. Results of reported experiments included: a spontaneous mutation frequency of 1-10 x 10'6; an increase in mutation frequency by 100-3000 times by treatment of cells with the mutagens ethyl methane sulfonate (EMS), nitrosoguanidine (MNNG), or ICR-l70; an optimal expression time for recovery of induced mutants of 5 days; no effect of cell density on the recovery of mutants at cell numbers of 1 x 10” - l x 106 per 100 mm culture dish; a mutation rate determined by Luria- Delbruck fluctuation analysis of 5-6.3 x 10'7 mutants per cell per generation; resistance of cell free extracts from one resistant cell pOpulation to inhibition of protein synthesis by diphtheria toxin; sensitivity of isolated mutant populations to the RNA virus, vesicular stomatitus virus, indicating general permeability was not affected. As indicated in a preliminary report of this work, Glover gt gt. (56) used a similar approach to select for diphtheria toxin resistant mutants in human diploid fibroblasts. Significantly different results were obtained for some induced mutation experiments. MATERIALS AND METHODS Cell Strains Diploid fibroblasts from human skin were used throughout all experiments. Cell strain 73-6 was derived from the foreskin of a normal human male and provided by Dr. David J. Segal, University of Alberta, Edmonton, Alberta, Canada. Cell strain XP7BE was derived from skin biOpsy specimens of a female patient with classical xeroderma pigmentosum including neurological abnormalities, and was obtained from the American Type Culture Collection (Rockville, Maryland). This XP strain is identified by the international nomenclature for XP strains (116), and belongs to complementation group D (117). Cells were suspended in 10% dimethylsulfoxide in PBS, sealed in glass ampules, and frozen in liquid nitrogen until needed for experimentation. Culture Medium Cells were grown throughout in modified Eagle's Minimum Essential Medium (MEM) (118) with Earle's salts (GIBCO, Grand Island, New York), supplemented with 50% increase of all essential amino acids except glutamine, 100% increase of all non-essential amino acids, 50% 29 30 increase of all the vitamins, and 1 mM sodium pyruvate. The concentration of bicarbonate was decreased to 1.5 g/l. Medium was sterilized by passage with positive pressure through Nucleopore filters (Nucleopore Corporation, Pleasanton, California), and was stored in the dark at 4°C. Before use, medium was supplemented with 10% fetal calf serum (GIBCO, Grand Island, New York, or Flow Laboratories, Inc., Rockville, Maryland) which was stored at -20°C, thawed, and heat inactivated at 56°C for 25 minutes prior to use. During experiments and when used for stock cultures a few days immediately before experi- ments began, medium was also supplemented with 100 units/ ml penicillin G and 100 ug/ml streptomycin. Culture Vessels and Incubation Conditions Stock cell cultures were grown for experiments in static culture attached to the surface of sterile glass bottles or 75 cm2 plastic flasks (Corning Glass Works, New York). Cells were subcultures 1:2 or 1:4 using 0.01% crystalline trypsin in PBS without calcium and magnesium ions. During experiments cells were grown in 9 cm plastic culture dishes (Falcon Plastics, Oxnard, California, or Corning, Corning, New York) or in 75 cm2 plastic flasks when appropriate. Cells were incubated at 37°C in humid air supplied with 5% C02. Under these conditions, cells 31 not contact inhibited have a mean generation time of about 24 hours. Cell and Colony Counts Trypsinized individual cells in suspension were counted using a hemacytometer. Macrosc0pic colonies were scored visually, when appropriate, or by rinsing with 0.85% saline, fixing with 95% ethanol, and staining with 2.5% Giemsa stain. Toxins Diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A were gifts from Dr. Stephen Li (National Institute of Environmental Health Sciences). Diphtheria toxin was a product of Connaught Medical Research Labora- tories (Toronto, Canada), lot D343, 2,000 floculating units (Lf) per ml, 19,000 guinea pig minimum lethal dose (MLD) per mg protein, and about 3 ug protein per Lf. Highly purified Pseudomonas aeruginosa exotoxin was originally a gift of Dr. Stephen Leppla of the U.S. Army Medical Research Institute of Infectious Diseases and has 5,000 MLD per mg protein. Diphtheria toxin was sealed in glass ampules in concentrated (2,000 Lf/ml) form and was stored frozen in liquid nitrogen until needed. Pseudomonas exotoxin A was stored frozen at -20°C until needed. 32 Plating Efficiencies and Cell Survival Determinations Plating efficiencies, "replating efficiencies", and survival determinations were made by plating cells in 10 ml medium in 9 cm plastic plates at cell densities expected to result in about 50 colonies per plate. Usually, six plates were used for each determination resulting in about 300 colonies scored for each data point. For mutation experiments, UV-survival plates were irradiated at the same time and under the same conditions as mutation plates and replating efficiency plates were treated the same way as mutation plates except no toxin was added. After 18-21 days, when macroscopic colonies were visible, plates were scored as described above. Establishment of Selective Conditions Cytotoxicity of Diphtheria Toxin Cells of strain 73-6 NF were plated for attachment at various cell densities into 9 cm plastic plates 6.5 hours before diphtheria toxin was added. With 10 ml of fresh medium added to the plates, toxin was added with a micropipette to give a final concentration of 0-1.0 Lf/ml. Four to twelve plates were used for each determination. After 48 hours incubation, the medium containing the toxin was removed, plates were rinsed with 10 ml PBS, and 10 ml 33 fresh medium was added to each plate. Cells were incubated for 21 days with one medium change 7 days after plating. Colonies were stained and scored as described. Relative survival was calculated as the ratio of the percent survival at different toxin concentrations to the percent survival in the absence of the toxin. Cytotoxicity of Pseudomonas Exotoxin A A qualitative determination was made of the toxicity of Pseudomonas exotoxin A to 73-6 NF fibroblasts. Cells were trypsinized and diluted to a density of about 1 x 106 cells/m1. One drOp of the cell suspension was innoculated in each of 12, 16 mm wells of a 24 well plastic culture dish (Costar, Cambridge, Massachusetts) containing 2 ml medium per well. Cells were allowed to attach over- night at which time the medium was changed and toxin was added with a micropipette to duplicate wells in concentra- tions from 0-0.l ug/ml. After 48 hours incubation, the medium containing the toxin was removed, cells were rinsed with 2 ml PBS per well, and 2 ml fresh medium was added to each well. Wells were scored visually at various times up to 3 weeks after plating for cell growth. Cross Resistance of DTr Colonies to Pseudomonas Exotoxin A To determine the concentration of diphtheria toxin to use for selection of only EF-2 mutants, DTr variants 34 selected at various concentrations of diphtheria toxin were isolated and tested for cross resistance to Pseudomonas exotoxin A. Both UV-irradiated and non- irradiated populations of 73-6 NF cells were used in the original selection procedure. For UV-irradiation, 2 x 105 cells were plated in each of 9, 9 cm plastic plates 6.5 hours before attached cells were exposed to 200 ergs/mm2 (20 J/mz) ultraviolet light irradiation from a germicidal lamp (General Elecyric G25T8-25W) which was positioned to deliver a dose rate of 20 ergs/mmZ/sec (2 J/mZ/sec). The UV dose was chosen to result in an estimated 90% cell killing. Cells were incubated for 3.5 days, subcultured to glass bottles, and incubated for an additional 3 days before replating for initial selection. Non-irradiated cells were treated in the same way except for the absence of UV-irradiation and fewer cells (1 x 105/9 plates) were initially plated. For replating, cells were trypsinized and plated at a density of 2 x 105 cells/plate 6.5 hours before medium was changed and diphtheria toxin was added at final concentrations of 0.001 Lf/ml, 0.01 Lf/ml, or 0.1 Lf/ml in 10 ml medium. Cells were incubated in the presence of the toxin for 24, 48, or 96 hours at which time toxin was removed with rinsing and fresh medium added as previously described. Six plates were used for each determination. Cells were incubated for 18-21 days until macroscopic colonies appeared. Colonies were scored 35 visually with or without staining depending on the frequency of recovery of DTr variants. Replating efficiency was determined from the average percentage of survivors in quadruplicate plates each with 200 cells plated. Once scoring was concluded, representative colonies from each toxin treatment were isolated with a glass cylinder, trypsinized, and inoculated into each of 4 wells of a 24 well plastic plate (Costar). Untreated cells from stock culture were similarly inoculated into duplicate 4 well columns. After overnight attachment, medium was removed and 2 m1 fresh medium was added per well. Two of the 4 wells for each treatment were left untreated, cells in one well were treated with 0.1 ug/ml Pseudomonas exotoxin for 48 hours, as described above, and cells in the final well were similarly treated with 0.1 Lf/ml diphtheria toxin for 48 hours. Wells were scored for cell killing on various days thereafter. Effect of Cell Density on Recovery of DTr Mutants To determine the effect of cell density on recovery of DTr mutants, UV-irradiated 73-6 NF cells were used to increase the frequency of resistant colonies. Cells were trypsinized and plated at a density of 8 x 105 cells per 9 cm plate 6.5 hours before UV-irradiation. With the medium removed from the plates, the attached cells were exposed to 113 ergs/mm2 ultraviolet light 36 irradiation from a short wave UV lamp (Mineralight R, UVSll, Ultra-Violet Products, Inc., San Gabriel, California) positioned to deliver a dose rate of 2.5 ergs/ mmz/sec (0.25 J/mZ/sec). Medium (10 ml) was added to each plate and cells were incubated for 15 days to allow expression of mutations. Cells were then trypsinized and replated at various cell densities from 1-8 x 105. After 6.5 hours for cell attachment, medium was changed and 0.1 Lf/ml diphtheria toxin was added to each plate. Cells were exposed to toxin for 48 hours at which time toxin was removed as previously described. Cells were incubated for about 21 days, stained and scored. Replating efficiency was determined from the average percentage of survivors in 6 plates. Expression of UV-Induced DTr Mutants The general protocol followed in this experiment is shown in Figure 2. Cells of strain 73-6 NF were trypsinized, counted and plated 6.5 hours before irradia- tion with UV-light from a germicidal lamp (G25T8-25W) as previously described. Enough cells were plated for each UV dose to give at least an estimated 3 x 106 survivors based on a 100% plating efficiency. Following UV treatment, cells for mutation analysis were incubated for two days at which time cells from each UV treatment were subcultured into 32 oz glass bottles at cell densities such that cells 37 .mummanounfim does: CH mcoflumuzfi HBO chDUCH uanHI>D mo cofluomamm m>Humufiucmsw How Hooououm .m ousmflm EsHpoE 30: com AHE\mq H.0v mmumam mumam 80 m mom mowcoaoo mmm cues mmcfln cofluomaom mHHoo mOH x v whoom .cflxou o>oEmm on saxou cod amHHoo mumam _ _ _ _ _ L S _ mama Hausa muses mg m mason m.m "Hooououm mafiumamom mHmmHmcm mammammMDSE cam GOHDMDDE Hm>fl>usm now How mpmammm >DH mason woman 1 . 1 mate ma .0H .s .m .66 muson m.w Tllll.mo0Huom coflmmmudxm mDOHHm> 38 had room for division. At 4, 7, 10, 14 and 21 days following UV treatment cells from each UV dose were pooled and the apprOpriate numbers of cells removed for replating for selection of mutants. The remaining cells were again delivered to bottles or 75 cm2 plastic flaks and incubated until the next replating at which time the procedure was repeated. Cells removed for replating were plated at a density of 4 x 105 cells per plate 6.5 hours before medium was changed and 0.1 Lf/ml diphtheria toxin was added to each plate. The total number of cells plated for the various UV treatments are shown in the tables accompanying the results. Toxin was removed after 48 hours as previ- ously described. Cells were incubated for 18-21 days with one change of medium 7 days after removal of toxin. Plates were stained and scored as described except for a few plates which were scored visually and from which representative DTr colonies were isolated and tested for cross resistance to Pseudomonas exotoxin A as previously described. UV survival and replating efficiencies were determined as previously described. Induction of DTr Mutations in XP Fibroblasts A comparison was made of spontaneous and UV- induced DTr mutations in XP7BE and 73-6 NF fibroblasts. The same procedure was followed as was used for the 39 expression time experiment previously described (see Figure 2) except that a short wave UV lamp (Mineralight R UVSll, Ultraviolet Products, Inc., San Gabriel, CA) was used, positioned to deliver a dose rate of 2.5 ergs/ mmZ/sec (0.25 J/mZ/sec) for irradiation of 73-6 NF cells, raised to deliver a dose rate of 1.25 ergs/mmz/sec (0.125 J/mZ/sec) for XP7BE cells, and the expression times allowed before replating were 4, 7, 10, and 15 days after UV treatment. The total numbers of cells plated for the various UV treatments are shown in the tables accompanying the results. Due to slower growth, some XP-DTr colonies were allowed up to 28 days incubation before scoring. RESULTS Establishment of Selective Conditions Cytotoxicity of Diphtheria Toxin The effects of different concentrations of diphtheria toxin on the colony forming ability of normal human fibroblasts was tested. As shown in Figure 3, diphtheria toxin is very toxic to human cells. The relative survival was progressively reduced by increasing concentrations of toxin from 0.00001 Lf/ml to 0.001 Lf/ml when applied for 48 hours. Toxin concentrations of 0.01 Lf/ml to l Lf/ml gave similar survivals of about 9 x 10—6. This frequency of survival is comparable to the spontaneous mutation frequencies seen in mutagenesis experiments (data to be presented) and indicates that these surviving colonies represent spontaneously occurring DTr variants in the cell population. The range 0.001-0.1 Lf/ml was chosen for further study. Cytotoxicity of Pseudomonas Exotoxin A To determine a toxic concentration of Pseudomonas exotoxin A to use in testing cross-resistance of DTr variants, a qualitative assessment was made. As shown in Table 2, 0.01 ug/ml is very toxic to human cells. Since EG-Z mutants should be resistant to a high range of toxic 40 41 Relative Survival l 1 1 J 0 -0000| .OOOI .OOI .Ol .I Diphtheria toxin (Lf/ml) Figure 3. Cytotoxicity of diphtheria toxin in human fibroblasts. 42 concentrations of Pseudomonas exotoxin, 0.1 ug/ml, which killed all cells tested in these experiments, was chosen for testing the cross-resistance of DTr variants. Table 2. Cytotoxicity of Pseudomonas Exotoxin A Toxin Conc. Cells Surviving Treatment (ug/ml) Well 1 Well 2 0 + + 0.00001 + + 0.0001 + + 0.001 +a +a 0.01 -b - 0.1 - - aMany cells killed; Growth of survivors slowed Two macrosc0pic colonies seen after 3 weeks Cross Resistance of Isolated DTr Colonies to ET-A In order to establish selective conditions that would enable only the growth of EF-2 mutants, colonies selected at different treatments with diphtheria toxin were tested for cross-resistance to a highly cytotoxic concentration of Pseudomonas exotoxin. As shown in Table 3, a high frequency of DTr variants is seen when lower concentrations of diphtheria toxin are used for the shortest times tested. Initial selection with 0.001 Lf/ml 43 ocaummumu How UOuMHOmw mmwcoaoo o: mucmmmnmmu AIVQ mcfixou such on acmumflmmu mum mmflcoHoo ucmumfimmu ammo >uo>m ch I I ~.m mm H.o I I I N.m mv H.o I I I N.m vm H.o I I I N.m mm Ho.o I I I m.m mv Ho.o I I I m.mm vm Ho.o I I QI m.m mm Hoo.o I o\H w\a Hmv mv Hoo.o I m\o m\o oama vm Hoo.o I {8 m3 3. mm To + o\o o\w N.nm mv H.o + o} ..{o 4.2 E To + m\o o\m ~.vv mm Ho.o + m\m o\m vm mv Ho.o + o\~ o\~ mma vm Ho.o + m\m m\m mm mm Hoo.o + o\a o\a mqm we Hoo.o + GE GE om: em :56 + .2 3. :75 35%.: 203333 HE\@: H.o HE\wA H.o muo>w>u5m Goa Hod Amunv cofluomamm How mamc m.m no» ucmpmflmmu moflcoHoo nee musmomxo saxou Hmchfluo new .~E\o omv mmflcoHoo woumHOmH mo umnEnz mo >ocmsqoum mo cede .ocoo ea >3 .mIem ou mmflcoHoo yea UquHOmw mo wocmumflmou mmouo .m manna 44 for 24 or 48 hours or with 0.01 Lf/ml for 24 hours results in recovery of 139-1130 colonies per 106 survivors in the UV treated population and as high as 1510 colonies per 106 survivors in the population not UV treated. However, under these selective conditions only 5/30 (16.7%) of all isolated colonies tested for cross-resistance to 0.1 ug/ml Pseudomonas exotoxin, 48 hours, exhibited cross-resistance. In addition, those same colonies which were killed by Pseudomonas exotoxin were also killed by more stringent selection with diphtheria toxin (0.1 Lf/ml for 48 hours). Using diphtheria toxin concentrations of 0.01 Lf/ml for 96 hours or 0.1 Lf/ml for 24, 48, or 96 hours for initial selection results in a lower frequency of recovery of DTr variants (27.2-44.2 per 106 survivors) but all isolated colonies tested (24/24) were cross-resistant to 0.1 ug/ml Pseudomonas exotoxin and resistant to 0.1 Lf/ml diphtheria toxin. Other diphtheria toxin concentrations used ini- tially resulted in intermediate values. For further experimentation, 0.1 Lf/ml diphtheria toxin applied for 48 hours was chosen for selection of DTr-EF-Z mutants. Effect of Cell Density on Recoverytof UV-Induced DTr Mutants With 113 ergs/mm2 UV-irradiation, resulting in about 33% survival, and a 15 day expression time, the frequencies of recovery of DTr mutants remain the same 45 with cell densities of l x 105 - 8 x 105 per 9 cm plate (Table 4 and Figure 4). In addition, there were no noticeable differences in cell morphology or colony size with the different cell densities. These results indicate that under these selective conditions, there is no cross- feeding effect (metabolic cooperation) resulting in loss of mutant recovery at higher cell densities as is seen with the 6-TGr/8-AGr mutation assay. Also, by using 0.1 Lf/ml diphtheria toxin for 48 hours for selection of mutants, at cell densities up to 8 x 105/p1ate, there is no relaxing of selective conditions due to dilution of the effective toxin concentration per cell thus potentially resulting in a higher recovery at higher cell densities. Because of these observations and the practicality of scoring the expected frequency of UV-induced DTr mutants at high UV doses, all mutagenesis experiments were done with 4 x 105 cells per 9 cm plate. Expression of UV—Induced DTr Mutants Five mutagen doses, 0-20 J/m2 UV-irradiation, were used in this experiment to detect any possible differences in the expression of DTr mutants in relation to severity of mutagen treatment. The cell survival following the UV-irradiation is shown in Figure 5. The recovery of DTr mutants as a function of expression time and UV dose is shown in Table 5 and Figures 6 and 7. The cell density of 46 Table 4. Effect of cell density on recovery of DTr mutants No. of No. of Plating No. of DTr Mutation cells per cells efficiency colonies frequency per plate (9 cm) plated (%) recovered 106 survivors (x106) 1 x 105 2.4 28.67 60 87.20 2 x 105 3.6 28.67 88 85.26 4 x 105 5.2 28.67 124 83.17 6 x 105 5.4 28.67 136 87.85 8 x 105 6.4 28.67 152 82.84 47 O. .352: HE Mo gm so .5350 :8 mo Dowmmm .v 0.33m m f0. 3 AEomV 22a .8 £3 .0 .3522 h 0 v n N o A d 1 d d d d O N O Q' 0 ‘0 0| Iad Kauanbaid 0000th 110 O In 9 O 9 snnwns 48 4 x 105 cells per 9 cm plate and selection with 0.1 Lf/ml diphtheria toxin for 48 hours were shown to give optimum recovery of DTr-ET-A mutants in previous experiments. The rate of expression of mutants was greater for lower doses of mutagen than for higher doses. However, for all UV doses tested, a maximum recovery of UV-induced mutations was seen at 10 days after UV treatment. Mutation frequen- cies at all UV doses steadily declined after 10 days to a point no higher than the spontaneous frequency at 23 days after UV. Eight colonies isolated from plates from 5, 10, and 14 days expression all showed cross-resistance to 0.1 ug/ml Pseudomonas exotoxin and continued resistance to 0.1 Lf/ml diphtheria toxin (data not shown). These results indicate that a phenotypic lag exists for the expression of UV-induced mutants at different doses of UV—irradiation express at different rates, and mutants are lost rapidly after the maximum expression time, possibly due to a selective disadvantage in the cell population. Dose-Response of UV-Induced DTr Mutants The dose-response curve from the experiment pre- viously described with UV doses from 0-20 J/m2 and a 10 day expression time is presented in Figure 8. The results showed that the DTr mutation frequencies increased linearly with increasing doses of UV—light plotted on a linear 49 IOO O Survival (°/o) L l l l a 5 I0 IS 20 UV dose ( J/mz) Figure 5. Survival of UV-irradiated normal human fibroblasts used in expression time experiment. Table 5. Expression of UV-induced DTr mutants. UV Expression No. of cells Replating Mutation frequency (J/mz) time platedd efficiency per 106 survivors (days) (X106) (8) (No. of mutants) 0 5 4.8 26.58a 11.75 (15) 5 5 4.8 19.83a 32.62 (31) 10 5 6.0 19.33 41.45 (47) 15 5 7.2 13.88C 21.98 (22) 20 5 7.2 11.17C 7.44 (6) 0 7 4.8 33.08a 10.08 (16) 5 7 4.4 30.57a 42.98 (58) 10 7 5.6 26.75a 122.80 (184) 15 7 6.0 24.28 63.84 (93) 20 7 6.8 18.06 23.61 (29) 0 10 4.8 31.00a 6.72 (10) 5 10 4.8 30.58a 55.18 (81) 10 10 4.8 30.50a 127.05 (186) 15 10 6.0 31.40a 219.21 (413) 20 10 5.2 32.25a 255.81 (429) 0 14 4.8 23.33a 0.89 (1) 5 14 4.8 25.42a 9.83 (12) 10 14 4.8 26.50a 27.52 (35) 15 14 6.0 21.25a 61.96 (79) 20 14 6.0 20.42a 216.30 (265) 0 23 4.8 25.33a 2.69 (3) 5 23 4.8 27.17a 8.43 (11) 10 23 4.8 26.00a 16.03 (20) 15 23 6.0 29.33a 18.75 (33) 20 23 6.0 32.75a 6.00 (11) a-CNumber of cells per 9 cm plate used: a b c d4 x 10“ 1200/6 1800/6 2400/6 cells per 9 cm plate 51 .mucmuse HBO pooschI>D mo coflmmmnmxm v. $33 oEF 3.9.2wa .mexa. 33>: .m musmflm ON. 0! Om . o .N ONN ocN 8N ooN con SJOAQMHS 90) )ad 4000an Ingram." jig DTr Mutation Frequency per IO6 Survivors 52 200 I5 J/rnz I00- 1 l l 0 5 7 I0 I4 23 300 20° I0 .I/ m2 Expression Time (days) Figure 7. Expression of UV-induced DTr mutants. 53 scale. These results indicate that UV-irradiation is an effective mutagen for inducing DTr mutations in human fibroblasts and, conversely, that DTr mutations in human fibroblasts are readily inducible by a known mutagen. DTr Mutagenesis in XP Fibroblasts Expression of UV-Induced Mutants An experiment was performed to compare the UV- light induced DTr mutation frequencies in 73-6 NF cells with XP7BE cells under the same experimental conditions. Multiple UV doses and expression times were used. Since XP cells are much more sensitive to UV-irradiation than normal cells, different UV doses were used for the two cell strains. Cell survival following UV-irradiation is shown in Figure 9. The recovery of DTr mutants as a function of expression time and UV dose is shown in Table 6 and Figure 10. The results for 73-6 NF cells are essen- tially the same as those obtained in an earlier experiment using a different UV source. XP7BE cells exhibited a longer period of time to express DTr mutations. The maximum frequency for the expression times chosen was not seen until 15 days after UV-irradiation. At this expres- sion time the frequency in 73-6 NF cells is declining from the maximum frequency obtained at 10 days expression. 54 300- . o g; 250)— C) .2 g o m 200- 00 S2 L- 8. I50- )5 8 Q) :3 3 LL“ I00!- C: .9 ..E 3 I 2 50" ,1 I n. ’l 5 $2” I 0 5 I0 I5 20 Figure 8. uv dose ( J/mz) Dose-response curve of UV-induced DTr mutations. *Solid line determined by linear regression analysis. 55 Survival (%) (I I 1 l l l 0 I0 25 50 75 I60 I25 I50 UVdosehrqs/mmz) Figure 9. Survival of UV-irradiated normal and XP fibroblasts used in determining expression time of DTr mutants. 556 Table 6. Expression of UV-induced DTr mutants in normal and XP fibroblasts. Cell UV Expression No. of cells Replating Mutation frequency Strain (ergs/mmz) Time platede efficiency per 10 survivors (days) (X106) (8) (No. of mutants) 73.6 NF 0 4 4.8 29.08a 18.63 (26) 75 4 6.0 19.06b 28.86 (33) 113 4 7.2 19.79C 71.58 (102) 150 4 6.8 18.83d 10.15 (13) o 7 4.8 20.50a 20.33 (20) 75 7 4.8 21.42a 103.10 (106) 113 7 5.6 21.06b 106.84 (126) 150 7 7.2 18.56b 73.34 (98) o 10 4.4 33.17a 16.44 (24) 75 10 4.8 34.83a 120.23 (201) 113 10 6.0 33.92a 201.45 (410) 150 10 5.6 30.58a 273.29 (468) XP7BE 0 4 4.8 21.75a 9.58 (10) 2.5 4 4.8 16.92: 9.85 (8) 7.5 4 6.0 18.94 18.48 (21) 12.5 4 7.2 11.75C 11.82 (10) 17.5 4 7.2 8.36 23.26 (14) 0 7 4.8 23.75a 15.79 (18) 2.5 7 4.0 24.67a 48.64 (48) 7.5 7 4.8 21.33a 15.63 (16) 12.5 7 5.2 15.50 7.44 (6) 17.5 7 7.2 9.61b 28.91 (20) 0 10 4.4 21.25a 1.07 (1) 2.5 10 3.6 23.42a 56.93 (48) 7.5 10 3.6 21.17a 135.15 (103) 12.5 10 2.4 20.68a 135.00 (67) 17.5 10 5.6 15.75a 79.37 (70) 0 15 4.8 20.83a 17.00 (17) 2.5 15 4.8 21.25a 58.82 (60) 7.5 15 5.2 18.58a 146.87 (142) 12.5 15 4.9 17.17a 182.44 (156) 17.5 15 3.6 24.58a 180.82 (160) a-d Number of cells per 9 cm plate used: a = 1200/6 b = 1800/6 c = 2400/6 d = 3600/6 e 4 x 10“ cells per 9 cm plate f 3.11 x 10“ cells per 9 cm plate 57 300 I I r UVdose , (eras/mm?!) 2 0 73-6NF I50 '1 (Normal) 200 - ISO - - 75 g I00 '1 \ -I Z . . (’3) 5. . 0 on ‘09 «— 1 f 1. O 4 7 IO l5 8 6‘ 5 a". E: 3" I 1* r7 T C .93 2 ' xp‘rae - .2 g 200 E I 0 IOO 50 O 4 7 l0 l5 Expression Time (days) Figure 10. Expression of UV-induced DTr mutations in normal and XP fibroblasts. 58 Dose-Response of UV-Induced DTr Mutants The dose response curves from the experiment described above with UV doses from 0-ll.3 J/m2 for 73-6 NF cells and O-l.75 J/m2 for XP7BE cells are shown in Figure 11. These data points are taken from the expres- sion times resulting in the maximum recovery of mutants for the two cell strains (10 days for 73-6 NF, 15 days for XP7BE). These results indicate that the linear dose- response curve for UV-induced DTr mutations in 73-6 NF cells is consistent with previous results, and that a similar dose-response curve is seen with XP7BE cells at much lower UV doses. Therefore, XP7BE group D xeroderma pigmentosum fibroblasts appear more sensitive to the mutagenic effects of UV-irradiation determined by the frequencies of recovery of DTr mutations. DTr Mutation Frequencies As A Function of Cytotoxicity of UV—Light Mutation frequencies and survival data from experiments shown in Figures 5, 8, 9 and 11 were used to show the relationship between UV-survival and UV-induced DTr mutations for XP (XP7BE) and normal (73-6 NF) fibroblasts (Figure 12). The frequencies of induced mutations in the two cell strains are similar at equal 59 300 I I I l I T7 l .2. 250- - O .2 > 5 U) 0 200 - O "' r— O XP7BE 73'6 NF 3') / (normal) O. 5‘ _ 3 3 u: I; c I’ 2 I00 - C3 I '5 ,’ 2 l 1’ s. b ’I’ p. C) 50 ”I, - ’l ’1 I l I L l l I O 5 25 5O .75 IOO I25 I50 UV dose (ergs/ mmz) Figure 11. Dose-response curved UV-induced DTr mutants in normal and XP fibroblasts. by linear regression analysis. *Solid lines determined 60 cytotoxic doses of UV-irradiation. These results indicate that there is no apparent difference between the two cell strains in the frequency of UV-induced DTr mutations when UV-light is considered in terms of cytotoxicity. DTr Mutation Frequency per IO6 Survivors Figure 12. 61 300 I I I I I 73-6NF (expt. 2) 73-6NF (expt. l) 200*- - 0 XP 78E 0 I005 4 C I l l l l 1 I00 75 50 25 IO 5 Survival (%) DTr mutation frequencies as a function of the cytotoxicity of UV-light. DISCUSSION Characterization of the DTr Mutation Assay Conditions for Selection of DTr Mutants In order to maximize selection for a single geno- typic class of DTr mutants which have altered EF-Z, it was necessary to establish selection conditions that would eliminate the phenotypically DTr permeability class vari- ants described by Moehring and Moehring (55,104-107). These variants, like naturally resistant mouse and rat cells, lack specific cell receptors for fragment B of diphtheria toxin or a functional DT—specific transport mechanism, but their EF-2 is as susceptible to toxin catalyzed ADP-ribosylation as sensitive cells. As shown by Moehring and Moehring (55), resistance in CHO-Kl cell permeability variants, but not in translational mutants with altered EF-2, can be overcome by using high concen- trations of diphtheria toxin which is internalized, likely, via normal endocytosis. In addition, permeability variants but not EF-2 mutants are killed by Pseudomonas exotoxin A which ADP-ribosylates EF-2 in an identical fashion as diphtheria toxin in sensitive cells, but has a different mode of entry into cells. These results provided the basis for the approach used in these studies to establish conditions for selection of DTr mutants with altered EF—2 62 ML 63 in human cells. Results in Table 3 show that DTr colonies originally selected at 0.01 Lf/ml DT for 96 hours toxin exposure or at 0.1 Lf/ml DT for 24, 48, 96 hours exposure, when isolated were all cross—resistant to a highly toxic concentration of Pseudomonas exotoxin. These results strongly suggest that selection with diphtheria toxin, under these condi— tions, results in survival only of DTr cells with EF-2 resistant to diphtheria toxin and Pseudomonas toxin cata- lyzed ADP-ribosylation. These DTr cells are thus similar to the CHO-Kl cell translational mutants described by Moehring and Moehring. Less stringent initial selection conditions resulted in a higher frequency of recovery of DTr variants but not all colonies were cross resistant to Pseudomonas exotoxin, or to a higher concentration of DT (0.1 Lf/ml) which, when used for initial selection, did result in survival of only cross-resistant cells, as described above. These DTr variants may represent permeability class variants or, merely, non-specific killing by DT. It is noteworthy that in the course of determining selection conditions, diphtheria toxin was removed from the growth medium after 24, 48, or 96 hours. The reason for this choice was that it was noticed in similar prelimi- nary experiments with Chinese hamster V79 cells (data not shown), that if DT is added to the cells throughout the 64 entire growth period of DTr mutant colonies, the growth rate of some cells is reduced, representing a practical disadvantage. These empirical observations appear to be supported by the recent description of EF—Z mutants in CHO cells by Gupta and Siminovitch (115) in which the growth rate of mutant cells was also significantly reduced in the presence of DT resulting from the fact that only 50% of the EF-2 seen in these mutants is resistant to ADP- ribosylation. We, therefore, attempted to establish selection conditions whereby sensitive cells would be quickly killed and DT could then be removed from the growth medium of resistant cells. Based on the results discussed above, selection with 0.1 Lf/ml DT at 48 hours exposure was chosen to use in subsequent experiments for selection of DTr mutants with presumably altered EF-2. The possibility exists that some DTr mutants selected at 0.1 Lf/ml DT, and which are thus cross- resistant to Pseudomonas exotoxin, are not EF-2 mutants but are deficient in transport mechanisms necessary for internalization of both toxins. However, this appears to be unlikely based on the following reasoning. Evidence exists that the two toxins enter cells through different mechanisms. Although they are enzymatically identical in the reaction catalyzed, the two toxins have different Species and cellular specificities and DTr permeability 65 variants of CHO-K1 cells are as sensitive to protein synthesis inhibition by Pseudomonas exotoxin as are normal, wild-type cells. Therefore, if it is then assumed that a DTr, ET-Ar phenotype occurring from lack of toxin inter- nalization mechanisms can result from just two or more independent mutational events, the probability of recover- ing such mutants would be the product of the two mutation frequencies, and thus, insignificant. The assumption of only two independent mutational events may actually be an underestimate since in naturally resistant mouse cells which lack specific membrane receptors for diphtheria toxin, resistance has been shown to act as a recessive trait in mouse and human hybrids (108). The possibility also exists that in at least some DTr variants, the resistant phenotype is a result of the cells inability to activate intact toxin molecules by proteolysis into Fragments A and B. However, this possi- bility appears unlikely in view of the fact that cells from a number of isolated DTr colonies have been shown to be cross-resistant to Pseudomonas exotoxin-A which does not appear to require proteolytic activation (111). Experiments to test this possibility could be designed based on the resistance of isolated DTr colonies to high concentrations of different toxin preparations containing varying proportions of "nicked" and intact toxin molecules. It is important to note that in the course of these 66 experiments it was observed that conditions for storage of diphtheria toxin are important in the ability to consis- tently reproduce results. Toxin stores at 4°C or at -20°C and repeatedly frozen and thawed for use was found to decrease in cytotoxicity and therefore effectiveness in selection of mutants. This problem was overcome by storing the toxin as described in Materials and Methods. Effect of Cell Density As shown in Table 4 and Figure 4 there was no difference in the recovery of DTr mutants at cell densities of l-8 x 105 per 9 cm plate. These results support those recently reported by Gupta and Siminovitch (40) also using human fibroblasts and those observed by Chang gt gt. (Dr. C.C. Chang, personal communication) in V79 cells with selection conditions similar to those used in these experi- ments. The results indicate that there is no cross- feeding effect as is seen in selection of 8—AGr or 6-TGr mutants resulting in loss of mutants and inaccurate quanti- tation of mutation frequencies at high cell densities and thus severely limits the size of mutation experiments using fibroblasts. It remains to be shown if the frequen- cies of recovery of DTr colonies would increase due to a potential dilution effect of diphtheria toxin, thus resulting in relaxed selection for mutants, if cells were plated at cell densities greater than 8 x 105 per 9 cm 67 plate. For subsequent experiments, a cell density of 4 x 105 cells per 9 cm plate was chosen as optimal in terms of minimizing the number of plates used and the efficiency of scoring an estimated number of induced mutants per plate. Expression of UV-Induced Mutants As shown in Tables 5 and 6 and Figures 6, 7, and 10, the expression of UV-induced DTr mutants differed with the severity of mutagen treatment and cell line used. In both experiments with normal fibroblasts, a maximum fre- quency of induced mutations was seen with selection begin- ning at 10 days after UV treatment followed by a rapid decline in mutation frequency with selection beginning on later days. The occurrence of a lag period before the pheno- typic expression of mutations in mammalian cells has been extensively studied in other selective systems (24,31,35, 39,51) and is believed to depend on a number of factors, including: mutation fixation, segregation lag, degrada- tion of non-mutant forms of cellular macromolecules of the synthesis of mutant forms, and the doubling time of mutant cells compared to the doubling time of non-mutant cells. It is reasonable to suggest that before the DTr mutants selected in these experiments can be phenotypically 68 expressed, a sufficient amount of the mutant form of EF-2 must be synthesized to support survival of cells in the presence of diphtheria toxin. In addition, pre-existing sensitive EF—2 molecules may have to be degraded to the extent that they do not significantly compete with the mutant form of EF-2 for ribosomal attachment sites in view of evidence that ADP-ribosylated EF-2 is still capable of binding to ribosomes and may have a decreased rate of dissociation. As recently discussed in detail by Jacobs and DeMars (39) any decrease in the fitness of mutant survi- vors in induced mutation studies, resulting in a longer doubling time as compared to non-mutant cells, may have a significant effect on the frequency of mutants observed at different expression times after mutagen treatment, especially when a replating protocol is followed, as it was in these experiments. The following example, similar to that given by Jacobs and DeMars, emphasizes this point. If it is assumed that (a) all DTr mutants in a population of cells are phenotypically expressed by day 10 following mutagen treatment, (b) the DTr mutant frequency is 10‘” on day 10, (c) that the average generation time of non-mutant cells is 24 hours, and (d) the average generation time of DTr mutants is 30 hours, then on day 15 the DTr mutant fre- quency would be 5 x 10's, and so on. The observed results 69 for the experiment shown in Figure 6 roughly fit this pattern. A difference in cell doubling time of 24 versus 30 hours would be difficult to notice under normal cell culture conditions. However, as mentioned earlier, a decrease in cell generation time is seen in at least some DTr, EF-Z mutants in Chinese hamster V79 cells in the presence of diphtheria toxin. It is reasonable to suggest that a reduced fitness of DTr mutants would also be observed with growth in the absence of toxin if only about 50% of the cells EF-2 is resistant to toxin inhibition, as with a co-dominant mutation, or if 100% of the cells EF-2 is resistant but the kinetics of protein synthesis is thus also affected. Events such as those discussed above could explain the observed results of the expression time experiments reported here. Further experiments would be necessary to test the accuracy of this hypothesis. A somewhat longer expression period was required for the maximum observed frequency of DTr mutants in XP7BE fibroblasts. The interaction of the cellular processes, described above, involved after UV treatment may differ between XP7BE and 73-6 NF cells, and therefore result in the observed difference in maximum expression times for recovery of mutants. The maximum recovery of DTr mutants was observed at 15 days following UV treatment, the 70 longest expression period chosen, in XP7BE cells. However, due to the large size of such experiments, only a limited number of expression time points could be evaluated and therefore further experiments would be required to deter— mine whether the maximum observed frequency is the precise maximum frequency attainable. Ideally, every day following UV treatment would be tested, but this is not practical. The argument that perhaps a maximum attainable frequency was not observed could explain why the maximum observed mutation frequency at 17.5 ergs/mm2 UV-irradiation in XP7BE cells was no higher than that for 12.5 ergs/mm2 UV-irradiation. It is possible that at 15 days expression not all mutants are phenotypically expressed at 17.5 ergs/ mm2 UV, or the mutation frequency may be declining as occurs after the maximum expression period in normal cells. It should be pointed out that the results reported for these experiments do not agree with the recently reported results of Gupta and Siminovitch (40) for the expression of induced DTr mutants in normal human fibro- blasts. Gupta and Siminovitch reported a maximum recovery of mutants at 5-8 days following treatment with EMS, MNNG, or ICR-l70 at doses resulting in about 18-36% survival. At 5-8 days expression in the experiments reported here, the observed mutation frequencies in normal fibroblasts were below the maximum attained at 10 days expression for all UV doses tested. It is unlikely that the difference 71 in mutagens used could account for this difference in observed expression times of induced mutants. There are, however, three factors which may influence any comparison of these two sets of data: (1) the frequency of dipr (DTr) mutants reported by Gupta and Siminovitch are from 3-10 times higher, depending on the mutagen used, than those obtained in the present experiments at similar levels of cell survival; (2) the spontaneous mutation frequencies in non-mutagen treated cells were not given by Gupta and Siminovitch to allow accurate determination of the mutagen induced frequency by subtraction; (3) 8 days was the longest expression time tested by Gupta and Siminovitch. Mutagenesis Dose-Response The results of this study indicate that UV-irradi- ation is an effective mutagen for the induction of DTr mutations in human fibroblasts and, conversely, that DTr mutations are induced by a known mutagen. The dose- response of UV-induced DTr mutations is linear and thus follows a single-hit mechanism also observed for UV- induced 8-AGr or 6—TGr mutations in Chinese hamster (24,31) and human fibroblasts (47). Comparison of Dose—Response in Normal and XP Fibroblasts The results shown in Figures 11 and 12 agree with those previously reported by Maher gt gt. (47) in group A 72 and Group C XP cell strains. These results indicate that XP7BE group D fibroblasts have higher induced mutation frequencies per unit UV-dose, but similar mutation frequen- cies per unit survival compared to normal cells. These results support the hypothesis that the excision repair function lacking in XP7BE cells is "error-free"; otherwise, a higher frequency of induced mutations would be expected in normal cells than in XP7BE cells at an equal cytotoxic dose of UV-light. Furthermore, these results support the hypothesis that humans having certain syndromes that predispose the individual to cancer will have higher induced mutation frequencies than non-susceptible indi- viduals. The Genetic Nature of DTr Mutants The DTr phenotype selected in these studies could be the result of gene or chromosomal mutation or, alterna- tively, an epigenetic change in gene expression but not in the composition of nuclear DNA. A number of properties of DTr variants are consistent with a genetic origin of the variant phenotype. It was shown in these studies, and elsewhere (40,55,115) that DTr mutants occur randomly in non—mutagenized populations, the frequency of occurrence is increased by UV-irradiation or other known mutagens, and the DTr phenotype is inherited by cell progeny and is 73 stable in the absence of selective agent (DT). In addition, a change in the activity of a Specific gene product, namely EF-2, has been demonstrated in DTr mutants in human fibroblasts selected under conditions similar to those used in these studies (40,55). These properties of DTr mutants and other charac- teristics of the DTr mutation assay make it well suited for tg vitro studies of somatic mutagenesis in human cells. SUMMARY Research in quantitative mutagenesis with cultured human cells has been limited by the availability of reliable genetic markers for such studies. The present study describes the development and characterization of a new mutation system for human diploid fibroblasts using resistance to diphtheria toxin as a genetic marker, and application of this new mutation assay to a comparative study of mutagenesis in normal and DNA excision repair deficient xeroderma pigmentosum fibroblasts. A concentration of diphtheria toxin to use in the selection of DTr mutants was determined whereby all surviving colonies were cross-resistant to Pseudomonas aeurginosa exotoxin-A indicating they represent a single class of mutants with altered elongation factor 2. Mutants are stable and heritable in the absence of the selective agent and are inducible by known mutagens. No cell density effect was observed in the recovery of DTr mutants. Relatively long expression times of 10 and 15 days were required for the maximum recovery of UV-induced mutants in normal and XP7BE xeroderma pigmentosum fibro- blasts, respectively. Maximum recovery of mutants was followed by a decline in mutation frequency on subsequent days evaluated for all UV doses. An apparent linear dose- response was observed for UV—induced mutations in both 74 75 normal and XP fibroblasts. 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