Proc. Nati. Acad. Sci. USA Vol. 82, pp. 5400-5403, August 1985 Cell Biology Chromosomal radiosensitivity during the G2 cell-cycle period of skin fibroblasts from individuals with familial cancer (chromatid gaps and breaks/cell-cycle-related radiosensitivity) RAM PARSHAD*, KATHERINE K. SANFORDtf, AND GARY M. JONESt *Pathology Department, Howard University College of Medicine, Washington, DC 20059; and tLaboratory of Cellular and Molecular Biology, National Cancer Institute, Building 37, Room 2D15, Bethesda, MD 20205 Communicated by Michael Potter, May 2, 1985

ABSTRACT We reported previously that human cells hanced G2-phase chromosomal radiosensitivity is associated after neoplastic transformation in culture had acquired an with both a high risk of cancer and with the neoplastic state. increased susceptibility to chromatid damage induced by In the present study, we have compared the extent of x-irradiation during the G2 phase of the cell cycle. Evidence chromatid damage after x-irradiation during G2 phase in suggested that this results from deficient DNA repair during G2 apparently normal skin fibroblasts from individuals with phase. Cells derived from human tumors also showed enhanced various types of neoplasms with that induced in skin fibro- G2-phase chromosomal radiosensitivity. Furthermore, skin blasts from normal donors. The individuals with neoplasms fibroblasts from individuals with genetic diseases predisposing were predominantly from families with a history ofneoplastic to a high risk of cancer, including ataxia-telangiectasia, Bloom disease, whereas the controls were normal individuals with- syndrome, Fanconi anemia, and xeroderma pigmentosum out evidence ofneoplastic disorders. The results suggest that exhibited enhanced G2-phase chromosomal radiosensitivity. enhanced G2-phase chromosomal radiosensitivity reflects a The present study shows that apparently normal skin fibro- genetic predisposition for neoplasia. Observations from a blasts from individuals with familial cancer-i.e., from fami- number of previous studies (2-4, 12-18) are consistent with lies with a history of neoplastic disease-also exhibit enhanced the proposition that this radiosensitivity results from either G2-phase chromosomal radiosensitivity. This radiosensitivity increased susceptibility of DNA to initial damage or to appears, therefore, to be associated with both a genetic deficient DNA repair during the G2 period of the cell cycle. predisposition to cancer and a malignant neoplastic state. Our recent study (9) supports the latter concept. Furthermore, enhanced G2-phase chromosomal radiosensitiv- ity may provide the basis for an assay to detect genetic susceptibility to cancer. MATERIALS AND METHODS Cell Lines. Normal skin fibroblasts from individuals with- X-irradiation of mammalian cells in G2 phase just prior to out evidence of neoplastic disease, who ranged in age from 8 mitosis produces chromatid aberrations, predominantly to 96 years (Table 1), were obtained from the American Type breaks and gaps, seen at the first posttreatment metaphase Culture Collection (CRL), and from T. Kakunaga (KD) (19) (1). Previously, we showed that mouse or human cells and R. Trimmer (RJH4) of this National Cancer Institute transformed spontaneously or by chemical carcinogens in Laboratory. Apparently normal skin fibroblasts from nine culture, compared with precursor or normal control cells, had individuals with diverse forms of cancer were obtained from increased chromatid damage after x-irradiation or fluorescent the Institute for Medical Research (Camden, NJ). At least six light exposure during the G2 or late S-G2 phases of the cell of these individuals were from families with a known history cycle (2-4). Exposure to fluorescent light (effective wave- of neoplastic disease (Table 1). For example, the 26-year-old length, 405 nm in the visible range; ref. 5), like x-irradiation, female with acute myelogenous leukemia (cell line AG 2655) produces chromatid damage that can be prevented to a large had two brothers and one sister with acute myelogenous extent by adding catalase to the culture medium or mannitol, leukemia; her mother had uterine cervical cancer; two a scavenger ofthe free hydroxyl radical (-OH) (5-7); the latter cousins had malignant reticuloendotheliosis; an uncle had may be generated from hydrogen peroxide (H202) through colon cancer; and her aunt was the 50-year-old female with the Fenton reaction (8). Thus H202 and -OH appear to be breast cancer (cell line AG 3778) (family 375 in refs. 20 and indirect or direct causative agents in the radiation-induced 21). The 11-year-old female with nevoid basal cell carcinoma damage. Cell lines derived from human tumors of diverse syndrome (cell line AG 3499) had a medulloblastoma excised tissue origin and histopathology also showed significantly at age 2 years, followed by radiotherapy and chemotherapy; higher frequencies of chromatid breaks and/or gaps than did 18 months later, numerous basal cell carcinomas appeared skin fibroblasts from normal donors when x-irradiated during with an ovarian fibroma at age 5 years, and subsequently G2 phase (9). Furthermore, skin fibroblasts from individuals other manifestations of basal cell carcinoma syndrome ap- with genetic disorders predisposing to a high risk of cancer, peared (22). At least five other family members had this including ataxia-telangiectasia, both homozygotes and syndrome. The 17-year-old male with liposarcoma of the heterozygotes (10), Bloom syndrome, Fanconi anemia, fa- scrotum, also an oral papilloma, benign fibrous papilloma of milial polyposis, Gardner syndrome, and xeroderma the nose, and achondroplasia (cell line AG 3308), had a pigmentosum, exhibited enhanced G2 chromosomal younger brother, the 15-year-old male (cell line AG 3302) radiosensitivity to both x-ray and fluorescent light (11-13, 43) with two primary tumors, a metastatic osteosarcoma, and a compared with skin fibroblasts from normal donors. From neurogenic (ganglion) tumor. The 7-year-old male with these observations on the cytogenetic response of cells to medulloblastoma (cell line AG 3000) showed an unusual radiation by x-ray or fluorescent light, it appears that en- response to radiation, developing a benign fibroma. His father had Hodgkin's disease. Although no family history of The publication costs of this article were defrayed in part by page charge neoplastic disorders was available on the remaining three payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4To whom reprint requests should be addressed. 5400 Downloaded by guest on September 24, 2021 82 5401 Cell Biology: Parshad et al. Proc. Natl. Acad. Sci. USA (1985)

Table 1. Chromatid damage in skin fibroblasts from normal donors and cancer patients after x-irradiation during G2 phase Family In vitro Gaps per 100 Breaks per 100 cells Cancer* history of passage of cells Donor age, 100 Cell line yr (sex) Diagnosis therapy neoplasia cells tested R = 0 R = 100 R = 0 R = Normal 9 0 13.1 1.0 2.1 CRL 1222 8 (M) 0.5 GM 0500 10 (M) Normal 14 0.5 6.5 1.0 Normal 25 0 4.7 0 1.9 KD 31 (F) 1.9 CRL 1221 40 (M) Normal 13 0 10.1 0.7 Normal 10 0 9.8 0 4.5 CRL 1224 40 (F) 1.7 CRL 1191 45 (M) Normal 7 0 6.2 0 Normal 6 0 8.7 0.6 2.0 CRL 1232 58 (M) 1.5 CRL 1188 63 (F) Normal 7 0 8.5 1.4 Normal 13 0 8.0 1.9 0.8 RJH 4 66 (M) 2.1 GM 1680 71 (F) Normal 12 2.9 5.7 0.7 96 Normal 14 3.1 10.6 0 2.4 GM 0731A (M) 11.1 AG 3000 7 (M) Medulloblastoma Rt + 9 1.7 54.6 0 Neuroblastoma C,R ? 12 0.5 132.0 0 53.5 AG 2202 9 (F) 18.5 AG 3499 11 (F) Nevoid basal cell C,R + 6 0 54.8 0 carcinoma syndrome 14.0 AG 3302 15 (M) Osteosarcoma, neurogenic C + 5 1.0 47.0 0 (ganglion) tumor Liposarcoma None + 6 1.5 53.5 1.5 20.5 AG 3308 17 (M) 15.9 AG 1732 19 (F) Chronic myelogenous R ? 12 1.7 55.1 0.9 leukemia, Wilms tumor in childhood 14.0 AG 2655 26 (F) Acute myelogenous C + 6 0.9 58.0 0.9 leukemia Breast cancer None + 8 2.5 83.5 1.5 24.0 AG 3778 50 (F) 16.0 AG 1360 68 (M) Multiple myeloma C 8 1.8 49.6 0.9 *C, chemotherapy; R, radiotherapy. tSkin biopsy was outside the field of localized radiation. in of donors, including the 68-year-old male, with multiple were achromatic lesions showing no dislocation spite certain clinical features sug- apparent chromatid discontinuity. By examining metaphase myeloma (cell line AG 1360), be assured that that the other two donors had a genetic background of cells within 1.5 hr after irradiation, we could gested at the time of x-irradiation. cancer susceptibility. For example, the 19-year-old female the cells were in G2 phase with chronic myelogenous leukemia, whose lymphocytes showed the Philadelphia marker chromosome (cell line AG RESULTS treatment for a Wilms' tumor 1732), had received radiation of chromatid breaks and when 4V/2 years old. The leukemia was diagnosed 9 years later Table 1 presents the frequency gaps R = 2.58 x 10-4 when she also had developed numerous pigmented nevi; a induced in late G2 phase by 100 R (1 was coulomb/kg) in skin fibroblasts from 11 normal donors and in constitutional predisposition to cancer suggested with cancer. The apparently normal skin fibroblasts from 9 individuals (23-26). Her father also had bladder 9-year-old No female with neuroblastoma (cell line AG 2202) had multiple malignant neoplasms; most, if not all, had familial cancer. mediastinum, interchanges or exchanges were observed. Skin fibroblasts individual primary tumors in adrenal, posterior low incidence of and liver. It has been suggested that this special form of from normal donors showed a relatively disseminated neuroblastoma, designated stage IV-S, is not, chromatid damage after G2-phase x-irradiation, irrespective nodule of donor age, sex, or in vitro passage level at time of test. An in fact, a malignant tumor, but rather a hyperplastic and 5 free of of mutant cells lacking a second event present in typical additional three individuals, aged 1, 3, yr, neuro- neoplastic disease but having Tay-Sachs disease or a defi- neuroblastoma cells. According to this hypothesis, lines GM 0077, blastoma IV-S may be a hereditary one-hit lesion (26). Cells ciency in glutathione synthetase activity (cell GM 3877, and GM 3878) also showed a low incidence of of all lines were predominantly diploid. with 100 R Experimental Procedure. Stock cultures were grown in chromatid damage after G2-phase x-irradiation with (chromatid gaps, 8.4, 1.9, 5.6; breaks, 1.4,4.8, and 4.4 per 100 Dulbecco's modified Eagle's medium supplemented diverse tissues of serum as described (3). For irradiation, cells, respectively). In spite of the origin 10% fetal bovine of the the incidence of both x cells in 2 ml of medium were inoculated into and histopathologies neoplasms, 0.7-1.0 105 and breaks was a 9 x 50 mm coverslip, and radiation-induced chromatid gaps significant- Leighton tubes, each containing with neo- 48 hr later according to methods de- ly higher in skin fibroblasts from the individuals cells were irradiated donors < 0.00001 for scribed (3). After irradiation, culture fluid was renewed plasms than that in cells from normal (P was both breaks and Chromatid aberrations in nonirradi- within 20-30 min and 0.1 ,ug of Colcemid per ml (GIBCO) gaps). from analysis, cells were pro- ated control cells of patients did not differ markedly added for 1 hr. For chromosome donors in situ on coverslips as described (27). Chromosome those of the normal (Table 1). cessed The incidence of radiation-induced chromatid damage in analyses were made on randomized coded preparations; four the and 100-200 metaphase the cells from individuals with neoplasms fell within range cultures were used for each variable, skin fibroblasts from individuals with were examined per variable. Statistical analysis was reported earlier for cells with a risk of cancer (10, 13), carried out by using the Wilcoxan rank sum test (28). genetic diseases associated high it was lower than that ofhuman Abnormalities scored as breaks showed distinct dislocation but as seen in Fig. 1, generally and misalignment of the chromatid fragment, whereas gaps tumor cells studied previously (9). Downloaded by guest on September 24, 2021 5402 Cell Biology: Parshad et al. Proc. Natl. Acad. Sci. USA 82 (1985) DISCUSSION relatives showed increased sensitivity to the DNA crosslink- ing agent, mitomycin C, but normal sensitivity to ultraviolet Families with a history of neoplastic disease have been (UV) and x-irradiation (31). Skin fibroblasts from two mem- studied extensively in efforts to detect markers of genetic bers of a family with Gardner syndrome showed hypersen- susceptibility to cancer or to obtain evidence of associated sitivity to the lethal effects of x-irradiation, UV irradiation, deficiencies in DNA repair. Fibroblasts from three members and mitomycin C, but they were normal in their capacity for of the family with acute myelogenous leukemia (including UV-light induced unscheduled DNA synthesis (32). En- lines AG 2655 and AG 3778 of the present study) in a hanced sensitivity to UV irradiation and the mutagenic effect colony-forming assay showed a significant increase in x-ray of the carcinogen 4-nitroquinoline 1-oxide was also observed sensitivity compared with cells from family members without in cultured skin fibroblasts or lymphoblastoid cell lines from cancer (29). There was also increased transformation on patients and unaffected members of families with hereditary exposure to simian virus 40, whereas among 10 first and cutaneous malignant melanoma (33-36). The enhanced pho- second degree cancer-free relatives, the test was normal (30). tosensitivity appeared not to be associated with abnormal These observations on radiosensitivity and transformation DNA repair synthesis, UV-light induced inhibition and re- suggested a possible defect in the repair of radiation- or covery of DNA synthesis, or a defect in nucleotide excision virus-induced DNA damage. However, results of assays for repair (33, 34); in the carcinogen-sensitive cells, DNA repair DNA repair replication, single-strand break rejoining, and synthesis also appeared to be normal (35). However, a removal of enzyme-sensitive sites in x-irradiated DNA were deficiency in repair of chemically induced DNA damage in the same in the cell lines from sensitive and normal family leukocytes of patients with or individuals genetically predis- members (29). In a large study of patients at high risk of posed to colorectal cancer has been reported (37). cancer, spontaneous sister chromatid exchanges were found The present study reports a common defect manifest as not to be a marker of cancer risk (21). In another study to enhanced G2-phase chromosomal radiosensitivity in individ- determine whether susceptibility to Wilms tumor is associ- uals with diverse forms of neoplastic disease. The cell lines ated with a defect in handling of DNA damage, lymphoblas- were mostly from relatively young members of families toid lines from Wilms tumor patients and their first-degree having a high incidence of cancer; therefore, they were probably all individuals genetically susceptible and at high cancer risk. Whether enhanced chromosomal radiosensitiv- 520{ ity also characterizes cells from individuals with sporadic cancer awaits further study. Most, but not all, of the Gaps individuals with neoplasms had received chemotherapy, 30Y *Breaks radiotherapy, or both. The possibility that this prior treat- ment influenced the response of their skin fibroblasts seems unlikely. The nonirradiated control cells showed no more 280 chromatid damage than was seen in cells from normal untreated donors. Also, the extent of radiation-induced 180 damage in cells from these patients was comparable to that reported previously (ref. 13 and Fig. 1) in cells from individ- = 160 uals with genetic disorders associated with a high risk of cancer who had not received chemotherapy or radiotherapy. 8 140 Furthermore, in another ongoing study, we have observed a 0. similar enhancement of G2-phase chromosomal radiosensi- I, 120 tivity in untreated patients with hereditary cutaneous malig- .0 nant melanoma (38). Moreover, a significant increase in chromatid breaks induced by the radiomimetic antibiotic, z 100 bleomycin, was reported in peripheral lymphocytes of un- treated patients with medullary thyroid carcinoma and in 80 their immediate family members compared with normal controls (39). It was further suggested that bleomycin sensi- 60 [ tivity may be useful in estimating an individual's risk for developing this type of cancer. 40 The induction of chromatid breakage in cells of G2 phase has been used as an assay for clastogenic effects of various 20 compounds (40). As noted earlier, an increased incidence of chromatid damage following G2-phase x-irradiation could 0. result from a greater susceptibility to the initial DNA damage a b c d or from a deficiency in DNA repair. Our recent results support the latter concept (9). According to the mononeme - Skin fibroblasts * theory, each chromatid contains a single continuous DNA FIG. 1. Comparison of chromatid damage induced by x-irradi- double strand. Therefore, chromatid breaks seen in the first ation (100 R) during G2 phase of skin fibroblasts from normal donors metaphase following x-irradiation represent unrepaired DNA (a), skin fibroblasts from individuals with genetic disorders associ- double-strand breaks. Chromatid gaps seen directly after ated with a high risk of cancer (b) (13), skin fibroblasts from cancer radiation probably result from unrepaired DNA single-strand patients (c), and human tumor cells (d) (9). The genetic disorders breaks arising directly or indirectly from incomplete nucle- represented, in order of increasing chromatid damage, were otide excision repair of DNA damage (9, 13-15, 41). In xeroderma pigmentosum variant, Gardner syndrome (GS), previous studies, the inability to detect by biochemical xeroderma pigmentosum, complementation group E (XP-E), GS, Bloom syndrome, XP-C, familial polyposis, ataxia telangiectasia methods deficient DNA repair in cells from individuals with heterozygotes (five individuals) and homozygotes (two individuals) familial cancer may result from using asynchronous cell (10, 13). Data on XP-A cells have not been included; for explanation, populations that consist primarily of G1- and S-phase cells. see ref. 13. The tumor cells were from malignancies ofdiverse tissues We also observed no difference in chromatid damage be- of origin and histopathology (9). tween cells from normal and cancer-prone individuals or Downloaded by guest on September 24, 2021 Cell Biology: Parshad et al. Proc. Natl. Acad. Sci. USA 82 (1985) 5403

between normal and tumor cells when exposed to fluorescent (1978) Proc. Natl. Acad. Sci. USA 75, 1830-1833. 8. A. (1982) Can. J. Physiol. Pharmacol. 60, 1330-1345. light during G, and S phases, whereas a significant difference Singh, late S-G2 phase (2, 9. Parshad, R., Gantt, R., Sanford, K. K. & Jones, G. M. (1984) was observed if cells were illuminated in Cancer Res. 44, 5577-5582. 4, 43). Exposure to fluorescent light, like x-irradiation, 10. Parshad, R., Sanford, K. K., Jones, G. M. & Tarone, R. E. produces chromatid damage that can be prevented to a large (1985) Cancer Genet. Cytogenet. 14, 163-168. extent by catalase or mannitol, thus implicating the intracel- 11. Bigelow, S. B., Rary, J. M. & Bender, M. A. (1979) Mutat. lular generation of H202 and the derivative free hydroxyl Res. 63, 189-199. radical as causative agents (6). 12. Taylor, A. M. R. (1978) Mutat. Res. 50, 407-418. Based on epidemiologic evidence, Knudson and Strong 13. Parshad, R., Sanford, K. K. & Jones, G. M. (1983) Proc. Natl. a two-mutation model for cancer development: Acad. Sci. USA 80, 5612-5616. (42) proposed Mutat. Res. 137-149. cancer, the first mutation occurs 14. Natarajan, A. T. & Obe, G. (1978) 52, in certain familial hereditary H. G. & J. S. (1974) Mutat. to cancer, and 15. Bender, M. A., Griggs, Bedford, in germinal cells predisposing the individual Res. 23, 197-212. the second mutation, producing neoplastic transformation, 16. Kihlman, B. A. (1977) Caffeine and Chromosomes (Elsevier occurs in somatic cells; on the other hand, in sporadic cancer Scientific, New York), pp. 227-246. both mutations occur in somatic cells. We propose that 17. Preston, R. J. (1982) Cytogenet. Cell Genet. 33, 20-26. enhanced G2-phase chromosomal radiosensitivity seen in 18. Evans, J. H. (1977) in Progress in Genetic Toxicology, eds. skin fibroblasts of individuals with the familial cancers under Scott, D., Bridges, B. A. & Sobels, F. H. (Elsevier/North- study as well as in cells from individuals with genetic Holland, Amsterdam), pp. 57-74. USA disorders predisposing to a high risk of cancer represents the 19. Kakunaga, T. (1978) Proc. Natl. Acad. Sci. 75, first mutational event associated with the germ line. Detec- 1334-1338. is important 20. Snyder, A. L., Henderson, E. S., Li, F. P. & Todaro, G. J. tion of these genetically susceptible individuals (1970) Lancet i, 586-589. for both cancer prevention and therapy because they are at 21. Cheng, W., Mulvihill, J. J., Greene, M. H., Pickle, L. W., high risk to develop secondary cancers following mutagenic Tsai, S. & Whang-Peng, J. (1979) Int. J. Cancer 23, 8-13. therapeutic procedures such as radiation and chemotherapy. 22. Heimler, A., Friedman, E. & Rosenthal, A. D. (1978) J. Med. Somatic cells transformed in culture by chemical carcino- Genet. 15, 288-291. gens (3, 4), tumor viruses (unpublished observations), and 23. Meadows, A. T. & Jarrett, P. (1978) J. Pediatr. 93, 889-890. spontaneously (2) acquire enhanced G2-phase chromosomal 24. Schwartz, A. D., Lee, H. & Baum, E. S. (1975) J. Pediatr. 87, Skin fibroblasts from individuals with ge- 374-376. radiosensitivity. G. V., Banfi, A., Harris, netic diseases predisposing to cancer, skin fibroblasts from 25. Meadows, A. T., D'Angio, J., Mike, and human C., Jenkin, R. D. T. & Schwartz, A. (1977) Cancer 40, the individuals with familial cancer reported here, 1903-1911. tumor cells of diverse tissue origin, all show enhanced 26. Knudson, A. G., Jr., & Meadows, A. T. (1980) N. Engl. J. G2-phase chromosomal radiosensitivity compared with cells Med. 302, 1254-1256. from normal donors. This enhanced radiosensitivity, which 27. Gantt, R., Parshad, R., Ewig, R. A. G., Sanford, K. K., appears to result from deficient DNA repair during the G2 Jones, G. M., Tarone, R. E. & Kohn, K. W. (1978) Proc. period of the cell cycle, thus seems to be associated with both Natl. Acad. Sci. USA 75, 3809-3812. a predisposition to cancer and a neoplastic state. According- 28. Snedecor, G. W. & Cochran, W. G. (1980) Statistical Methods ly, enhanced G2-phase chromosomal radiosensitivity may (University Press, Ames, 10), pp. 208-213. N. B. M., Mulvihill, J. J. & Paterson, the basis for an assay to detect genetic susceptibility 29. Bech-Hansen, T., Sell, provide M. C. (1981) Cancer Res. 41, 2046-2050. to cancer. 30. McKeen, E. A., Miller, R. W., Mulvihill, J. J., Blattner, W. A. & Levine, A. S. (1977) Lancet ii, 310. We are grateful to Dr. John J. Mulvihill, Clinical Epidemiology 31. Imray, F. P., Relf, W., Smith, P. J. & Kidson, C. (1984) Branch, National Cancer Institute, Bethesda, MD; Dr. Anna T. Lancet i, 1148-1151. Meadows, Division of Oncology, The Children's Hospital, Philadel- 32. Little, J. B., Nove, J. & Weichselbaum, R. R. (1980) Mutat. phia, PA; Dr. Dorothy Warburton, Department of Human Genetics Res. 70, 241-250. and Development, Columbia Presbyterian Medical Center, New 33. Ramsey, R. G., Chen, P., Imray, F. P., Kidson, C., Lavin, York, NY; and Dr. Stephen Vasso, Department of Pathology, Our M. F. & Hockey, A. (1982) Cancer Res. 42, 2909-2912. Lady of Lourdes Hospital, Camden, NJ, for providing biopsy 34. Smith, P. J., Greene, M. H., Devlin, D. A., McKeen, E. A. & material or cell lines to the Institute for Medical Research and Paterson, M. C. (1982) Int. J. Cancer 30, 39-45. particularly for providing us with information and references con- 35. Smith, P. J., Greene, M. H., Adams, D. & Paterson, M. C. cerning their patients. We are also grateful to Dr. Raymond Gantt of (1983) Carcinogenesis 4, 911-916. this National Cancer Institute Laboratory for his helpful suggestions 36. Howell, J. N., Greene, M. H., Corner, R. C., Maher, V. M. & and to Dr. Robert E. 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Preston, R. J. (1980) Mutat. Res. 679, 71-79. Jones, G. M. & Baeck, A. E. (1979) Photochem. Photobiol. 42. Knudson, A. G., Jr., & Strong, L. C. (1972) J. Natl. Cancer 29, 971-975. Inst. 48, 313-324. 6. Parshad, R., Taylor, W. G., Sanford, K. K., Camalier, R. F., 43. Sanford, K. K., Parshad, R. & Gantt, R. (1985) in Free Gantt, R. & Tarone, R. E. (1980) Mutat. Res. 73, 115-124. Radicals, Aging, and Degenerative Diseases, ed. Johnson, 7. Parshad, R., Sanford, K. K., Jones, G. M. & Tarone, R. E. J. E., Jr. (Liss, New York), in press. Downloaded by guest on September 24, 2021