Defects 40-infected Skin Fibroblasts from Individuals ...

Elevated Expression of T-Antigen in Simian Papovavirus 40-infected Skin Fibroblasts from Individuals with Cytogenetic Defects A. S. Lubiniecki, W. A. Blattner and J. F. Fraumeni, Jr. Cancer Res 1977;37:1580-1583. Published online May 1, 1977.

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(CANCER RESEARCH

37, 1580-1583,May 1977]

Elevated Expression of T-Antigen in Simian Papovavirus 40infected Skin Fibroblasts from Individuals with Cytogenetic Defects1 A. S. Lublniecki,2 W. A. Blattner, and J. F. Fraumeni, Jr. MeloyLaboratories,Inc., Springfield,Virginia22151[A. S. LI, and EnvironmentalEpidemiologyBranch,NationalCancerInstitute,NIH,Bethesda,Maryland 20014 (W. A. B.,J. F. F.J

16, 18, 24). The remainder included 2 cases of Turner's syndrome (18), 2 cases of ataxia telangectasia (10), and 1 caseoftnipboidy (17).Sincethesestudies were mainlyofcell A number of cytogenetic conditions were examined for lines with extra or unstable chromosomes, had used various expression of simian papovavinus 40 T-antigen in vitro. Skin fibmoblasts from patients with Turner's syndrome and methodologies, and did not have a large on adequately characterized normal control population, a more complete tnisomy 18 syndrome and most cell lines from Klinefelten's investigation seemed warranted. syndrome, tnisomy 13 syndrome, chromosomal transboca Studies from our laboratory have led to modifications in tions, chromosome 21 deletions, and single cases of 18qâ€” the T-antigen assay system that minimize variability and and 4pâ€”exhibited elevated T-antigen expression, com permit generation of reproducible data (8, 9). A statistically pared to a clinically and cytogenetically normal control significant increase in T-antigen expression was observed population. Thus, T-antigen expression was generally ele in SV4O-infected skin fibmoblasts from 10 Fanconi's anemia vated in cells with increased, decreased, on rearranged patients compared to a normal population of 76 individuals genetic material involving many different chromosomes. Variation in T-antigen expression among cell lines may (13). For a determination of whether increased expression of SV4O genetic information is a general characteristic of reflect two factors. Individual cell line factors may account cytogenetic disorders, this assay system was applied to skin for differences within homogeneous clinical groups, fibroblasts from patients with Turner's syndrome, Klinefel whereas population factors appear to account for differ ten's syndrome, various autosomal tnisomy and deletion ences between the various clinical groups and the control syndromes, and patients and carriers with chromosome population. translocations. The observation of elevated T-antigen expression in di verse cytogenetic conditions suggests that this phenome non may be a manifestation of chromosomal aberration MATERIALS AND METHODS unrelated to cancer susceptibility. Cell Lines. Most of the human skin fibmoblastlines were established from punch biopsy specimens as previously INTRODUCTION described (18). Cell lines 2278 (GM-5), 2279 (GM-73), 2280 SUMMARY

A number of laboratories have investigated SV4O' T-anti gen expression and colony formation (transformation) in skin fibroblasts from patients with cytogenetic disorders. These studies have generally shown increased expression of SV4O genetic information in infected cells from cytoge netically abnormal individuals but were usually based on small numbers of cell lines representing a limited spectrum of disorders. Examination of 14 such reports (1, 2, 5, 10, 14, 16-19, 22-24, 26, 27) revealed that 52 distinct cell lines from 13 different cytogenetic conditions and 61 distinct normal cell lines were studied . The abnormal cell lines studied were primarily from 17 patients with Fanconi's anemia(1 , 2, 5, 14, 17, 18, 22, 23, 27), 19 patients with Down's syndrome (1, 14, 17-19, 22, 24, 26), and 11 patients with other tmisomies (14, I This

work

was

supported

by

NIH

Contracts

NO1-CP-43216

and

61011 from the National Cancer Institute. a To 3 The

whom

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Received December 13, 1976; accepted February 14, 1977.

N01-CP

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43),

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(GM-230),

2292 (GM-327),

2290

(GM-284),

and 2294 (GM-479) were

from the Human Genetic Mutant Cell Repository, Institute for Medical Research, Camden, N. J. Cell lines 2267 (CRL 1246), 2375 (CCL-2i), 2377 (CCL-65), 2381 (CCL-i 17), and 2383 (CCL-i23)

were from the American

Type Culture Col

lection, Rockville, Md. All cells were grown in Dulbecco's modified Eagle's minimum essential medium plus 10% (v/v) fetal bovine serum as previously described (9). The kamyo types of peripheral leukocytes were assessed by the method of Moorehead et a!. (15), and fibmoblasts were assessed by the method of Sun et al. (21). Virus. The origin, propagation, and titration of the SV4O small plaque strain used have been described earlier (8). T-Antigen Assay. This technique has been reported pme viously (9). Briefly, 3 to 4 replicate cultures of each cell line were infected with 100 plaque-forming units of SV4O virus pen cell. After a 3-hr adsorption period, cultures were incu bated for 3 days at 37Â°in the presence of sufficient rabbit CANCERRESEARCHVOL. 37

1580


SV4O T-Antigen Expression in Cytogenetic Defects anti-SV4O serum to prevent secondary infection. At this time cultures were fixedand stainedby indirect immunofluores cent methods. In each culture, 100 microscopic fields (an average of 3000 cells) were examined for T-antigen expnes sion. The mean proportion of T-antigen-containing cells, weighted according to the number of cells examined per replicate, was calculated (p,@ and mathematically trans formed by the arc sine square root function (sin-' .,JpJ. This quantity was regarded as the estimate of T-antigen expres sionfora givencellline. The sin-'@,Jp,,, transformwas used to provide homogeneity of variances required for panarnet nc statistical analysis of these data. Using these methods we found that T-antigen expression in cells from 76 healthy, karyobogically unremarkable mdi viduals was normally distributed and not significantly influ enced by age,sex,orethnicbackground(13). The probabil

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the mean value of this representative healthy population (11.5 Â±4.0) was calculated from the cumulative normal probability function (z test). Assay results do not vary significantly between expemi ments (9,13);thereforesingledeterminations of 3 to 4 replicate cultures per cell line were adequate to assess T antigen expression accurately. Nonetheless, we used inter nabcontrols in each experiment. Tumor cell lines A172 and A498 were selected since their levels of T-antigen expres sion represented the high and low extremes, respectively, and did not change with extensive serial cultivation (9). Skin fibroblast lines were assayed at their lowest available serial passage. This was between passage bevels5 and 10 for lines established in this laboratory or within 3 to 5 passages of receipt from external repositories.

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TURNER'S KLINEFELTER'SAUTOSOMAL SYNDROME

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TRISOMY

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TRANS-

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LOCATION

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CARRIERS

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Chart 1. T-antigen expression in SV4O-infected skin fibroblasts from individuals with cytogenetic anomalies. T-antigen expression is presented as p@and sin' ..Jp,,,. Probabilities were calculated from the cumulative normal frequency distribution with the mean value (11.5 Â±4.0) of sin-' ..,/p@ for a population of 76 normal individuals (13). 0, males; 0, females. Karyological information is contained within thesymbol(i.e., which chromosomes are translocated, deleted, or

trisomic). Numbersadjacentto the symbolsreferto the cell line designationcode.

MAY 1977

1581


A. S. Lubiniecki et a!. different experiments and that the level of T-antigen expres sion generally remains constant oven several cell passages (9). Since differences between cell lines are greater than T-antigen expression in SV4O-infected skin fibroblasts those accounted for by experimental variation, it appears from 6 of 8 cases of Turner's syndrome exceeded the upper that at least 2 biological factors control the expression of T 99% confidence limit for the healthy control population antigen in cells. Individual cell line factors seem to account (Chart iA). One of the 6 patients also had acute myebogen ous leukemia(Case0453).The remaining2 casesexceeded for differences within homogeneous clinical groups, whereas population factors account for differences be the 95% confidence limit. T-antigen expression was normal tween normal individuals and those with cytogenetic abnon in 3 cases (not shown) that phenotypicalby resembled malities. Individual variation may explain the elevation in all Turner's syndrome but had eupboid kanyotypes. 8 patients with Turner's syndrome in this study, whereas 2 Four of 5 cases of Klinefelter's syndrome (Chart iB) patients previously reported from another laboratory had showed significantly elevated T-antigen expression , includ ing a case with breast cancer (Case 1654). In 1 case (Case normal values (18). These observations suggest that cell lines differ in their 2280) the extra X chromosome was transbocated onto 1 regulation of T-antigen expression. Experiments utilizing chromosome 14. All 5 cases of tnisomy 18 and 2 of 3 cases of radioactively labeled SV4Ovirus do not support the hypoth tnisomy 13 had significantly elevated levels of T-antigen esis that cells containing cytogenetic anomalies absorb vi expression (Chart iC), whereas 1 case of trisomy 7 and 1 of nus more efficiently than do normal cells (Ref. 1; J. M. Kelley 3 cases of tnisomy 13 examined had normal levels. Two of the tnisomy 13 cases (Cases 2281 and 2380) contained D;D and A. S. Lubiniecki, unpublished observations). Therefore, it is possible that as yet undefined biological factors in cell transbocations. T-antigen expression was significantly elevated in 3 of 6 lines with cytogenetic abnormalities explain these differ ences. These biochemical changes could be of fundamen patients with malformations resulting from translocations tab significance in considering the mechanism by which (Chart iD). Four of 5 transbocation carriers had significantly cytogenetic abnormalities predispose to cancer. elevated levels of T-antigen expression (Chart iE). None of Another question arising from these data concerns the these cell lines contained extra genetic information in addi relationship of elevated T-antigen expression to increased tion to their respective transbocations. cancer risk. Patients with Fanconi's anemia and Down's Among the chromosome deletion syndromes examined syndrome are prone to acute leukemia (6, 11). Turner's (Chart iF), marked elevation was observed in single cases with 18qâ€”and 4pâ€”(Wolf's syndrome) and in 2 of 3 cases syndrome is associated with various tumors derived from neural crest tissue (25); Klinefelter's syndrome is associated with monosomy 21. Results on 1 patient with cmi-du-chat with male breast cancer (7); and tnisomy 18 is associated syndrome were normal, but the kamyotype [80% with premalignant lesions of Wilms' tumor (4). All 5 syn 45,XY,t(5;22) and 20% 46,XY,5pâ€”] was not typical. dromes exhibit elevated SV4O T-antigen expression but so do cytogenetic disorders that are not presently associated DISCUSSION with high cancer risk. Although some of these disorders are too nameor lethal to permit an analysis of cancer risk, the This study examined T-antigen expression in skin fibro finding of elevated T-antigen expression in diverse cytoge blasts from patients with a spectrum of tnisomy, deletion, netic syndromes suggests that it may be a manifestation of and transbocation disorders involving both autosomal and chromosomal aberration that is unrelated to cancer suscep sex chromosomes. Significantly increased expression (P < tibility. 0.05) was found in 30 of 39 cell lines from such patients, and It remains possible, however, that elevated T-antigen only 1 case failed to exceed the normal mean. These find expression in cytogenetic syndromes is a predisposing fac ings extend our previous reports of elevated T-antigen ton for certain neoplasms in combination with other risk expression in skin fibroblasts from patients with Fanconi's factors such as the estrogen hormonal imbalance that leads anemia (13) and in many but not all tnisomy 21 cell lines.4 to breast cancer in Klinefeltem's syndrome. This notion is Thus, elevated T-antigen expression is seen in a wide van consistent with the finding of increased colony formation ety of chromosomal anomalies. and T-antigen expression in kanyobogicably normal families A major concern here is the variation of T-antigen expres prone to leukemia and certain other cancers (3, 12, 20). sion within groups of patients with a particular cytogenetic Perhaps the SV4O assay measures a variety of molecular disorder. Among individuals with transbocations differences abnormalities analogous to but not necessarily identical in T-antigen expression may reflect the specific chromo with the different DNA repair defects seen in patients with somes involved, but the variation within relatively homoge xenoderma pigmentosum. Further experiments are pres neous categories (e.g. , Turner's syndrome, Klinefelten's ently underway in this laboratory that should lead to clarifi syndrome, tnisomy 18, and monosomy 21) suggests that noncytogenetic factors within individual cell lines also meg cation of the relationship between T-antigen expression and ulate the level of T-antigen expressed. This is supported by susceptibility to cancer. the observations that different aliquots of the same cell line gave relatively homogeneous results in the same and in ACKNOWLEDGMENTS RESULTS

4 A.

S.

Lubiniecki,

preparation.

W.

A.

Blattner,

and

J.

F.

Fraumeni,

Jr.,

manuscript

in

The authors thank Dr. Harvey Dosik, Dr. Wladimir Wertelecki, Dr. Henry Lynch, and Dr. Kenneth Garver for submitting skin biopsies, cell cultures,

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SV4O T-Antigen Expression in Cytogenetic Defects and clinical diagnoses for these studies. The authors are grateful to Mark Gunnell, Thomas Archer, Elizabeth Alexander, and Russell Hanson for their technical assistance; to Cecily Sun and Connie Hemphill for cytogenetic analysis; and to Celeste Martin for secretarial assistance.

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