M. MIYAKI, ET AL. AC, PJ and normal cells were seeded ml kanamycin. The fibroblasts were detached from the dish by treatment at room temperature

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1 INCREASED SENSITIVITY OF FIBROBLASTS OF SKIN FROM PATIENTS WITH ADENOMATOSIS COLI AND PEUTZ-JEGHERS' SYNDROME TO TRANSFORMA- TION BY MURINE SARCOMA VIRUS*1 Michiko MIYAKI,*2 Noriko AKAMATSU,*2 Makoto ROKUTANDA,*2 Tetsuo ONO,*2 Hiroshi YOSHIKURA,*3 Masao SASAKI,*4 Akira TONOMURA,*4 and Joji UTSUNOMIYA*5 Department of Biochemistry, Tokyo Metropolitan Institute of Medical Science,*2 Department of Genetics, Institute of Medical Science, University of Tokyo,*3 and Department of Cytogenetics, Medical Research Institute*4 and Department of Surgery and Polyposis Center,*5 Tokyo Medical and Dental University Skin fibroblasts from family members of cases of adenomatosis coli (AC) and Peutz-Jeghers' syndrome (PJ) were examined for susceptibility to morphological transformation by Moloney murine sarcoma virus (MSV) of rat 78Al cells, which was found in the present study to contain both dualtropic and ecotropic murine leukemia virus (MLV). The fibroblasts from individuals with manifest AC (22 subjects) and manifest PJ (5 subjects) were up to 5 times more susceptible to transformation by rat 78Al-MSV (MLV) than those from normal individuals (7 subjects). Nonmanifest members of AC families (8 subjects) showed various sensitivities: the fibroblasts from 3 family members had a sensitivity as low as that of normal cells, and those of 5 individuals exhibited increased sensitivity similar to that of manifest cases of AC. The AC and PJ cells that were sensitive to 78Al-MSV (MLV) were also susceptible to transformation by other pseudotypes of Moloney MSV, although they were not transformed by infection of standard Moloney MSV. None of the fibroblasts were transformed by xenotropic MLV alone. The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate enhanced transformation by 78Al-MSV (MLV) in both AC and normal cells. Key words: Sensitivity to murine sarcoma virus-human fibroblast cell- Adenomatosis coli-peutz-jeghers' syndrome Adenomatosis coli (AC), whch is characterized by multiple adenomas and a high incidence of cancer in the large bowel,10,16) has been shown to be an autosomal dominant trait. Peutz-Jeghers' syndrome (PJ) is another heterogeneous type of hereditary gastrointestinal polyposis which is characterized by scattered hamartomatous poplys and specific mucocutaneous pigmentation.7) PJ has been described as a high risk condition for malignancy in various organs.20,21) Participation of environmental factors in genetic predisposition to cancer has been demonstrated by the fact that skin fibroblasts from cases of recessively inherited disorders have increased sensitivity to X-ray and UVlight irradiation and to various chemical carcinogens.19) High susceptibility to morphological transformation by the DNA and RNA tumor viruses, SV40 and Kirsten murine sarcoma virus (MSV), has also been demonstrated in cells from individuals with *1 This work was supported in part by grants for cancer research from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare. This study was presented at the 37th Annual Meeting of the Japanese Cancer Association, Tokyo, August 1978.

2 neoplasia and genetic abnormalities.8,12) A similar high susceptibility to Kirsten MSV has been demonstrated in cells from patients with AC.15) However, a study has not yet been made on PJ. We have examined the transformation of skin fibroblasts from both manifest and nonmanifest members of families having AC, and of families having PJ in Japan using several pseudotypes of murine sarcoma virus. The present paper shows that fibroblasts from individuals with manifest symptoms of these diseases have high susceptibility to morphological transformation by MSV. MATERIALS AND METHODS Origin of Fibroblasts Fibroblasts were obtained by biopsy of skin taken from the left elbows of 20 patients with manifest AC (AC cells), of 2 patients of Gardner's syndrome, of 7 nonmanifest members of families of patients with AC (nonmanifest AC), of 5 patients having PJ syndrome (PJ cells) and of 7 normal control subjects (normal cells). The clinical backgrounds of these individuals are shown in Table II. The cells were cultivated in Eagle's minimum essential medium (MEM) supplemented with M. MIYAKI, ET AL. tion of the fluid was used for measuring the frequency of transformation of human fibroblasts. The 78Al virus sample was found to contain XC+ ecotropic murine leukemia virus (MLV) and XC+ dualtropic MLV by examination of the host range of the two isolates from SC-1 cells and S + L-mink cells as indicated in Table I. The total dualtropic MLV and MSV (MLV) titer of the fluid from 78Al cells was 15,000 FFU/0.2ml when assayed on S + L-mink cells,13) and 430 FFU/0.2ml when assayed on normal mink cells. The pseudotypes of Moloney MSV containing xenotropic MLV, amphotropic MLV and dualtropic MLV were rescued from S + L-mink cells by superinfection with NZB-IU-1-MLV,5) wild mouse-derived amphotropic MLV,5) and HIX-MLV.2) The XC plaque formation of these viruses was measured as described elsewhere.17) NZB-mouse-derived xenotropic MLV6) was ob- tained from a culture of duck cells. Viral Transformation of Human Fibro- AC, PJ and normal cells were seeded blasts ml kanamycin. The fibroblasts were detached from the dish by treatment at room temperature for 15min with 0.016% Pronase E (Kaken Chemicals, Tokyo) and 0.02% EDTA. These reagents were removed by centrifugation of the resulting cell suspension, and then the cells were passaged. These fibroblasts were used within the first 10 passages. Origin of Viruses 78Al-MSV (MLV) was obtained from the 24-hr culture medium of rat cell line 78Al1) transformed by Moloney MSV- (MLV), by centrifuging the medium at 1,000g for 30min and then at 10,000g for 10min. The virus Table I. Characterization of Virus Isolates from 78Al Culture Fluid Gann

3 TRANSFORMATION OF AC AND PJ CELLS BY MSV (MLV) washed and infected with 0.2ml of 1-, 5-, 25- and 125-fold diluted MSV (MLV) suspensions. After incubation for 11 days with a change of medium every third day, the cells were fixed with 10% formaldehyde and the number of foci was counted. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) (Consolidated Midland Chemical Co. Brewster, N. Y.) was added to the culture medium three days after infection with MSV (MLV). Immunofluorescence Staining of MSV (MLV)- infected Cells with Antiserum against p30, a Major Group-specific Antigen of MLV The ml of 78Al-MSV (MLV) and cultivated for 10 days with a medium change every third day. The culture containing the transformed cells was replated at 1/4 cell density on a cover glass and cultivated for 6 days. The cells were washed with phosphate-buffered saline and fixed with cold acetone. The cover glass was incubated with anti-p30 rabbit serum for 1hr at room temperature, washed, and subsequently incubated with anti-rabbit goat serum conjugated with fluorescein. RESULTS Frequencies of Morphological Transformation of AC, PJ and Normal Cells blasts were infected with murine sarcoma virus (murine leukemia virus), MSV (MLV), of rat 78Al cells, transformed cells appeared after 5 or 6 days. The transformed cells had a roundish spindle-shaped form (Fig. 1, a and b) and the morphologically altered cells were highly stained by anti-p30 rabbit serum (Fig. 1c) indicating expression of the viral genome by these cells. After cultivation of the infected cells for 11 days with medium change every third day, all cell lines had nearly reached confluence clear. The cells were fixed and the number of foci was counted. On prolonged cultivation, some of the rounded cells became detached from the dish and the number of morphologically transformed cells decreased. Since the foci were rather diffuse, their number was counted on plates which gave about 100 foci per dish after infection with 5-, 25- or 125-fold diluted virus suspension and extrapolated to the value of 1-fold dilution. Nearly single-hit kinetics (1 to 1.5 hit) of frequency of focus formation were observed. Pre-treatment of the cells with polybrene increased the frequency of transformation by 100-fold, but did not affect the relative frequency of AC, PJ and normal cells. Fig. 1. a, AC fibroblasts transformed morpho- b, PJ fibroblasts transformed morpho- c, immunofluorescence staining of trans-

4 M. MIYAKI, ET AL. Table II. Clinical Backgrounds of Fibroblasts and Their Susceptibility to Transformation by 78Al-MSV (MLV) Gann

5 TRANSFORMATION OF AC AND PJ CELLS BY MSV (MLV) Infection of normal fibroblasts with 0.2ml of 78Al-MSV (MLV) suspension after treating the cells with polybrene induced 120 to 440 foci per dish, whereas a similar treatment of cells from manifest AC induced 560 to 1,700 foci (Table II). Fibroblasts from cases of Gardner's syndrome showed a transformation frequency of 850. Family members of AC cases showed various sensitivities: one group gave cells which showed sensitivity as low as that of normal cells (300 to 400 foci) and cells from another group exhibited increased sensitivity to 78Al-MSV (MLV) similar to that of manifest cases of AC (600 to 1,100 foci). The cells from individuals with manifest PJ showed high sensitivity to focus formation by 78Al-MSV (MLV), like those from individuals with manifest AC, as indicated in Table II. Frequencies of Morphological Transformation of AC, PJ and Normal Cells by Pseudotypes of Moloney MSV (MLV) The 78Al-MSV (MLV) obtained from rat 78Al cells which had been transformed by Moloney MSV (MLV)1) contained both ecotropic MLV and dualtropic MLV (Table I). To confirm which of these MLV's participates in the morphological transformation of human fibroblasts, three kinds of pseudotype of Moloney MSV containing xenotropic MLV, amphotropic MLV and dualtropic MLV, as well as standard Moloney MSV (MLV) were examined for their ability to transform AC, PJ and normal cells. All three pseudotypes could transform the human fibroblasts, while standard Moloney MSV with ecotropic MLV could not (Table III). The infectivities of these MLV's measured on S + L-mink cells (Table III) indicated that transformation of human fibroblasts is supported by the xenotropic or dualtropic MLV. While, these human fibroblasts were not transformed by xenotropic MLV alone. The relative frequencies of transformation of AC, PJ and normal cells caused by infection with the three pseudotype viruses in the present experiment were similar to those obtained with 78Al-MSV (MLV), suggesting that high susceptibility to viral transformation is a general property of AC and PJ cells. Effect of the Tumor Promoter TPA on Morphological Transformation It has been shown that TPA enhanced the transformation of fibroblasts by adenovirus4) or EB virus,22) suggesting that TPA acts on transformation steps rather than promotes cell growth. To examine whether the effect of tumor promoter on the transformation of Table III. Susceptibility of Normal, AC and PJ Cells to Transformation by Pseudotypes of Moloney MSV treatment with polybrene as described in "Materials and Methods." After cultivation for 12 days, the number of foci was counted. The number of foci on S+L- mink cells indicates the titer of xenotropic or dualtropic MLV. Values are averages of duplicate experiments. Virus obtained from a) rat 78Al cells, b) xenotropic NZB-IU-MLV, c) wild mouse-derived amphotropic MLV, d) dualtropic HIX-MLV, e) Moloney-MLV.

6 Table IV. Effect of TPA on the Transformation of Normal and AC Cells by 78Al- MSV (MLV) TPA at 1ng/ml was added to the cell cultures 3 days after infection of the diluted 78Al-MSV (MLV), and the medium was changed every third day. Numbers of foci were counted 12 days after infection, and extrapolated to obtain the value for 1-fold dilution. M. MIYAKI, ET AL. AC cells differs from that on normal cells, TPA was added to the cultures after infection with MSV (MLV). The effective dose of TPA was determined by measuring the maximal incorporation of [3H]2-deoxyglucose into cells. AC and normal cells exhibited almost the same response to various concentrations of TPA and the maximal incorporation was observed with TPA at 1 to 2ng/ml. When TPA was added at 1ng/ml to the culture medium 3 days after infection with 78Al-MSV (MLV), the transformed cells in TPA-treated cultures were rounder and the foci were clearer than those in untreated cultures. The number of foci in cultures of both AC and normal cells doubled (Table IV), suggesting that AC cells have the same sensitivity as normal cells to the effect of TPA as a promoter of viral transformation. TPA alone did not cause focus formation in AC or normal cells. DISCUSSION In this work we confirmed that fibroblasts from cases of manifest AC were up to 5 times more susceptible to morphological transformation by MSV than those from normal subjects, judging by counts of transformed foci. We found that PJ cells were also susceptible to viral transformation, no work has been done on this problem previously. PJ has been considered to be not precancerous, but one of us had previously found by a long-term follow-up study20) that patients with PJ have a high risk condition for various types of malignancy. The present results seem to support this clinical observation on PJ. The difference in susceptibility to 78Al- MSV (MLV) between AC and normal cells in this experiment was less than that reported for Kirsten MSV.15) This gap seems to depend on the method, the former being determined by counting the number of foci and the latter by titration end point. In every experiment using 78Al-MSV (MLV) and the three pseudotypes of Moloney MSV containing xenotropic, amphotropic and dualtropic MLV, the differences in sensitivity to transformation between AC, PJ and normal cells were almost the same. However, standard Moloney MSV (MLV) which had no xenotropic MLV did not cause detectable transformation. These results indicate that the transformation of human fibroblasts by MSV requires xenotropic or dualtropic MLV. The xenotropic MLV alone did not cause morphological transformation of AC and PJ cells, but the susceptibility of these cells to infection by MLV remains to be examined. Focus formation is affected by the growth conditions of the cells, but the growth rate of the AC cells did not differ from that of the normal cells in a medium containing 15% serum. Even in a medium with 1% serum, the growth rate of AC cells tested here was almost the same as that of normal cells, in contrast to the results reported previously.14) However, the saturation density of the AC cells was 10 to 20% higher than that of the normal cells as described elsewhere.9) The increased susceptibility of AC cells to 78Al-MSV (MLV) does not seem to be due to an increased growth rate, although increased saturation density cannot be Gann

7 TRANSFORMATION OF AC AND PJ CELLS BY MSV (MLV) neglected as a possible cause. TPA enhanced the focus formation in both AC and normal cells but did not affect the growth rate. The higher transformation frequency of the AC cells than the normal cells was observed even in the presence of TPA, which has been demonstrated to enhance the expression of transformation-related phenotypic properties.3) This indicates that the increased transformation frequency in AC cells may be caused by increased phenotypic expression. However, the difference in the frequency between AC and normal cells, which is observd even in the presence of TPA, may result from not only the expression but also from some other process of transformation. Although the mechanism of high sensitivity of the AC or PJ cells to virus is not understood yet, several diseases associated with a high risk of cancer and spontaneous chromosomal aberrations have been reported to be highly sensitive to viral transformation. For example, cells from cases of Fanconi's anemia8,12) and ataxia telangiectasia8) and from cases with chromosomal aberrations11) are sensitive to MSV and SV40, and these observations suggest that the process causing chromosomal abnormality may be related to the viral transformation. It may be noted here that some AC cells exhibited a high frequency of reciprocal translocation of chromosomes.18) Further studies are required to determine whether or not the process of integration of the viral genome is enhanced in AC cells when they are infected with virus. We are grateful to Dr. K. Suzuki and Dr. T. Tajima for providing some biopsy specimens of normal and AC skin and to Dr. R. Hirai for MLV. We also thank Dr. M. Hatanaka for examining transformed foci and Dr. T. Katayama for encouragement. (Received June 23, 1980/Accepted Aug. 21, 1980) REFERENCES 1) Bernard, C., Boiron, M., Lansneret, J., (1967). 2) Fishinger, P. J., Nomura, S., Bolognesi, D (1975). 3) Fisher, P. B., Goldstein, N. I., Weinstein, 4) Fisher, P. B., Weinstein, I. B., Eisenberg, D., Ginsberg, H. S., Proc. Natl. Acad. Sci. 5) Hartley, J. W., Rowe, W. P., J. Virol., 19, 6) Hirai, R., Yuasa, Y., Yamamoto, T., Virology, 7) Jeghers, H., McKusik, V. A., Kermit, H., 1036 (1949). 8) Klement, V., Freedman, M. H., McAllister, R. M., Nelson-Rees, E. A., Heubner, R. J., 9) Kopelovich, L., Pfeffer, L. M., Bias, N., 10) Lockhart-Mummerg, J. P., Dukes, C. E., 11) Lubuniecki, A. S., Blattner, W. A., Frau- 14) Pfeffer, L., Lipkin, M., Stutman, O., Kopelo- 15) Pfeffer, L. M., Kopelovich, L., Cell, 10, 16) Reed, T. E., Neel, J. V., Am. J. Hum. Genet., 17) Rowe, W. P., Pugh, W. E., Hartley, J. W., 18) Sasaki, M. S., "DNA Repair Mechanisms," ed. P. C. Hanawalt, E. C. Freiberg, and Press, New York. (1978). 20) Utsunomiya, J., Gocho, H., Miyanaga, T., Hamaguchi, A., Kashimura, A., Aoki, N., Komatsu, I., Johns Hopkins Med. J., 136, 21) Utsunomiya, J., Proc. 7th Int. Symp. Princess 22) Yamamoto, N., Hausen, H., Nature (London),