@ 1991 Wiley-Liss,
Inc.
Cytometry 12748-756 (1991)
Viral and Cellular Oncogene Expression During Progressive Malignant Transformation of SV40 Transformed Human Fibroblasts' Charles L. Goolsby? Marianne Steiner, and Jeffrey Nemeth Department of Pathology, Northwestern University Medical SchooliVA Lakeside Medical Center, Chicago, Illinois 60611 Received for publication December 28, 1990; accepted April 26, 1991
In vitro investigation of the multistep neoplastic progression which occurs during transformation of human cells has been hindered by resistance of human cells to both immortalization and tumorigenicity (Mut. Res. 199; 273, 1988). Previously our laboratory established a cell line, HSF4-Tl2, by transfection of normal human foreskin fibroblasts with the plasmid pSV3-neo which contains the early genes of simian virus 40 (SV40). A multistep progression in karyotypic alterations and transformed phenotype occurred resulting in a neoplastic cell line that was immortal, transformed, and tumorigenic. We have examined changes in the SV40 proteins, large T (T-antigen) and small t (t-antigen) antigens, and in the cellular protein, p53, during progressive transformation of these cells. Total viral protein expression relative to total cellular protein increased following im-
Elucidation of the sequential multistep process that occurs in establishment of human cancer is critical to a n understanding of the development of malignant disease. In vitro studies of multistep transformation may aid in gaining a n understanding of these processes (20,28). Our laboratory established a cell line, HSF4T12, by transfection of normal human fibroblasts with the early genes of simian virus 40 (SV40). This cell line underwent immortalization, a multistep progression in both transformed phenotype and altered karyotype, and formed progressive tumors when cells were injected into implanted Gelfoam sponges in nude mice (20). The early genes of SV40 comprise two proteins, large T antigen (T-antigen) and small t antigen (t-antigen) (51). T-antigen is a phosphorylated protein with a molecular weight of approximately 94 Kd. It exhibits
mortalization of HSF4-Tl2 as did the ratio of T-antigen to t-antigen. Interestingly, no significant change in DNA content accompanied immortalization. However, during the progressive in vitro transformation of HSF4-T12 which occurred primarily post-immortalization, DNA index increased to 1.6 but only small additional increases in T-antigen expression were seen. No consistent or critical role for t-antigen in development of the tumorigenic phenotype was found in this system. Key terms: Papovavirus transformation, Human diploid fibroblast transformation, Multistep neoplasia, Tumorigenicity, Simian virus 40, In vitro transformation, Immunofluorescence, Total cellular protein, Flow cytometry, Bivariate analysis
DNA binding, ATPase, helicase activities (12,50), and has a clastogenic effect on human cells (42). T-antigen binds to at least two cellular proteins (13,31,35) and plays a critical role in transformation (1,7,36,40). However, expression of T-antigen is insufficient for expression of a number of transformation markers (44), and for HSF4-Tl2 (20) and similar systems (49), immortalization is a rare event. Induction of the tumorigenic phenotype in human cells following SV40 infection has
'This work was supported in part by the Veteran!; Administration (Medical Research Service), NIH Biomedical Research Support Grant No. RR05370, and the Northwestern Memorial Foundation. 'Address reprint requests to: Dr. Charles L. Goolsby, VA Lakeside Medical Center, Room 711,333 East Huron Street, Chicago, IL 60611.
SV40 T-ANTIGEN, t-ANTIGEN, AND P53 EXPRESSION
749
been rare (26). t-antigen is a small protein of molecular level of viral gene expression are correlated with either weight 17 Kd (51). Expression of t-antigen has been increasing expression of in vitro transformation markshown to enhance transformation of host cells by T- ers or with development of the tumorigenic phenotype antigen (61, to disrupt the actin cytoskeleton (21), and in this system and would be consistent with additional to be involved in tumor induction and progression (9); cellular changes being required for the neoplastic binding of t-antigen to two cellular proteins has been transformation of human fibroblasts by the early genes reported (41,46). But, despite what is known, T-anti- of SV40. gen’s role in the transformation process is still not MATERIALS AND METHODS clearly understood (50). Cells and Culture Methods The effects of T-antigen involve interaction with a number of cellular proteins. The best characterized of The details of the establishment and characterizathese are complexes with p53 and the Rb gene product tion of the cell line, HSF4-Tl2, and of tumors derived (13,31,35). Loss or mutation of p53 sequences have from HSF4-Tl2 (HSF4-Tl2 TU#s) are described elsebeen correlated with the later stages of colorectal can- where (20). The cell strain HSF4 was kindly provided cer (3,39,52). The p53, T-antigen, and myc proteins by Dr. David Chen, Genetics Group, Los Alamos Nahave been associated with immortalization (25,30). p tional Laboratory, Los Alamos, New Mexico. Cultures 53 protein is increased in a number of transformed cell were grown in a-minimum essential medium (a-MEM, lines (5,11,27,45) and appears to be involved in estab- GIBCO, Grand Island, NY) plus 10% fetal bovine selishment of the tumorigenic phenotype (15,531. How- rum (FBS, GIBCO, Grand Island, NY). SVT2 cells were ever, mutation of p53 is required for this transforming obtained from the American Type Tissue Culture Colactivity in vitro (22) and expression of wild type p53 lection (ATTC, Rockville, MD) and were grown in Dulcan suppress the transformed phenotype (18).Associa- becco’s modified essential medium (D-MEM, GIBCO, tion of p53 with the GOiGl transition (38) and in- Grand Island, NY) plus 10% FBS. 100 unitsiml penicreased levels of p53 with restimulation of serum cillin, 100 pgiml streptamycin, and 0.25 pgiml amphostarved cells (43) have been reported. However, ele- tericin (GIBCO, Grand Island, NY) were added to all vated expression of wild type p53 has been reported to media, and cultures were grown in a 5% CO, humidiblock mouse cell lines in GOiGl (341, and Baker et al. fied incubator. HSF4-Tl2 and HSF4-T12 TU# cultures (4) showed growth suppression of human colorectal car- were carried in this media with 250 pgiml active gencinoma cells by wild type p53. A recent report indicates tamicin (G418, GIBCO, Grand Island, NY). All culT-antigen induction or activation of a protein kinase tures were grown in plastic T-75 flasks (Corning Glass which phosphorylates p53 (48), and the p53iT complex Works, Corning, NY). has been shown to bind to DNA polymerase a (19). Cellular Fixation and Staining Thus, the role(s) that p53 and p53iT complexes play in the transformation seen following expression of the Staining of cell suspensions for T-antigen, t-antigen, early genes of SV40 is not clear. and p53 was by the method of Jacobberger et al. (24) Although Laffin et al. (29) examined changes in viral with slight modifications. Suspensions were stained protein expression and p53 following SV40 infection of with 1 pg of primary antibody in 1 ml PBS containing human diploid fibroblasts, the studies were restricted 0.1% serum albumin (PBA)/l% normal goat serum to pre-crisis and crisis cells. In this study, early viral (NGS). PAb 419 (Ab-1, Oncogene Science, Manhasset, protein and p53 expression were examined in HSF4- NY) was used for staining large T- and small t-antiT12 a t various stages of transformation. Pre- and post- gens, and PAb 1801 (Ab-2, Oncogene Science, Manhascrisis, long term in vitro culture during which progres- set, NY) for staining p53. Mouse IgG isotype control sive transformation occurred, and tumorigenic phases (Coulter Source, Marietta, GA) and a substitution of are included. In order to understand this multistep pro- PBA/1% NGS were used as negative controls. Secondcess, changes in viral gene expression must be ascer- ary antibody was 10 pg of FITC conjugated goat antitained. Flow cytometric analyses have been employed mouse IgG immunoglobulin (Coulter Source, Marietta, to correlate expression of T and p53 with total protein GA). For correlation of immunofluorescence measureand DNA content. Western analyses were used to con- ments with total DNA content, cells were counterfirm these results independently and to ascertain stained with 50 pgiml propidium iodide (Calbiochem, changes in t-antigen. Significant increases in T-anti- San Diego, CA). Alternatively, for correlation of immugen expression were found to accompany immortaliza- nofluorescence with total protein content, cells were tion. However, the progressive increase in expression resuspended in 0.1 M Tris buffer (pH = 8.3) containing of a number of vitro phenotypic markers of transfor- 0.6 mg/ml sulphorhodamine 101 (KODAK, Rochester, mation which occurred following immortalization was NY) (16). Cells grown on coverslips were fixed in 0.5% formalaccompanied by only small increases in T-antigen expression relative to total cellular protein. No selection dehyde (EM Grade, Polysciences, Inc., Warrington, PA) for either increased T-antigen or t-antigen expression followed by acetone for 3 min. Coverslips were stained was seen in the tumors derived from HSF4-Tl2 cells. with 150 pl of 1 pg/ml primary antibody in PBA/1% Thus, these data do not support that changes in the NGS. Secondary antibody was 10 pg/ml FITC conju-
GOOLSBY ET AL.
750
Table 1 Measurement of In Vitro Transformation Parameters and In Vivo Tumorigenicitya Cell straidline HSF4
PDS 30
HSF4-T12
66
Growthb 1% 0.1% 0.22 0.10
CF" 10% 47.0
1%
Inc.
Cytometry 12748-756 (1991)
Viral and Cellular Oncogene Expression During Progressive Malignant Transformation of SV40 Transformed Human Fibroblasts' Charles L. Goolsby? Marianne Steiner, and Jeffrey Nemeth Department of Pathology, Northwestern University Medical SchooliVA Lakeside Medical Center, Chicago, Illinois 60611 Received for publication December 28, 1990; accepted April 26, 1991
In vitro investigation of the multistep neoplastic progression which occurs during transformation of human cells has been hindered by resistance of human cells to both immortalization and tumorigenicity (Mut. Res. 199; 273, 1988). Previously our laboratory established a cell line, HSF4-Tl2, by transfection of normal human foreskin fibroblasts with the plasmid pSV3-neo which contains the early genes of simian virus 40 (SV40). A multistep progression in karyotypic alterations and transformed phenotype occurred resulting in a neoplastic cell line that was immortal, transformed, and tumorigenic. We have examined changes in the SV40 proteins, large T (T-antigen) and small t (t-antigen) antigens, and in the cellular protein, p53, during progressive transformation of these cells. Total viral protein expression relative to total cellular protein increased following im-
Elucidation of the sequential multistep process that occurs in establishment of human cancer is critical to a n understanding of the development of malignant disease. In vitro studies of multistep transformation may aid in gaining a n understanding of these processes (20,28). Our laboratory established a cell line, HSF4T12, by transfection of normal human fibroblasts with the early genes of simian virus 40 (SV40). This cell line underwent immortalization, a multistep progression in both transformed phenotype and altered karyotype, and formed progressive tumors when cells were injected into implanted Gelfoam sponges in nude mice (20). The early genes of SV40 comprise two proteins, large T antigen (T-antigen) and small t antigen (t-antigen) (51). T-antigen is a phosphorylated protein with a molecular weight of approximately 94 Kd. It exhibits
mortalization of HSF4-Tl2 as did the ratio of T-antigen to t-antigen. Interestingly, no significant change in DNA content accompanied immortalization. However, during the progressive in vitro transformation of HSF4-T12 which occurred primarily post-immortalization, DNA index increased to 1.6 but only small additional increases in T-antigen expression were seen. No consistent or critical role for t-antigen in development of the tumorigenic phenotype was found in this system. Key terms: Papovavirus transformation, Human diploid fibroblast transformation, Multistep neoplasia, Tumorigenicity, Simian virus 40, In vitro transformation, Immunofluorescence, Total cellular protein, Flow cytometry, Bivariate analysis
DNA binding, ATPase, helicase activities (12,50), and has a clastogenic effect on human cells (42). T-antigen binds to at least two cellular proteins (13,31,35) and plays a critical role in transformation (1,7,36,40). However, expression of T-antigen is insufficient for expression of a number of transformation markers (44), and for HSF4-Tl2 (20) and similar systems (49), immortalization is a rare event. Induction of the tumorigenic phenotype in human cells following SV40 infection has
'This work was supported in part by the Veteran!; Administration (Medical Research Service), NIH Biomedical Research Support Grant No. RR05370, and the Northwestern Memorial Foundation. 'Address reprint requests to: Dr. Charles L. Goolsby, VA Lakeside Medical Center, Room 711,333 East Huron Street, Chicago, IL 60611.
SV40 T-ANTIGEN, t-ANTIGEN, AND P53 EXPRESSION
749
been rare (26). t-antigen is a small protein of molecular level of viral gene expression are correlated with either weight 17 Kd (51). Expression of t-antigen has been increasing expression of in vitro transformation markshown to enhance transformation of host cells by T- ers or with development of the tumorigenic phenotype antigen (61, to disrupt the actin cytoskeleton (21), and in this system and would be consistent with additional to be involved in tumor induction and progression (9); cellular changes being required for the neoplastic binding of t-antigen to two cellular proteins has been transformation of human fibroblasts by the early genes reported (41,46). But, despite what is known, T-anti- of SV40. gen’s role in the transformation process is still not MATERIALS AND METHODS clearly understood (50). Cells and Culture Methods The effects of T-antigen involve interaction with a number of cellular proteins. The best characterized of The details of the establishment and characterizathese are complexes with p53 and the Rb gene product tion of the cell line, HSF4-Tl2, and of tumors derived (13,31,35). Loss or mutation of p53 sequences have from HSF4-Tl2 (HSF4-Tl2 TU#s) are described elsebeen correlated with the later stages of colorectal can- where (20). The cell strain HSF4 was kindly provided cer (3,39,52). The p53, T-antigen, and myc proteins by Dr. David Chen, Genetics Group, Los Alamos Nahave been associated with immortalization (25,30). p tional Laboratory, Los Alamos, New Mexico. Cultures 53 protein is increased in a number of transformed cell were grown in a-minimum essential medium (a-MEM, lines (5,11,27,45) and appears to be involved in estab- GIBCO, Grand Island, NY) plus 10% fetal bovine selishment of the tumorigenic phenotype (15,531. How- rum (FBS, GIBCO, Grand Island, NY). SVT2 cells were ever, mutation of p53 is required for this transforming obtained from the American Type Tissue Culture Colactivity in vitro (22) and expression of wild type p53 lection (ATTC, Rockville, MD) and were grown in Dulcan suppress the transformed phenotype (18).Associa- becco’s modified essential medium (D-MEM, GIBCO, tion of p53 with the GOiGl transition (38) and in- Grand Island, NY) plus 10% FBS. 100 unitsiml penicreased levels of p53 with restimulation of serum cillin, 100 pgiml streptamycin, and 0.25 pgiml amphostarved cells (43) have been reported. However, ele- tericin (GIBCO, Grand Island, NY) were added to all vated expression of wild type p53 has been reported to media, and cultures were grown in a 5% CO, humidiblock mouse cell lines in GOiGl (341, and Baker et al. fied incubator. HSF4-Tl2 and HSF4-T12 TU# cultures (4) showed growth suppression of human colorectal car- were carried in this media with 250 pgiml active gencinoma cells by wild type p53. A recent report indicates tamicin (G418, GIBCO, Grand Island, NY). All culT-antigen induction or activation of a protein kinase tures were grown in plastic T-75 flasks (Corning Glass which phosphorylates p53 (48), and the p53iT complex Works, Corning, NY). has been shown to bind to DNA polymerase a (19). Cellular Fixation and Staining Thus, the role(s) that p53 and p53iT complexes play in the transformation seen following expression of the Staining of cell suspensions for T-antigen, t-antigen, early genes of SV40 is not clear. and p53 was by the method of Jacobberger et al. (24) Although Laffin et al. (29) examined changes in viral with slight modifications. Suspensions were stained protein expression and p53 following SV40 infection of with 1 pg of primary antibody in 1 ml PBS containing human diploid fibroblasts, the studies were restricted 0.1% serum albumin (PBA)/l% normal goat serum to pre-crisis and crisis cells. In this study, early viral (NGS). PAb 419 (Ab-1, Oncogene Science, Manhasset, protein and p53 expression were examined in HSF4- NY) was used for staining large T- and small t-antiT12 a t various stages of transformation. Pre- and post- gens, and PAb 1801 (Ab-2, Oncogene Science, Manhascrisis, long term in vitro culture during which progres- set, NY) for staining p53. Mouse IgG isotype control sive transformation occurred, and tumorigenic phases (Coulter Source, Marietta, GA) and a substitution of are included. In order to understand this multistep pro- PBA/1% NGS were used as negative controls. Secondcess, changes in viral gene expression must be ascer- ary antibody was 10 pg of FITC conjugated goat antitained. Flow cytometric analyses have been employed mouse IgG immunoglobulin (Coulter Source, Marietta, to correlate expression of T and p53 with total protein GA). For correlation of immunofluorescence measureand DNA content. Western analyses were used to con- ments with total DNA content, cells were counterfirm these results independently and to ascertain stained with 50 pgiml propidium iodide (Calbiochem, changes in t-antigen. Significant increases in T-anti- San Diego, CA). Alternatively, for correlation of immugen expression were found to accompany immortaliza- nofluorescence with total protein content, cells were tion. However, the progressive increase in expression resuspended in 0.1 M Tris buffer (pH = 8.3) containing of a number of vitro phenotypic markers of transfor- 0.6 mg/ml sulphorhodamine 101 (KODAK, Rochester, mation which occurred following immortalization was NY) (16). Cells grown on coverslips were fixed in 0.5% formalaccompanied by only small increases in T-antigen expression relative to total cellular protein. No selection dehyde (EM Grade, Polysciences, Inc., Warrington, PA) for either increased T-antigen or t-antigen expression followed by acetone for 3 min. Coverslips were stained was seen in the tumors derived from HSF4-Tl2 cells. with 150 pl of 1 pg/ml primary antibody in PBA/1% Thus, these data do not support that changes in the NGS. Secondary antibody was 10 pg/ml FITC conju-
GOOLSBY ET AL.
750
Table 1 Measurement of In Vitro Transformation Parameters and In Vivo Tumorigenicitya Cell straidline HSF4
PDS 30
HSF4-T12
66
Growthb 1% 0.1% 0.22 0.10
CF" 10% 47.0
1%
