Shi-Qi Wu

In vitro radiation-induced neoplastic progression of low-grade uroepithelial tumors

Recent interest has focused on the identification of molecular genetic mechanisms in multistep neoplastic transformation. In vitro exposure of simian virus 40 (SV40)-immortalized human uroepithelial cells (SV-HUC) that are environmentally relevant to bladder carcinogens has been shown to produce tumorigenic transformation, as assessed by the ability of cells exposed to a carcinogen to form xenograph tumors with heterogeneous cancer phenotypes ranging from very aggressive, invasive high-grade carcinomas to superficial low-grade indolent tumors. In addition, exposure of a low-grade indolent tumor generated in the SV-HUC system, MC-T11, to the same carcinogens results in neoplastic progression as assessed by the production of high-grade aggressive cancers. In the present study, we show neoplastic progression of MC-T11 after in vitro exposure to a single dose of 6 Gy X rays. In addition, we show that the chromosome deletions, including losses of 4q, 11p, 13q and 18, observed in these radiation-induced tumors are similar to those observed in carcinogen-induced tumors, thus supporting the hypothesis that the experimental cell system, not the transforming agent, dictates the genetic losses required for tumorigenic transformation and progression.

Nonrandom chromosome losses in tumorigenic revertants of hybrids between isogeneic immortal and neoplastic human uroepithelial cells

Somatic cell hybrid analysis was used to examine the role of recessive cancer genes in tumorigenic transformation in vitro of human uroepithelial cells (HUC). Hybrids between nontumorigenic pseudodiploid SV40-immortalized HUC (SV-HUC) and two aggressive grade III transitional cell carcinomas (TCC) produced in nude mice after in vitro exposure of SV-HUC to 3-methylcholanthrene (MC) were completely suppressed for tumorigenicity at early passage. Tumorigenic reversion occurred after five or more passages in culture and was always accompanied by chromosome losses. Overall, the tumorigenic revertants showed statistically significant losses of chromosomes 1, 4, 5, 9q, 12, 14q, and 17 (all P≤0.05) as compared to losses in suppressed hybrids. In addition, hybrid reversion was accompanied by losses that left specific tumors with a single remaining homolog of certain chromosomes (i.e., 3, 5q, 11p, 17p, and 18q). These losses were also considered significant because of the likelihood that genes on these chromosomes were reduced to homozygosity. Many of the significant losses (i.e., 5q, 9q, 11p, and 17p) were of chromosomes that are frequently lost in clinical TCC. Thus, these results support the hypothesis that these chromosomes contain genes whose loss leads to HUC tumorigenesis.

Marker chromosome stability associated with neoplastic transformation of human uroepithelial cells.

Chromosome studies were performed on three independently derived tumor cell lines established from carcinomas induced in nude mice after innoculation of SV40 immortalized human uroepithelial cells that had been treated with methylcholanthrene. Tumor 1 was an undifferentiated carcinoma, while tumors 7 and 9 were both squamous carcinomas. After six to eight passages in vitro the tumor cells were each reinoculated into other nude mice to yield secondary tumors (1.1 and 7.1). Chromosome studies on both primary and secondary tumors demonstrated the same distinctive chromosome markers. Tumors 1 and 1.1 shared the same histopathology in addition to the same modal chromosome number and identical chromosomal duplications and deficiencies; the same was true of tumors 7 and 7.1. Tumor 9, which did not yield a secondary tumor, nevertheless showed the same chromosome pattern in different passages. The stability of the characteristic marker chromosomes in the three tumor cell lines distinguishes these malignant lines from the nonmalignant SV40 transformed parent line from which the three tumors derived because the parent line was characterized by extreme marker instability. This suggests that the stable marker chromosomes that characterize the tumor cell lines may be critical for their tumorigenicity, and that evolution of an adaptive neoplastic genome may select for cytogenetic stability as long as there are no new selective pressures.

Chromosome losses in tumorigenic revertants of EJ/ras-expressing somatic cell hybrids

Tumorigenic transformation of SV40-immortalized human uroepithelial cells (SV-HUC) after transfection with EJ/ras was previously reported to be a rare event. To test the hypothesis that ras transformation requires loss of suppressor genes, somatic cell hybrids were generated between a rare tumorigenic transformant and an isogeneic nontumorigenic EJ/ras transfectant obtained in the same experiment. Both parental cell lines, as well as all hybrid progeny, expressed mutant p21 ras protein, but injections of three such independent hybrids into athymic nude mice at passage (P) 4 demonstrated that tumorigenicity was suppressed at 20 of 22 sites. Two tumors developed, after a relatively long 17-week latent period, as compared with a 4-week latent period for the tumorigenic parent. All three hybrids produced tumors at P8, but these showed different latent periods (3-14 weeks). Revertant hybrid tumors were high-grade carcinomas. Cell lines derived from these tumors expressed mutant p21 ras and retained at least 1 EJ/ras integration site. Karyotypic analysis of six independent hybrid tumor revertants showed that each had a unique clonal karyotype. Losses of two or more homologues of 1p, 3p, 4, 8, 10p, 11p, 13q, and 18 were identified in one or more tumorigenic revertants. Losses of all these chromosomes were previously associated with transformation of SV-HUC by EJ/ras, but were also associated with chemical transformation of SV-HUC in tumors that did not express mutant ras. Genetic losses involving most of these chromosomes have also been identified in clinical bladder cancers (i.e., 1p, 3p, 8, 11p, 13 and 18q). These data show that expression of EJ/ras does not negate or significantly alter requirements for multiple genetic losses in HUC tumorigenesis.

Transformation in vitro of Human Uroepithelial Cells

Normal human uroepithelial cells can now be routinely cultured in vitro, immortalized by SV40 T antigen oncoprotein gene, and tumorigenically transformed after exposure to oncogenic agents including the human bladder carcinogen, 4-aminobiphenyl (ABP) and its metabolites and mutant EJ/ras to carcinoma phenotypes that resemble human bladder cancers. Neoplastic transformation of HUC in vitro is accompanied by chromosome changes that recapitulate many cytogenetic changes reported in clinical bladder cancers.