Chinghai Kao

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.

Establishment and characterization of a SV40 T-antigen immortalized human bronchial epithelial cell line

Dear Editor: Events surrounding the transformation of normal human epithelial cells to the frankly malignant phenotype are being studied by numerous investigators. These studies are exemplified by reports describing the genetic events associated with the neoplastic development of colorectal carcinoma (3). The study of the transformation of normal human colonic epithelium to carcinoma has been facilitated by the availability of preneoplastic tissue in the form of adenomatous disease, which ranges from the hyperplastic state to premalignant. These tissues are readily available because of their ease of identification through colonoscopy, and the low morbidity related to that procedure. Studies of colonic tissue ranging from early adenomas, intermediate adenomas, late adenoma, to frank carcinoma have identified both activation of oncogenes and mutational inactivation of tumor suppressor genes, with the latter predominating (3). Unlike colorectal carcinoma, preneoplastic tissue is not readily available from human lung tissue. Although squamous metaplasia is thought to precede the development of lung cancer, this is a histological diagnosis, and not readily identifiable through a bronchoscope. Furthermore, bronchoscopy is associated with significant morbidity, and not done on a routine basis as a screening procedure. Thus, an in vitro model is necessary to identify the genetic changes associated with multistep bronchogenic carcinogenesis. We report the successful immortalization of normal human bronchial epithelial cells to an immortalized bronchial epithelial cell line by strontium chloride transfection of a plasmid carrying the replication origin defective large T antigen SV40 genome. Our objective was to establish an immortalized HBE cell line so as to have a culture system which would allow extended replication of HBE ceils, and thus facilitate the study of human lung tumor cell biology. A tracheobronchial specimen was obtained from a 20-year old female at the time of autopsy, and cultured as previously described (2,7). Briefly, the epithelial layer from human tracheobronchial specimens was separated from the underlying tissue with forceps, minced into approximately 1 mm 2 explants and plated (approximately 30 explants/100 mm dish) onto type I rat tail collagen-gelcoated dishes in approximately 1 ml of media. Ammonia-reconstituted collagen gels were pre-equilibrated in supplemented Hams F12 medium for at least 48 hours prior to use. The culture media consisted of Hams F12, supplemented with 2% FBS, insulin, transferrin, EGF, dextrose, hydrocortisone, and a mixture of nonessential amino acids. This supplemented medium, referred to as F 12 + 2 FBS, also contained Gentamycin and fungizone. Cultures were grown in humidified incubators at 37 ° C in an atmosphere of 5% CO2 and received media changes 2 to 3 times per week. Passage of explants was performed using repeated 4 minute exchanges of HEDH (Ca ++and Mg++-free HBSS with 0.02% EDTA, 8% dialyzed fetal bovine serum and 25 mM HEPES) at 37 o

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.