Catherine A. Reznikoff

20q gain associates with immortalization : 20q13.2 amplification correlates with genome instability in human papillomavirus 16 E7 transformed human uroepithelial cells

Breast, bladder, colon, and ovarian carcinomas show frequent low level 20q gain and less frequently high level 20q13.2 amplification, but the significance of these 20q amplifications in transformation has not been defined. Using karyotypic and comparative genomic hybridization (CGH) analyses, chromosome losses and gains were analysed in six newly immortalized human uroepithelial cell (HUC) lines transformed by Human Papillomavirus 16 (HPV16) E7. Results showed clonal chromosomes with 20q11->qter gain in all six lines. CGH revealed a peak of 20q13.2 amplification in two cell lines. FISH with whole chromosome 20 paint showed expanded chromosome regions (ECRs) and double minute chromosomes (DMs) that contained chromosome 20 material in cell lines with 20q13.2 amplification. FISH with probes from the center of the 20q13.2 human breast cancer amplicon showed as many as 24 signals in cells with 20q13.2 amplification. The acquisition of genome instability in these E7-HUCs did not correlate with TP53 mutation, as all E7-HUCs contained only wildtype TP53. These results suggest that low level 20q gain is associated with overcoming cellular senescence in E7 transformed cells (P-value=2×10−7), but does not confer genome instability, while high level 20q13.2 amplification is associated with chromosome instability. Loss of 10p (P-value=3×10−5) was also important in immortalization of E7-transformed HUCs. Thus, these results have profound implications for interpreting the significance of high versus low level 20q gains in human cancers.

GROWTH AND CHARACTERIZATION OF NORMAL HUMAN UROTHELIUM IN VITRO

SummaryA method for initiating rapidly growing cultures of normal human transitional cells from ureter and embryonic bladder specimens has been developed and quantified. A new microdissection technique was used to nonenzymatically separate the urothelium. The use of enriched medium containing 10 μg/ml insulin, 5 μg/ml transferrin, and 1 μg/ml hydrocortisone resulted in improved growth. The use of thin collagen gel substrates (0.6 ml/60 mm petri dish) resulted in 97% attachment of explants compared to 77% attachment on plastic. Explants grown on thicker collagen (2 ml/60 mm petri dish) showed, in addition to better attachment, enhanced growth of cells as determined both by measurements of colony size and cell density. Cultures of transitional cells that were initiated using explants could be passed three to five times using 0.1% EDTA for dispersion. Autoradiography of [3H]thymidine-labeled cells showed an initial phase of rapid cell division in primary explant cultures and restimulation of cell division in passaged cultures. Transmission electron microscopy showed that the cells growing out from the explants were continuous with the stratified urothelium maintained in the original explant. Stratification of transitional cells occurred in cultures of both ureter and embryonic bladder cells. Surface cells were joined near their apices by junctional complexes. Desmosomes and Golgi vesicles were present in all cells. Passage in culture did not alter the morphological characteristics of cells.

Replicative Senescence in Human Uroepithelial Cells

PURPOSE Normal human uroepithelial cells (HUCs) proliferate rapidly in culture during early passage and then spontaneously undergo replicative senescence. We previously reported that the cyclin D1-CDK4/6 inhibitor, p16INK4a, is elevated at senescence in HUCs. Hence, we proposed that p16INK4a may play a critical role in mediating senescence in this cell type. In the current study, we further characterized the senescent state in HUCs. We also tested the possible roles of changes in other cell cycle proteins, including p53, p21WAF1, pRb, and cyclin D1 in HUC senescence. METHODS Normal HUCs cultured from explants of ureteral mucosa were used for these studies. Senescence associated-beta-galactosidase activity (SA-beta-gal) was used to identify cells in senescence. Flow cytometric analysis was used to determine changes in cell cycle distribution at senescence. Response of cells to serum stimulation was determined by Northern analysis of c-fos. Western analysis was used to assess changes in p53, p21WAF, p16INK4a, cyclin D1 and plasminogen activator inhibitor-1 (PAI-1) levels at senescence. RESULTS beta-gal-positive HUCs were blocked at G1/S in senescence and failed to show c-fos induction in response to serum stimulation. As previously reported, senescent HUCs also showed elevated p16INK4a. However, unlike human fibroblasts, neither p53 nor p21WAF1 elevation accompanied HUCs senescence. PAI-1 levels were also not elevated in HUC senescence. CONCLUSION These findings support a model in which elevation of p16INK4a, but not p53 or p21WAF1 plays a critical role in HUC replicative senescence. These findings elucidate the tumor suppressor mechanism of p16INK4a and the frequent loss of either p16INK4a or pRb in invasive human bladder tumors.

Antiproliferative activities of interferons against human bladder carcinoma cell lines in vitro

The antiproliferative effect of interferons against 5 human bladder carcinoma cell lines, RT112, T24, RT4, 647V and HT1197, was determined in vitro. Each of these human bladder carcinoma cell lines except 647V was sensitive to human interferons in liquid media. The antiproliferative effect of interferons was observed only upon continuous exposure, not after 1 hour. Partially purified, naturally produced interferon beta was more inhibitory of cell growth than naturally produced interferon alpha. Interferon alpha 54, 76, 61, 6L and 1 purified to homogeneity were as effective as naturally produced, partially pure interferon alpha. Although interferon beta, produced by recombinant DNA technology and purified to homogeneity, was not equivalent in effectiveness to naturally produced interferon beta, its antiproliferative activity was greater than interferon alpha 54 for 3 of 4 cell lines tested. Antimitotic effects may underlie, at least in part, the potential therapeutic activity of interferons for bladder carcinoma.

Endogenous oncornaviruses in chemically induced transformation. I. Transformation independent of virus production.

Abstract The C3H/10T1/2 cell line, established from mouse embryo fibroblasts, is highly sensitive to postconfluence inhibition of division and is susceptible to malignant transformation by chemical carcinogens in culture. Normal, chemically transformed and spontaneously transformed clones of C3H/10T1/2 cells were examined for the production of infectious murine leukemia virus (MuLV) and MuLV and mouse mammary tumor virus (MTV) antigens. Criteria for the expression of oncornaviruses included the production of: (1) MuLV and MTV gs-antigens, (2) MuLV GL and GT cell surface antigens, (3) DNA polymerase-containing particles, and (4) infectious virions. Normal C3H/10T1/2 cells were free of oncornaviruses by all four of these criteria. Transformation of these cells to malignancy by chemical carcinogens did not result in an increased production of MuLV or MTV products; hence, these cells demonstrated the same virus-free phenotype as the parental cell line. Attempts to rescue transforming viral information from transformed C3H cell lines by superinfection with leukemia viruses were unsuccessful. Repressed endogenous MuLV (but not MTV) could be induced from all cell lines by treatment with 5-iododeoxyuridine (IUdR). Induction of MuLV in normal cells occurred in three phases: I, an initial transient production of low-titered virus; II, a 3–4-week interval where virus production fell to a lower level and then gradually increased. This was accompanied by the recruitment of virus-free cells in the culture to produce MuLV gs-antigens and DNA polymerase-containing particles; and III, the constitutive production of high-titered virus. Transformed cells were more sensitive to treatment with IUdR than normal C3H/10T1/2 cells. These cells underwent a more rapid induction of MuLV, contained a larger fraction of cells that expressed MuLV functions, continued the production of MuLV throughout the second phase of induction, and had an earlier onset of the third phase of high-titered MuLV production. Oncornaviruses induced from IUdR-treated cells showed diversity in their host range and in their ability to produce XC plaques; none of these viruses, however, were capable of transforming normal C3H/10T1/2 fibroblasts in culture.

Dominant genetic alterations in immortalization: Role for 20q gain

Gain of 20q has been observed in many cancer types, including bladder cancers. However, the biological significance of low‐copy‐number 20q gain in human cancer pathogenesis has not yet been defined. We reported that immortalization of human uroepithelial cells (HUC) transformed with human papillomavirus 16 (HPV 16) E7 is associated with single‐copy 20q gain (P = 2 × 10‐7). We also observed 20q13.2 amplification in some cell lines, but only after 20 passages. Thus, we hypothesized that low‐copy gain of 20q gene(s) contributes in a dominant way to bypassing HUC senescence. To test this hypothesis, we fused precrisis E7‐transformed HUCs (pcE7s) with three independent immortal E7‐HUCs that acquired a single‐copy 20q gain at immortalization. In one of these lines, a single‐copy gain of 20q and a 10p12.1–pter loss were the only cytogenetic alterations. Immortal cell hybrids were obtained with all three crosses. Southern analysis for unique HPV16 insertion sites, as well as fluorescence in situ hybridization (FISH) with whole chromosome 20 painting probes (WCP20) for marker chromosomes in the immortal clones, confirmed the hybrid and independent nature of representative immortal clones. In contrast, when we used the same protocol, no immortal somatic cell hybrids were obtained when HPV16 E6 immortal HUC (E6‐HUC) that showed 3p and 9p losses, but no 20q gain, were fused with precrisis E6‐transformed HUC (pcE6s). This latter observation is consistent with many results demonstrating that recessive changes are required for cell immortalization. Therefore, the new results reported herein for the first time demonstrate that dominant changes can contribute to bypassing senescence, and that such genes may be located on 20q. Genes Chromosomes Cancer 26:304–311, 1999.

On the statistical analysis of allelic-loss data

This paper concerns the statistical analysis of certain binary data arising in molecular studies of cancer. In allelic-loss experiments, tumour cell genomes are analysed at informative molecular marker loci to identify deleted chromosomal regions. The resulting binary data are used to infer properties of putative suppressor genes, genes involved in normal cell cycling. Various factors can complicate this inference, including background loss of heterozygosity, spatial (that is, within chromosome) dependence of the binary responses, non-informativeness of markers, covariates such as protein levels or tumour histology, heterogeneity of cells within tumours, and measurement error. We focus on the first three factors, discussing methods for statistical inference that separate background loss from significant loss. We outline the extension to other inferences, such as comparison questions and the relationship to covariates. Using characteristic features of tumourigenesis, we present a framework for the stochastic modelling of allelic-loss data, and build models within this framework; in particular, we propose a simple model that has chromosome breaks at locations of a Poisson process, and preferential selection cells with inactivated suppressor genes. We illustrate these methods on allelic-loss data from induced rat mammary tumours and human bladder cancers.

Quantitative changes in cytoskeletal and nuclear actins during cellular transformation

Actin, a highly conserved protein comprising cell stress fibers and other cellular structures, is found in both the cytoplasm and nucleus of cells and responds to both epigenetic signals and altered gene expression occurring during tumorigenesis. We have previously shown that changes in the cytoplasmic F‐ and G‐actin ratios reflect bladder cancer risk. To determine whether nuclear actin is also altered and how nuclear and cytoplasmic actin alterations are interrelated in transformation, an in vitro model of carcinogen‐induced transformation consisting of 2 human uroepithelial cell lines immortalized by infection with SV‐40 was studied. One line, HUC‐PC, is tumorigenic in nude mice after incubation with the carcinogen 4‐ABP, the other, HUC‐BC, is not. Cytoplasmic and nuclear F‐ and G‐actin were determined by QFIA on individual cells using fluorochrome‐labeled phallicidin and DNase, I, respectively. Before exposure to 4‐ABP, the PC cells had lower cytoplasmic F‐actin content, higher cytoplasmic G‐actin content, but similar levels of nuclear G‐ and F‐actin in comparison to the BC cells. After incubation with 4‐ABP, F‐actin decreased and G‐actin increased in both cytoplasm and nuclei of PC cells and cytoplasmic F‐actin fibers were lost, but only cytoplasmic actin was altered in the BC cells. Northern blot analysis showed the expression of the β‐actin gene was only approximately 20% lower in 4‐ABP‐treated PC cells than in untreated controls, indicating the cellular change in actin was attributed to a shift between F‐and G‐actin proteins rather than to net actin synthesis. Int. J. Cancer, 70:423–429, 1997.

Serial cultivation of normal rat bladder epithelial cells in vitro.

Recent advances in culture techniques for human urothelial cells have led to the development of an improved method for growing primary rat bladder epithelial cells. We report here the conditions developed for large-scale in vitro growth and serial cultivation of normal diploid rat bladder epithelial cells. Primary cultures were initiated by attachment of bladder mucosal explants to type I collagen gels. A rapid outgrowth of epithelial cells from the explants occurred when cultured in a hormone-supplemented medium with epidermal growth factor. These primary outgrowths were passaged by nonenzymatic dispersion with 0.1 per cent ethylenediaminetetracetic acid and replating onto new gels. The capacity for routine serial passaging and maintenance of rat bladder epithelial cells required the presence of epidermal growth factor, a requirement not observed with human urothelial cells. The characteristics of the cultured rat bladder epithelial cells were similar to human urothelial cells in: ultrastructural and phase-contrast morphologic properties, showing junctional complexes, desmosomes, stratification and an apical glycocalyx; the absence of stromal cell contamination; and the ability to be serially passaged. Spontaneous cell-line formation was observed with the rat bladder epithelial cells, but has not been found with the human urothelial cells. With the method that we have developed, the number of rat bladder epithelial cells generated from a single bladder of a 4 to 6 week old rat was increased 100-fold from about 7 X 10(5) cells to 7 X 10(7) viable cells within 3 weeks of culture. The capability of culturing normal, primary rat bladder epithelial cells on this scale has not been reported previously and will facilitate comparative studies of the biological and molecular characteristics of the mammalian urothelium. Furthermore, this culture system will be useful for carcinogenesis studies, including metabolic activation of carcinogens and cellular transformation in vitro.

Minimal deletion of 3p13-->14.2 associated with immortalization of human uroepithelial cells.

Immortalization and tumorigenic transformation of many human cell types, including human uroepithelial cells (HUCs), are frequently associated with loss of genetic material from the short arm of chromosome 3 (3p). In addition, losses of 3p have been observed in many human cancers including renal cell carcinoma, lung cancer, breast cancer, and bladder cancer. Genetic studies suggest that there are at least two regions on 3p in which tumor suppressor genes might be located, but the precise location of these genes is not known. We studied chromosome 3 losses that were specifically associated with immortalization of five independent human papilloma virus 16 (HPV16) E6‐ or E7‐transformed HUCs. Cytogenetic analysis showed that the smallest common region of deletion was 3p14.1→14.2. Fluorescence in situ hybridization using a 3p13→14‐specific yeast artificial chromosome (YAC) contig showed the precise localization of the breakpoints to be in 3p13 and 3p14.2, thus defining the smallest common overlap of 3p deletions in HPV16 E6‐ or E7‐immortalized HUCs. These results suggest the presence in this region of genes involved in the control of senescence in vitro and possibly tumorigenesis in vivo. Genes Chromosomes Cancer 21:39–48, 1998.