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The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC-E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-α and the TGF-βs, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma. [ABSTRACT FROM PUBLISHER]

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.

Morphologic transformation and tumorigenicity are separate cellular phenotypes in transformed 10T1/2 cells. We have investigated the levels of expression of genes for c-myc, c-H-ras, c-K-ras, c-N-ras, TGF beta and Rb in 42 morphologically transformed 10T1/2 cell lines, in an attempt to define the molecular mechanisms governing morphologic transformation and tumorigenicity in the 10T1/2 cell system. The 10T1/2 cell lines investigated generally overexpressed mRNAs for c-myc, c-H-ras, and TGF beta relative to the levels expressed by wild-type 10T1/2 cells (levels of expression > 1.5-fold that of wild-type 10T1/2 cells). In contrast, only half of these cell lines overexpressed mRNAs for c-N-ras and/or Rb relative to wild-type 10T1/2 cells, and only 25% overexpressed c-K-ras mRNA. The mean levels of mRNA expression for each of c-K-ras, c-N-ras and TGF beta genes in tumorigenic cell lines were significantly greater than the mean levels of expression in non-tumorigenic cell lines, suggesting an association between tumorigenicity and the levels of expression of these specific genes. In contrast, levels of expression for c-myc, c-H-ras and Rb genes were not correlated with tumorigenicity. Cell lines that coexpressed high levels of c-K-ras, c-N-ras and TGF beta genes were likely to be tumorigenic (11/12 cell lines were tumorigenic), whereas cell lines that coexpressed low levels of these genes were unlikely to be tumorigenic (1/10 cell lines were tumorigenic). High expression of TGF beta was sufficient for tumorigenicity in the absence of high levels of expression of c-K-ras and c-N-ras (5/5 cell lines were tumorigenic). Elevated expression of either c-K-ras or c-N-ras alone was insufficient for tumorigenicity, however, coordinate overexpression of both c-K-ras and c-N-ras was associated with tumorigenicity irrespective of the expression status for TGF beta (13/15 cell lines were tumorigenic). These results suggest that overexpression of c-myc, c-H-ras and TGF beta are commonly associated with, and possibly mechanistically related to, the process of morphologic transformation in 10T1/2 cells. In addition, these results suggest that progression from morphologic transformation to tumorigenicity in 10T1/2 cell lines is frequently accompanied by overexpression of c-K-ras and c-N-ras, and by enhancement of the level of overexpression of TGF beta.

Clonal lines of transformed rat liver epithelial cells, derived from a single population of cloned diploid rat liver epithelial (stem-like) cell line (WB-F344) by exposure in vitro to N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), produce hepatocellular carcinomas, hepatoblastomas and adenocarcinomas in syngeneic rats (Tsao and Grisham, Am. J. Pathol., 127, 168-181, 1987). In this study we show that these clonal lines demonstrate near-diploid (GN clones) or near-triploid (GP clones) aneuploidy and the universal occurrence of non-random chromosomal abnormalities. Marker chromosomes that involved four autosomes--a non-reciprocal translocation involving chromosomes 1 and 7 (t1q43;7q34), and addition of DNA of unknown origin to the pericentromeric regions of chromosomes 4 and 10--occurred in all of the cells of all transformed clones and in the cells of tumors that grew from them. New marker chromosomes involving the same regions of chromosomes 4 and 7 were found in several cell lines established from independent tumors. The preservation of marker chromosomes in tumor cells in the face of random loss and gain of other chromosomes suggests that these non-random aberrations were necessary for tumor formation. The presence of marker chromosomes was associated with increased expression of the c-myc gene (located at q34 on chromosome 7), the c-H-ras gene (located at q41-43 on chromosome 1) and the c-K-ras and TGF alpha genes (both located at unknown sites on chromosome 4), which we have previously shown to be highly correlated with tumorigenicity in these same transformed clonal lines (Lee et al., Cancer Res., 51, 5238-5244, 1992).

Wildtype 10T1/2 cells and 42 clonally derived morphologically transformed 10T1/2 cell lines were evaluated for expression of p53 mRNA and protein. Wildtype 10T1/2 cells expressed detectable levels of p53 mRNA and multiple immunologically distinct forms of the p53 protein, including both PAb240- and PAb246-reactive conformations of the protein. However, DNA sequence analysis revealed no mutations within the coding sequence of the expressed p53 gene(s) in wildtype 10T1/2 cells. Morphologic transformation of 10T1/2 cells resulted in clonal cell lines that overexpressed p53 mRNA and total p53 protein (PAb421-reactive) relative to the level of expression in wildtype 10T1/2 cells. Immunoprecipitation analysis demonstrated the expression of both PAb246-reactive and PAb240-reactive conformations of the p53 protein in all morphologically transformed 10T1/2 cell lines evaluated, irrespective of the tumorigenic potential of the individual line. Nonetheless, expression of PAb240-reactive p53 protein at levels equal to or higher than that found in wildtype 10T1/2 cells was associated with tumorigenicity in morphologically transformed 10T1/2 cell lines, suggesting that the relative contribution of PAb240-reactive p53 protein to the total cellular p53 protein represents an important determinant for the expression of the tumorigenic phenotype. Our results demonstrate that altered expression of p53 represents a common feature of morphologically transformed 10T1/2 cell line, suggesting that alteration of the expression of the p53 tumor suppressor gene may represent an early and necessary step in the morphologic transformation of 10T1/2 cells. Additionally, overexpression of PAb240-reactive p53 protein may influence the tumorigenic potential of morphologically transformed 10T1/2 cell lines.

Hepatocyte proliferation and damage to DNA were characterized during the initiation phase of carcinogenesis in livers of rats that had received a single administration of the methylating agent methyl(acetoxymethyl)nitrosamine (DMN-OAc). Quiescent non-proliferating hepatocytes in intact livers did not appear to be susceptible to initiation by DMN-OAc, whereas proliferating hepatocytes in the S phase appeared to have greatest risk. To characterize the phenomenology of S-phase-dependent initiation further, the fractions of hepatocytes in the S and M phases of the cell cycle were enumerated at various times after treatment with DMN-OAc. Hepatocytes treated when in G1 experienced a delay of up to 20 h in the onset of S phase and a reduced rate of entry into the S and M cycle phases. Hepatocytes treated when in S phase experienced considerable delay in progression to mitosis due to part to inhibition of DNA replication. Hepatocytes treated when in late S/G2 also demonstrated a delay in progression into mitosis. The levels of 7-methylguanine and O6-methyldeoxyguanosine were quantified in the nuclear DNA of proliferating hepatocytes. The kinetics of removal of these lesions appeared to be first-order (half-life = 24 h). Hepatocyte risk of initiation was modeled by a function which summed over time the product of the fraction of hepatocytes in the S phase and the fraction of residual, unrepaired damage to DNA. For hepatocytes treated when in early G1, the time-weighted frequency of premutagenic DNA damage that was present during DNA replication was estimated to be less than half of that for hepatocytes treated when in early S. The results suggest that cell-cycle-dependent variation in sensitivity to initiation of hepatocarcinogenesis may be, in part, due to efficient removal of potentially carcinogenic lesions from DNA during an extended G1. The apparent high sensitivity of hepatocytes in late S/G2 suggests the contribution of additional factors.

Mouse lung adenoma were induced in adult female BALB/c mice by chronic i.p. urethane injection. Sialoadenectomy of the mice prior to carcinogen treatment did not alter the frequency or size of lung adenoma. However, sialoadenectomized mice exhibited a decrease in the proportion (18 versus 52%) of tumours which exhibited a low nuclear to cytoplasmic ratio and which grew along alveolar septa. Sialoadenectomy was also related to a complementary increase in the proportion (82 versus 48%) of tumours with large vesicular nuclei, prominent nucleoli and generally increased phenotypic features of malignancy. Replacement of epidermal growth factor (EGF) in sialoadenectomized mice restored the tumour-type ratio observed in non-sialoadenectomized mice. These data are discussed with respect to the possible roles of EGF in mouse lung alveologenic carcinoma formation and the cell type of origin for the tumours observed.