Metastatic sublines of an SV40 large T antigen immortalized human prostate epithelial cell line.

BACKGROUND: The available human prostate cancer cell lines that are metastatic in athymic nude mice all have complex, highly aneuploid karyotypes. Other prostatic cells immortalized by transforming genes of SV40 or HPV and converted to tumorigenicity by additional genetic manipulation are not reported to be metastatic. METHODS: Tumorigenic sublines of human prostate epithelial cells previously immortalized by transfection with the SV40T antigen gene were obtained by sequential passage in male athymic nude mice. These sublines were evaluated histopathologically for tumorigenicity and metastasis in athymic nude mice after subcutaneous, intraperitoneal, and intraprostatic injection. Each subline was characterized by standard (GTG-banding) cytogenetic and FISH analysis, and RNase protection assays for androgen receptor expression. RESULTS: Two sublines produced metastases in lungs and the diaphragm of most mice after either intraprostatic or intraperitoneal injection. The M2205 subline formed large local tumors after intraprostatic injection. Cytogenetic aberrations present in the metastatic sublines, but not in the tumorigenic, nonmetastatic lines or the parental P69SV40T line, included dup(11)(q14q22), der(16) t (16;19) (q24;q13.1), which resulted in the loss of the short arm and proximal long arm of chromosome 19 (19q13.1-->19pter), and loss of the Y chromosome. None of the sublines expressed the androgen receptor. CONCLUSIONS: These cytogenetically defined, SV40T-immortalized human prostate epithelial cell lines, with distinct biological behaviors in vivo, provide additional tools for the genetic analysis of the emergence of metastatic capacity.

Type-1 insulin-like growth factor receptor reexpression in the malignant phenotype of SV40-T-immortalized human prostate epithelial cells enhances apoptosis.

The authors have previously shown that the type 1 insulin-like growth factor receptor (IGF-1R) is decreased in the transformation from benign to malignant human prostate epithelial cells in vivo. Further, in a well-described human SV40-T immortalized human epithelial cell system beginning with the immortalized, but rarely tumorigenic P69SV40-T cell line, to the highly tumorigenic and metastatic M12 subline, there is a similar decrease in IGF-1R number from 2.0 x 10(4) receptors per cell to 1.1 x 10(3) receptors per cell. When the IGF-1R was reexpressed in the M12 subline using a retroviral expression vector, M12-LISN, to a receptor number similar to that of the P69SV40-T parental cell line, the authors demonstrated a marked decrease in colony formation in soft agar in the M12-LISN cells vs the M12 control cells (p < or = 0.01), and a decrease in vivo tumor growth and metastases when injected either subcutaneously or an intraprostatic location (p < or = 0.01). This decrease in tumor volume was not because of a decrease in proliferative capacity, but was associated with an increase in apoptosis in baseline cultures and in response to the apoptotic-inducing agents 6-hydroxyurea, retinoic acid, and transforming growth factor beta 1.

Reexpression of the type 1 insulin-like growth factor receptor inhibits the malignant phenotype of simian virus 40 T antigen immortalized human prostate epithelial cells.

Type 1 insulin-like growth factor receptor (IGF-1R) expression is decreased in prostate cancer compared to that in noncancerous prostate epithelium. We have demonstrated that as the simian virus 40 T antigen (SV40T) immortalized human prostate epithelial cell line, P69SV40T, undergoes transformation from a poorly tumorigenic to a malignant phenotype, the M12 subline, there is a significant decrease in IGF-1R expression. In the present study, we examine the effects of reexpression of the IGF-1R on the malignant phenotype of M12 cells. The IGF-1R was reexpressed in M12 cells using a retroviral vector containing a 7-kilobase coding sequence for the IGF-1R, LISN, to create several clones of the M12-LISN cell line. As a control, M12 cells were also infected with a retroviral vector (LNL6) without the 7-kilobase IGF-1R insert (M12-LNL6 clones). Functional assays were performed with two separate clones each of M12-LNL6 and M12-LISN cells. Each clone of M12-LISN cells regained the proliferative response to IGF that was lost in the transition from P69SV40T cells to M12 cells. In addition, M12-LISN clones had a significantly decreased growth rate compared to the M12-LNL6 cells when injected s.c. in athymic/nude mice (P < 0.001). Tumorigenicity, as assessed by anchorage-independent growth of colonies in soft agar, was also decreased by 75% in the M12-LISN clones compared to that in the M12-LNL6 control cells. These data demonstrate that reexpression of the IGF-1R in a malignant human prostate epithelial cell line results in decreased tumor growth and decreased anchorage-independent colony formation independent of an increased proliferative response to IGF. Reexpression of the IGF-1R may be associated with reacquisition of the regulation of cellular proliferative and differentiation functions mediated by the IGF-1R that are lost as prostate epithelial cells undergo conversion to a malignant phenotype.

Evaluation of cathepsin D and epidermal growth factor receptor in prostate carcinoma.

Differential reactivity for cathepsin D (cath-D) and epidermal growth factor receptor (EGFR) was compared in 102 archival cases of human primary prostatic carcinoma and nine prostate carcinoma metastases by immunohistochemical techniques using commercially available antibodies (Ciba-Corning, Triton Diagnostics Division, Alameda, CA). Western immunoblotting confirmed that the anti-cath-D and anti-EGFR antibodies recognized the appropriate-sized proteins in extracts of human prostatic carcinoma cell lines. For immunohistochemical analysis, the primary prostate carcinomas ranged from Gleason's combined scores of 2 to 9. High-grade prostatic intraepithelial neoplasia was coexistent in 79 of the cases. Immunohistochemical staining was scored by summing the intensity of staining (0 to 3+) weighted by the percentage of tumor staining at each intensity (H score, theoretical range 0 to 300). Heterogenous moderate to strong reactivity with anti-cath-D was detected in 96 of 102 cases of primary prostate carcinoma (94%), with a mean H score of 176.5. EGFR reactivity was much less common and less strong, with 41 of 102 primary prostate carcinomas staining (40%) at a mean H score intensity of 29.2. The immunohistochemical (H) scores of cath-D and EGFR reactivity both significantly correlated with the Gleason's combined score of the tumors. There was no significant correlation between the cath-D and EGFR scores. Ninety-nine percent of the examples of prostatic intraepithelial neoplasia were reactive with anti-cath-D, with no clear correlation between the intensity of staining of prostatic intraepithelial neoplasia and the adjacent carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumorigenicity of SV40 T antigen immortalized human prostate epithelial cells: association with decreased epidermal growth factor receptor (EGFR) expression.

Our primary objectives were to: 1) develop a system for the study of prostatic tumor evolution; and 2) examine the role of the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) pathway in prostate tumor progression. Adult human prostate epithelial cells previously immortalized by transfection with the SV40 T antigen gene (P69SV40T) produced tumors in only 2/18 mice with a 6 month latency period. Reinjection of cells recovered from these tumors after 1 or 2 cycles of growth in nude mice produced tumors in 2/4 and 2/3 mice with markedly decreased latent intervals of 12, 25, 25 and 25 days each. The chromosomal complement of each tumor was human, consistently pseudodiploid, and retained the Y chromosome. In both anchorage-independent and adherent cell growth assays, EGF stimulated proliferation by approximately 2-fold in both the parental P69SV40T line and the tumor sublines. The tumor sublines expressed less EGFR protein than the parental line, as assessed by Western immunoblotting and flow cytometric analysis. Immunoprecipitation revealed increased production of the 18 and 25 kDa TGF-alpha precursors parallel to decreases in detectable EGFR. The growth of both the parental P69SV40T line and the tumor sublines was inhibited by a neutralizing antibody to TGF-alpha under serum-free defined conditions. Inclusion of the TGF-alpha neutralizing antibody consistently inhibited the proliferation of the tumor sublines more than P69SV40T in both proliferation and [3H]thymidine incorporation assays. This finding suggests that the increased tumorigenicity and decreased latent interval observed among the human prostate tumor cells is partially due to activation of the TGF-alpha/EGFR autocrine network.