An expression cassette carrying 426 basepairs of the rat probasin (PB) gene promoter and 28 basepairs of 5'-untranslated region is sufficient to target the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically to the prostate in transgenic mice. The PB-CAT transgene was expressed in three of five (60%) independent lines of mice, and this expression, as reported previously for the endogenous rat gene, was male specific, restricted primarily to the lateral, dorsal, and ventral lobes of the prostate, with only very low levels of CAT activity detected in the anterior prostate and seminal vesicles. The developmental and hormonal regulation of the transgene also paralleled that reported for the rat gene, with a 70-fold increase in CAT activity in the mouse prostate observed between 2-7 weeks of age, a time corresponding to sexual maturation. PB-CAT activity in the prostate declined after castration to 3.5% of the precastration level, and the CAT activity in castrated males approached precastration levels when mice were supplemented with testosterone. Transgene expression in castrated males was not induced by dexamethasone. Coinjection of PB-CAT with a chicken lysozyme gene matrix attachment region resulted in their cointegration and further restricted the pattern of PB-CAT to the dorsolateral prostate, with suppressed expression observed in the ventral prostate. These studies demonstrate that a minimal rat probasin promoter can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion.

Enhanced expression of the epidermal growth factor receptor (EGFR) or its ligands, epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) can increase signalling via receptor-mediated pathways which may lead to excessive proliferation and cellular transformation. Such autocrine regulation of growth has been demonstrated for prostate cancer cell lines in culture but its role in prostate cancer in vivo has not been established. To assess the potential of such a mechanism, we have examined the pathway components in prostate carcinomas (CaP) in comparison with non-malignant benign prostatic hyperplasias (BPH). In the present study, we investigate the dosage, structure and expression of EGF, TGF-alpha and EGFR genes in a series of 34 human prostate samples and 3 prostate cancer cell lines. All of the samples contained transcripts from each of the genes. The expression of pre-pro-TGF-alpha mRNA and pre-pro-EGF mRNA were significantly higher in CaP (n = 13) than BPH (n = 21) specimens (p < 0.05). The androgen-responsive prostatic carcinoma cell line, LNCaP, expressed high levels of EGF mRNA while the androgen-independent DU145 and PC-3 cell lines expressed high levels of TGF-alpha mRNA and EGFR mRNA. In general, overexpression of these mRNAs was not associated with amplification or detectable gene rearrangement; only DU145 cells demonstrated any alteration in these genes, with apparent amplification of the TGF-alpha gene. Relative to BPH, all prostate carcinomas and cell lines studied had elevated levels of mRNA for one or both mRNA coding for the ligands for EGFR.(ABSTRACT TRUNCATED AT 250 WORDS)

IGF binding protein (IGFBP)-3 inhibits cell growth and promotes apoptosis by sequestering free IGFs. In addition IGFBP-3 has IGF-independent, proapoptotic, antiproliferative effects on prostate cancer cells in vitro. Expression of the large T-antigen (Tag) under the long probasin promoter (LPB) in LPB-Tag mice results in prostate tumorigenesis. To investigate the IGF-dependent and IGF-independent effects of IGFBP-3 on prostate tumor growth, we crossed LPB-Tag mice with cytomegalovirus (CMVBP-3) and phosphoglycerate kinase (PGKBP-3) mice that overexpress IGFBP-3 under the cytomegalovirus promoter and the phosphoglycerate kinase promoter, respectively, and also I56G/L80G/L81G-mutant IGFBP-3 (PGKmBP-3) mice that express I56G/L80G/L81G-IGFBP-3, a mutant, that does not bind IGF-I but retains IGF-independent proapoptotic effects in vitro. Prostate tumor size and the steady-state level of p53 were attenuated in LPB-Tag/CMVBP-3 and LPB-Tag/PGKBP-3 mice, compared with LPB-Tag/wild-type (Wt) mice. A more marked effect was observed in LPB-Tag/CMVBP-3, compared with LPB-Tag/PGKBP-3, reflecting increased levels of transgene expression in CMVBP-3 prostate tissue. No attenuation of tumor growth was observed in LPB-Tag/PGKmBP-3 mice during the early tumor development, indicating that the inhibitory effects of IGFBP-3 were most likely IGF dependent during the initiation of tumorigenesis. At 15 wk of age, epidermal growth factor receptor expression was increased in LPB-Tag/Wt and LPB-Tag/PGKmBP-3 tissue, compared with LPB-Tag/PGKBP-3. IGF receptor was increased in all transgenic mice, but pAkt expression, a marker of downstream IGF-I action, was increased only in LPB-Tag/Wt and LPB-Tag/PGKmBP-3. After 15 wk of age, a marked reduction in tumor growth was apparent in LPB-Tag/PGKmBP-3 mice, indicating that the IGF-independent effects of IGFBP-3 may be important in inhibiting tumor progression.

Probasin, a rat prostatic protein, is statistically related to members of a protein family that includes serum, cellular, and nuclear proteins. In vivo, probasin appears both in the secretions and in the nucleus of prostatic epithelial cells. Using primer extension and S1 nuclease protection assays we detected only one probasin mRNA. Thus, the localization of this protein to two separate cellular regions must be encoded by this one mRNA. Furthermore, in vitro translation of synthetic probasin mRNA demonstrated that a protein containing a signal peptide and a protein lacking a signal peptide were synthesized by initiation at different AUG codons. These data are consistent with a mechanism of translational regulation of a eukaryotic bifunctional mRNA.

The mitogenic activity of epidermal growth factor (EGF) is mediated by a cell surface receptor (EGF-R) which has been identified in human prostate tissues. Because of conflicting reports on the relative levels of EGF-R in prostate tumors as measured by binding of radiolabelled EGF, we have examined EGF-R expression at the level of the specific messenger RNA using a sensitive RNase protection assay. Expression of the mRNA for EGF-R was higher in carcinoma (CaP, N = 38) than in benign prostatic hyperplasia (BPH, N = 35) samples (p less than 0.01). The highest levels of EGF-R mRNA were found in the human prostatic carcinoma cell lines, PC-3 and DU145. Among the CaP samples, there was an association of higher EGF-R mRNA levels with higher tumor extent and dedifferentiation. Since EGF has also been found in prostatic tissues, the enhanced expression of the EGF-R gene may play a role in the growth of prostate tumors, possibly by an autocrine pathway.