Paracrine Regulation of Human Prostate Cancer Cell Growth and Function

Carruba, Giuseppe (1994) Paracrine Regulation of Human Prostate Cancer Cell Growth and Function. PhD thesis, University of Glasgow.

Abstract

This thesis mainly deals with steroid-growth factor interaction in the regulation of growth and steroid metabolism of LNCaP, DU145 and PC3 human prostate cancer cell lines. Growth response to both androgens and oestrogens and to TGFalpha and TGFbeta1 were investigated; in addition, content and status of steroid (AR and ER) and growth factor (EGFR and TGP?R) receptors were assessed using multiple approaches, including ligand binding assay, immunocytochemistry and reverse transcriptase-PCR (RT-PCR). Furthermore, immunofluorescent staining was used to evaluate expression of EGF, TGFalpha and TGFbeta1 and of the 27 kDa heat shock protein (hsp27) as a marker of oestrogen sensitivity. On the other hand, patterns of both testosterone (T) and oestradiol (E2) metabolism were studied using incubation of cultured cells with labelled steroid precursor and reverse phase-HPLC (RP-HPLC) analysis of precursor degradation and formation of metabolic products. Possible influence of both TGFalpha and TGFbeta1 on rates and direction of metabolism of both steroids was also determined. Growth of LNCaP cells was significantly stimulated by physiological concentrations of the two major androgens (T and dihydrotestosterone: 30.2% and 33.8% respectively, P<0.03) and of E2 (65.8%, P=0.009), using stringent culture conditions. Interestingly, increasing concentrations of E2 (0.01-100 nM) induced a significant inhibition of the proliferative activity of PC3 cells (55.2% at 100 nM E2, P<10-6), while neither androgen SUMMARY significantly affected growth of this cell line. In contrast, DU145 cells proved insensitive to the addition of either androgens or oestrogens. Presence of androgen binding sites in prostate tumour cell lines was ascertained using radioligand binding assay and RT-PCR approaches. High affinity AR were detected in both soluble and pellet fractions of LNCaP and DU145 cells, whilst PC3 cells showed only nuclear AR. These results were also supported by PCR system, where ampifiable AR mRNA was found in all three cell lines. Multiple evidence for high affinity sites of oestrogen binding in LNCaP cells was obtained: i) biochemical assay allowed the detection of high affinity, low capacity binding sites in both soluble and nuclear cell fractions; ii) immunocytochemical and immunofluorescent assays showed a consistently intensive staining for both ER and PgR, as well as hsp27; iii) the RT-PCR system documented the presence of normal or a variant ER mRNA in PC3 cells; the latter, which lacks the entire exon 4, has been recently characterised in our laboratories in human mammary carcinoma cells. Presence of ER in PCS cells was also documented by biochemical and cytochemical assays as well as, indirectly, by hsp27 staining. However, the relative estimate of ER expression displayed levels significantly and consistently lower than those found in LNCaP cells. This finding was confirmed using the RT-PCR approach, where transcript levels for both normal and variant ER mRNA were proportionally lower than in LNCaP cells. Conversely, DU145 cells were found to be consistently ER-negative using biochemical and cytochemical assays, hsp27 staining and RT-PCR system. The evidence that the E2-induced growth was completely reversed in LNCaP cells by the addition of the pure antioestrogen ICI-182,780, clearly suggests that E2 acts via its own receptor. The possibility that the inhibitory effect exerted by E2 on growth of PC3 cells could be mediated via an increase of TGFalpha production was also supported by the fact that use of a neutralising antibody raised against TGFbeta1 produced a three-fold increase of cell growth; this effect was almost completely abolished after addition of 100 nM E2. However, Northern blot analysis did not reveal any increase of TGFbeta1 mRNA following E2 administration in this cell line. Growth of PC3 cells was significantly stimulated by TGFalpha (36% at 50 ng/ml, P<0.003) and inhibited by TGFbeta1 (55% at 5 ng/ml, P<10-6) after 48 hours exposure in routine medium. Proliferative activity of DU145 cells was minimally affected by TGFalpha, but significantly inhibited by TGFbeta1 (28% at 5 ng/ml, P<0.009). In contrast, LNCaP cells proved to be poorly sensitive, at least in the short-term, to either growth factor. Radioreceptor assay showed presence of high affinity binding sites for EGF in all three cell lines and for TGFalpha in DU145 and PC3 cells, whilst no detectable site of TGFalpha binding was found in LNCaP cells. DU145 cells displayed the highest EGFR content, LNCaP the lowest; TGFalphaR were expressed in greater amounts in PC3 than in DU145 cells. EGFR binding data were also confirmed using Western blot analysis, the DU145 cells having EGFR expression levels higher than PC3 and LNCaP cells. In addition, immunofluorescent staining revealed high amounts of both EGFR and TGFalpha, and fairly high EGF in DU145 and, to a lesser extent, in LNCaP cells; by contrast, PC3 cells exhibited low levels of both receptor (EGFR) and ligands (EGF, TGFalpha), but appreciable levels of endogenous TGFbeta1. Overall, these results suggest a differential sensitivity to TGFalpha and TGFbeta1 by prostate cancer cells; TGFalpha response seems to be not proportional to the EGF-R content of individual cell lines, while TGFbeta1 response appears to be inversely related to the androgen-sensitivity of cells. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Robin Leake
• Keywords: Biochemistry, Endocrinology
• Date of Award: 1994
• Depositing User: Enlighten Team
• Unique ID: glathesis:1994-74965
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 14:57
• Last Modified: 27 Sep 2019 14:57
• URI: https://theses.gla.ac.uk/id/eprint/74965