Non-malignant Prostate Cells Research Articles

Cellular repair of oxidatively induced DNA base lesions is defective in prostate cancer cell lines, PC-3 and DU-145.

Mutagenic oxidative DNA base damage increases with age in prostatic tissue. Various factors may influence this increase including: increased production of reactive oxygen species, increased susceptibility to oxidative stress, alterations in detoxifying enzyme levels or defects in DNA repair. Using liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry, we show increased levels of oxidative DNA base lesions, 8-hydroxyguanine (8-oxoG), 8-hydroxyadenine (8-oxoA) and 5-hydroxycytosine (5OHC) over the baseline in PC-3 and DU-145 prostate cancer cells following exposure to ionizing radiation and a repair period. Nuclear extracts from PC-3 and DU-145 prostate cancer cell lines are defective in the incision of 8-oxoG, 5OHC and thymine glycol (TG) relative to the non-malignant prostate cell line. Consistent with reduced expression of OGG1 2a, incision of 8-oxoG is reduced in PC-3 and DU-145 mitochondrial extracts. We also show a correlation between severely defective incision of TG and 5OHC and reduced levels of NTH1 in PC-3 mitochondria. The antioxidant enzymes, glutathione peroxidase (GPx), catalase and superoxide dismutases (SOD1, SOD2), have altered expression patterns in these cancer cell lines. Genetic analysis of the OGG1 gene reveals that both PC-3 and DU-145 cell lines harbor polymorphisms associated with a higher susceptibility to certain cancers. These data suggest that the malignant phenotype in PC-3 and DU-145 cell lines may be associated with defects in base excision repair and alterations in expression of antioxidant enzymes.

Novel stimulatory role for insulin-like growth factor binding protein-2 in prostate cancer cells.

The insulin-like growth factor (IGF) axis is a complex system composed of 2 mitogenic ligands, IGF-I and -II, 2 receptors, IGF-1R and IGF-2R, and 6 binding proteins, IGFBP-1 to -6. The IGFBPs exert their actions through their regulation of IGF bioavailability for IGF receptors. In addition, some IGFBPs have also been found to have direct cellular actions independent of IGFs. IGFBP-2 is a major IGFBP in the prostate and in seminal fluid. IGFBP-2 levels, which are elevated in many malignancies, are markedly increased in prostate cancer, and show a positive correlation with prostate specific antigen (PSA) and prostate tumor aggressiveness. We investigated the potential role of IGFBP-2 in the pathogenesis of prostate cancer by evaluating its ability to stimulate growth and the expression and activity of the nuclear enzyme, telomerase. We found IGFBP-2 to have a modest suppressive effect on the growth of primary cultures of normal prostate epithelial cells and a potent IGF-antagonistic effect in these cells, similar to previous reports on the inhibitory nature of this molecule. Surprisingly, IGFBP-2 had a potent stimulatory effect on growth of LAPC-4 prostate cancer cells, an effect that was more pronounced in the absence of androgens. IGFBP-2 growth stimulation of LAPC-4 cells was completely blocked by MAP-kinase inhibitors and partially blocked by PI3-kinase inhibitors. IGFBP-2 stimulation of LAPC-4 cell growth seen in serum-free conditions was lost in the presence of 10% FBS. IGFBP-2 also had a growth stimulatory effect on DU 145 prostate cancer cells. IGFBP-2 significantly stimulated telomerase activity in LAPC-4 cells in the absence of androgens. In addition, IGFBP-2 significantly stimulated hTERT expression and telomerase activity in DU 145 cells. Thus, we demonstrated an inhibitory effect of IGFBP-2 on non-malignant prostate cells, but showed it to be stimulatory for prostate cancer cells in a MAP-kinase and androgen-modulated process. In conclusion, IGFBP-2 may play a role in the progression, but not in the initiation of the prostate cancer disease process, suggesting the existence of a molecular switch transitioning IGFBP-2 from a growth inhibitor to a pro-carcinogenic molecule.

Osteoprotegerin secretion from prostate cancer is stimulated by cytokines, in vitro

Osteoprotegerin (OPG), a member of the tumor necrosis receptor family, is produced by various tissues and inhibits osteoclast differentiation and activity. Since the metastasis of prostate cancer to bone often induces osteosclerosis, the possibility that these tumor cells secrete OPG is of interest. We have investigated whether the prostate cancer cell lines LNCaP, PC-3, and DU-145 produce and secrete OPG in vitro and if the production might be regulated by cytokines involved in remodeling of bone. OPG transcripts were detected by RT-PCR in all cell lines. OPG in culture media was analyzed by ELISA. In all three lineages, treatment with tumor necrosis factor-α and interleukin-1β dose dependently (5–5000 pM) stimulated the OPG secretion. Treatment with tumor necrosis factor-β in increasing concentrations (1–1000 pM) stimulated OPG secretion in PC-3 but had no effect on the DU-145 and LNCaP cells. Dexamethasone (100 pM) had a small, but not significant, inhibitory effect on OPG secretion from DU-145 and LNCaP. In human non-malignant prostate cells, used as controls, there was no effect of IL-1 or TNFs on the secretion rate of OPG.

Focal adhesion kinase, Rap1, and transcriptional induction of vascular endothelial growth factor.

Signals from a cell's environment are sensed by receptors, which activate pathways that, in turn, transmit the signals to the nucleus, informing decisions on growth, angiogenesis, and other cell functions. Transcription of vascular endothelial growth factor (VEGF), a potent angiogenic factor, can be induced by cell-cell contact. In the current work, we sought to determine if this induction is dependent on transformation of cells to a malignant phenotype and subsequently to determine which signaling molecules mediate activation of VEGF transcription. Normal and transformed prostate epithelial cell lines were examined at various cell densities to simulate the effect of increased cell contact on expression of VEGF messenger RNA. Transformed cells were also cotransfected with a VEGF promoter-reporter construct and with constructs that express dominant negative or activated versions of signal transduction proteins hypothesized to be involved in the cell-cell contact process, and reporter activity was assessed at various cell densities. All P values are two-sided. Direct cell-cell contact, but not extracellular matrix components, resulted in transcriptional activation of a VEGF promoter-reporter construct in malignant (P<.0001) but not in nonmalignant (P =.37) prostate cells. This process was mediated via a mitogen-activated protein kinase (MAPK); it required the activity of focal adhesion kinase (FAK), Rap1, and Raf and was Ras independent. In addition, transcriptional activation of a Ras-sensitive Elk-1 chimeric reporter by cell-cell contact suggests that Rap1 is a key factor in regulating the specificity of convergent MAPK-signaling pathways arising from different upstream extracellular stimuli. Cell contact induction of VEGF transcription via FAK and Rap1 provides a novel Ras-independent, but transformation-dependent, mechanism for stimulus-specific regulation of tumor VEGF expression via MAPK.

MOLECULAR DETECTION OF PROSTATE CELLS IN EJACULATE AND URETHRAL WASHINGS IN MEN WITH SUSPECTED PROSTATE CANCER

Purpose To determine whether prostatic cells were normally present in ejaculate and if the sensitivity and specificity of the detection of malignant prostate cells in ejaculate and urethral washings from men with suspected prostate cancer could be improved using the more sensitive molecular technique of reverse transcriptase-polymerase chain reaction (RT-PCR). Materials and Methods RT-PCR for prostate-specific antigen (PSA), prostate-specific membrane antigen (PSM) and Apoliprotein D (3 putative prostate-specific and/or cancer-specific markers) was performed on RNA extracts of ejaculate (80) and urethral washings (52) from 77 men with suspected prostate cancer and 12 young controls (<30 years of age) and urines from 5 men who had radical prostatectomies and 10 women. Results PSA, PSM and Apolipoprotein D expression was detected in ejaculates and urethral washings from both patient and control groups. No differences were observed in the results obtained for 58 men with suspected or 19 men with confirmed prostate cancer or the 18 vasectomized men within the patient group. Urines from the 5 men who had radical prostatectomies and 10 women were all negative for PSA, but PSM was detected in 2 female urines and in 3 radical prostatectomy samples. As few as 10 LNCaP prostate tumor cells could be detected by PSA RT-PCR when added to female urine. Conclusion We have established a sensitive method of detecting prostatic cells in ejaculate and urethral washings and shown that PSA RT-PCR is a reliable indicator of prostate cells in these samples. However, RT-PCR for PSA, PSM and Apoliprotein D were not useful for discriminating malignant from non-malignant prostate cells.

Characterization of insulin-like growth factor-binding protein-related protein-1 in prostate cells.

Insulin-like growth factor-binding protein-related protein-1 (IGFBP-rP1; also known as Mac25, TAF, and PSF) is a member of the IGFBP superfamily. It is a cysteine-rich protein that shares structural and functional similarities with the conventional IGFBPs. In situ hybridization of prostate tissue sections show intense IGFBP-rP1 messenger ribonucleic acid (mRNA) expression in normal stroma and glandular epithelium. There was a significant loss of detectable IGFBP-rP1 mRNA in metastatic prostate tissue. IGFBP-rP1 mRNA (Northern blots) and protein (immunoblots) were detectable in primary cultures ofprostatic stromal and epithelial cells as well as in the immortalized nonmalignant prostatic human epithelial cells, P69, and in the P69 metastatic subline, M12. IGFBP-rP1 expression was not detectable in the prostatic cancer cell lines PC-3, DU145, and LNCaP. IGFBP-rP1 expression was regulated in P69 cells but not in M12 cells. Protein and mRNA expression was up-regulated by IGF-I, transforming growth factor-beta, and retinoic acid. The observations that IGFBP-rP1 expression is significantly diminished in prostate tumorigenesis and is regulated in nonmalignant prostate cells suggest IGFBP-rP1 is important in normal prostatic cell growth.