Faculty Bibliography

Neuroendocrine (NE) cells are enigmatically found in association with human prostate cancers and their numbers are reported to increase in advanced and hormoneresistant tumors. The origin of this cell type and the reason for their appearance in prostate tumors remains unresolved. Previously, Bang et al. (Proc Natl Acad Sci USA 1994;91:5330) reported that dibutyryl adenosine 3',5'-cyclic phosphate (db-cAMP), an agent that upregulates intracellular cAMP, was able to induce a NE cell-like phenotype of cultured human prostate cancer cells, including the androgen-sensitive LNCaP line. Here we report that chronic incubation of LNCaP cells in a medium containing 10% charcoal-stripped fetal bovine serum (CSFBS) likewise induces NE differentiation of these cells. Within 5 days of switching low density cultures of LNCaP cells to this modified medium, the cells growth arrest and acquire an altered morphology with numerous cytoplasmic secretory granules and elongated processes that resemble cultured neurons. This morphology predominates at 10 days with complete transformation seen by 20 days of culture. Electron microscopic analysis of sections of CS-FBS maintained cells showed the presence of abundant dense core secretory granules characteristic of NE cells. Immunohistochemical staining identified the upregulation of the expression of NE markers bombesin, neuron-specific enolase, and S-100 in this modified culture medium. Once established, the NE cell-like phenotype was found to be reversible upon replacement with a medium containing unmodified fetal bovine serum, but not by direct supplementation of CS-FBS medium with dihydrotestosterone (DHT) (I nM). DHT supplementation did, however, suppress the development of the NE cell-like phenotype when it was present at the initiation of exposure to CS-FBS medium. In contrast to db-cAMP treatment, which did not affect prostate specific antigen (PSA) or androgen receptor (AR) expression of LNCaP cells, NE-differentiated LNCaP cells derived in this hormone-deficient medium showed marked downregulation of PSA and AR expression. These in vitro results further support the concept that prostate cancer cells can tranform in vivo to cells with a NE phenotype and suggest that this transformation might be accelerated in patients by certain therapies for prostate cancer.

Rapid expression cloning and differential RNA display identifies a gene, named prostate tumor inducing gene-1 (PTI-1), that is differentially expressed in prostate cancer versus normal prostate and benign prostatic hypertrophy. PTI-1 encodes a truncated and mutated human elongation factor 1 alpha, and its 5' untranslated region (UTR) shares significant homology with the 23S rRNA gene of Mycoplasma hyopneumoniae. PCR with human genomic DNAs, using PTI-1 5' UTR-specific primers, suggests that this sequence is part of the human genome. Furthermore, reverse transcription (RT)-PCR, with one primer specific to the 5' UTR region and the other to the elongation factor 1 alpha coding region, amplifies PTI-1 transcripts from total RNA of various human tumor cell lines and blood samples from prostate carcinoma patients. RT-PCR products with the predicted size and sequence of PTI-1 are detected in RNAs from cell lines of human prostate, breast, and colon carcinomas. This RT-PCR product is shown by Southern blotting and sequence analyses to contain the junction sequence between the 5' UTR and the coding region of the PTI-1 gene. Furthermore, RT-PCR analysis indicates that the PTI-1 gene is also expressed in prostate carcinoma patient-derived blood samples. On the basis of serial dilution experiments, PTI-1 can detect 1 prostate carcinoma cell in 10(8) cells not expressing PTI-1. In this context, PTI-1 represents a sensitive marker for detecting human prostate cancer in the bloodstream. This study confirms the authenticity of the PTI-1 gene and documents its potential clinical utility as a sensitive and specific indicator of prostate cancer progression.

PURPOSE/OBJECTIVE:We examined the effects of prostatic manipulations, including flexible cystoscopy and transrectal needle biopsy, on the enhanced reverse transcriptase polymerase chain reaction assay in 57 men. MATERIALS AND METHODS/METHODS:The reverse transcriptase polymerase chain reaction assay was performed on 25 patients with clinically localized stages T1 to T2cN0M0 prostate cancer before and 30 minutes after cystoscopy. In addition, blood specimens from 32 patients with elevated serum prostate specific antigen and/or abnormal digital rectal examinations were tested immediately before and at 30 minutes after transrectal ultrasound guided prostate needle biopsy. RESULTS:We detected no difference between polymerase chain reaction results obtained immediately before and 30 minutes after cystoscopy in 25 men. Transrectal needle biopsy had no effect on the polymerase chain reaction results in 30 of 32 men. However, 2 men had positive reactions on post-biopsy specimens only. Pathological results of the biopsy revealed benign prostatic hyperplasia and prostatic intraepithelial neoplasia, respectively, in these 2 men. CONCLUSIONS:We conclude that cystoscopy has no clinically significant effect on the reverse transcriptase polymerase chain reaction assay. However, prostatic needle biopsy may cause a positive polymerase chain reaction for PSA in the immediate post-biopsy period.

RT-PCR, if targeted against a prostate-specific marker, can be a highly specific and sensitive assay to detect the presence of occult prostate cancer cells at sites far distant from the primary tumor. The low false-positive rate (0.8%) observed when combining most published studies and the high rate of detection of prostate cells in metastatic patients (88%) found in well-defined clinical studies address the basic requirements of a clinically effective diagnostic modality. Additionally, all reports indicate that RT-PCR positivity for PSA or PSMA increases with increased stage of prostate cancer, suggesting that RT-PCR assays may have value in staging the patient presumed to have clinically localized disease. Indeed, data from two institutions have shown the unique contribution that RT-PCR technology might lend to this most vexing problem. With the suggestive data already published, we remain optimistic that molecular staging will become a reality in the future. After all, the variability in techniques of RT-PCR (as well as differences in targets and samples assayed) used by the various laboratories now investigating this method could themselves be responsible for many of the differences reported. There is no question that standardization among laboratories is essential before clinical utility of molecular staging can be proved. Indeed, this endeavor represents the major aim of the RT-PCR consortium in the United States. Nevertheless, at our own medical center, we are struck by our finding that if a preoperative RT-PCR for PSA is negative, the patient can be assured of a successful operative outcome in 88% of cases. We also are impressed by the data showing that if the serum PSA level is greater than 10 ng/mL and the RT-PCR assay is positive, 90% of patients undergoing radical surgery have adverse pathology reports (and, in fact, have already experience operative failure in nearly half the cases). There is increasing consensus that RT-PCR assays afford prognostic information that is also unique. Three institutions have similar data comparing operative or preoperative RT-PCR strategies (employing all three potential sample sources) with treatment outcome. This may suggest the validity of RT-PCR as a staging modality or, alternatively, suggest that a truly metastatic phenotype is identified in patients with circulating PSA-synthesizing cells, or in patients harboring PSA-synthesizing cells in node or marrow tissues. In either case, RT-PCR appears already to provide information not available before the use of this technology. In fact, in the only series comparing RT-PCR with other, more established predictors (PSA, Gleason score) in a multivariate analysis, RT-PCR status provided the best prognostic information. Prostate manipulation does, in fact, appear to affect the RT-PCR assay. Although digital rectal examination and cystoscopy do not seem sufficiently invasive to release prostate cells into the circulation, a small number of individuals undergoing transrectal ultrasound biopsy will experience this phenomenon. This observation provides a cautionary note relative to timing of sample collection for RT-PCR assays. Furthermore, a significant fraction of patients undergoing prostate manipulation during radical surgery may experience shedding of prostate cells into the operative field and release of some into the peripheral circulation. This has not been universally observed and, even if true, may not share the significance of spontaneous RT-PCR positivity (before prostatic manipulation). In the one instance, a potentially metastatic phenotype is responsible for RT-PCR positivity, whereas in the other instance, PSA or PSMA synthesizing cells may have no metastatic potential at all. Further studies on the biologic half-life of such cells are required before any clinical judgement can be made regarding this phenomenon. In summary, there appears to be consensus that RT-PCR technology can differentiate controls from patients with metas

BACKGROUND:The bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers. Towards this end, we have synthesized an anti-bcl-2 gene therapeutic reagent based on ribozyme technology and have tested its effectiveness against bcl-2 mRNA in vitro and in vivo. METHODS:A divalent hammerhead ribozyme was constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA. A disabled ribozyme lacking catalytic activity was also constructed as a control reagent for our experiments. The ribozymes were tested for endonucleolytic activity against synthetic and natural bcl-2 mRNAs. Simple transfection procedures were then utilized to introduce the ribozymes into cultured prostate cancer cells (LNCaP derivatives). We measured the effects of the ribozymes on endogenous expression of bcl-2 mRNA and protein in these cells as well as their ability to induce apoptosis. RESULTS:The functional but not the disabled ribozyme was able to rapidly degrade bcl-2 mRNA in vitro, without the requirement for any other cellular protein or factor. When directly transfected into LNCaP cell variants, it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment. This activity was sufficient to induce apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line. For the high-bcl-2-expressing variant, however, it did restore the ability to genetically respond to a secondary apoptotic agent, phorbol ester, as evidenced by the renewed ability of phorbol ester to induce NGF1A mRNA in these cells. CONCLUSIONS:This study supports the potential utility of an anti-bcl-2 ribozyme reagent for reducing or eliminating bcl-2 expression from hormone-refractory prostate cancer cells and for killing prostate cancer cells. As such, it is the first step toward an effective gene therapy against hormone-refractory human prostate cancers.

PURPOSE/OBJECTIVE:We determined whether pre-radical prostatectomy prostate specific antigen (PSA) doubling time could predict pathological stage at radical prostatectomy or PSA failure postoperatively. We also sought to compare PSA doubling times from men with prostate cancer treated with radical prostatectomy to a group treated with radiation therapy. MATERIALS AND METHODS/METHODS:Detailed followup was available for 150 patients with clinically localized prostate cancer who underwent radical prostatectomy from January 1993 to August 1995. PSA doubling time was calculated for all patients with 3 or more pre-radical prostatectomy PSA levels using linear regression. We assessed the association between PSA doubling time and PSA failure, pathologic stage at radical prostatectomy, final PSA before treatment and Gleason score. We compared our PSA doubling time values and distribution to a published series of patients with prostate cancer who had undergone radiation therapy. RESULTS:A total of 56 patients had 3 or more PSA values before treatment. Median followup was 17.3 months. PSA doubling time did not correlate with PSA failure, final PSA or Gleason score, but it did with pathological stage at radical prostatectomy (p = 0.0035 for positive margins, p = 0.025 for positive seminal vesicles). Our PSA doubling time and PSA failure rates did not differ from the radiation therapy population with similar followup times. CONCLUSIONS:Although studies from the radiation literature have shown PSA doubling time to be useful in predicting PSA failure after treatment for prostate cancer, our results do not confirm this finding. We did find a correlation with pathologic stage at radical prostatectomy, and so longer followup with more patients may confirm this in the future. We also found no significant differences in PSA doubling time between our patients and a group treated with radiation. At least for this parameter, patients with prostate cancer referred for radical prostatectomy and radiation therapy may be similar.

PURPOSE/OBJECTIVE:We previously demonstrated than an enhanced reverse transcriptase-polymerase chain reaction assay for prostate specific antigen (PSA) can predict final pathological stage in radical prostatectomy patients. The potential role of the assay in predicting serum PSA recurrence after radical prostatectomy was explored. MATERIALS AND METHODS/METHODS:We evaluated 100 radical prostatectomy candidates by reverse transcriptase polymerase chain reaction preoperatively, and status was compared to serum PSA, Gleason score and final pathological results. Potential surgical failure was defined as tumor at the surgical margin or extending into the seminal vesicle. Patients were monitored postoperatively by serum PSA every 4 months. Kaplan-Meier analysis was used to evaluate the correlation between reverse transcriptase polymerase chain reaction and disease recurrence, defined as a PSA of 0.2 ng/ml. or greater. RESULTS:Enhanced reverse transcriptase polymerase chain reaction for PSA had a stronger correlation with potential surgical failure than preoperative serum PSA or Gleason score (relative risks 15.2, 5.9 and 3.2, respectively). The correlation between these modalities and PSA recurrence was evaluated during a mean followup of 13.6 months (range 5 to 26). Of 36 patients with positive reverse transcriptase polymerase chain reactions 9 had failure by PSA compared to 3 of 64 (4.7%) with negative polymerase chain reactions (p<0.0286). The relative risk for failure by reverse transcriptase polymerase chain reaction was 3.6. Gleason score and serum PSA had higher correlations with postoperative PSA elevations (relative risk 13.2 and 7.6, respectively). A Cox regression analysis model demonstrated that reverse transcriptase polymerase chain reaction for PSA can be used in conjunction with Gleason score and provides statistically significant risk information. CONCLUSIONS:Enhanced reverse transcriptase polymerase chain reaction for PSA is a statistically significant predictor of potential failure by pathological analysis and of disease recurrence by PSA. Longer followup data are required to define further the role of the assay in the management of patients with prostate cancer.

OBJECTIVE:To assess the potential role of a recently developed reverse transcriptase-polymerase chain reaction (RT-PCR) assay for prostate-specific antigen (PSA), that detects circulating prostate cells in patients with prostate cancer, in the management of clinically localized cancer. PATIENTS AND METHODS/METHODS:A total of 138 men (mean age 62.5 years, range 49-70) scheduled for radical retropubic prostatectomy had an RT-PCR assay before surgery. The results were compared with the final pathological stage of disease, the results from local imaging techniques, serum PSA levels, digital rectal examination (DRE) and Gleason score. RESULTS:Enhanced RT-PCR for PSA was the best predictor of potential surgical failures; 70% of patients with positive surgical margins or invasion into the seminal vesicle were identified pre-operatively by a positive RT-PCR assay (odds ratio = 12.0, positive predictive value = 64%, negative predictive value = 87%). RT-PCR was able to identify pre-operatively patients with adverse pathology, despite low serum PSA values (< 4.0 ng/mL). In patients with high PSA level (> 10 ng/mL), RT-PCR discriminated between potentially curable candidates and those with established extraprostatic disease. CONCLUSIONS:RT-PCR for PSA adds unique prognostic information when considering patients for radical surgery. The final role for the RT-PCR assay is as yet undefined; however, the ability to detect potential surgical failures pre-operatively using a molecular approach should have a significant impact on the management of patients with prostate cancer.