Molecular load of pathologically occult metastases in pelvic lymph nodes is an independent prognostic marker of biochemical failure after localized prostate cancer treatment.

PURPOSE: Thirty percent of patients treated with curative intent for localized prostate cancer (PC) experience biochemical recurrence (BCR) with rising serum prostate-specific antigen (sPSA), and of these, approximately 50% succumb to progressive disease. More discriminatory staging procedures are needed to identify occult micrometastases that spawn BCR. PATIENTS AND METHODS: PSA mRNA copies in pathologically normal pelvic lymph nodes (N0-PLN) from 341 localized PC patients were quantified by real-time reverse-transcriptase polymerase chain reaction. Based on comparisons with normal lymph nodes and PLN with metastases and on normalization to 5 x 10(6) glyceraldehyde-3'-phosphate dehydrogenase mRNA copies, normalized PSA copies (PSA-N) and a threshold of PSA-N 100 or more were selected for continuous and categorical multivariate analyses of biochemical failure-free survival (BFFS) compared with established risk factors. RESULTS: At median follow-up of 4 years, the BFFS of patients with PSA-N 100 or more versus PSA-N less than 100 was 55% and 77% (P = .0002), respectively. The effect was greatest for sPSA greater than 20 ng/mL, 25% versus 60% (P = .014), Gleason score 8 or higher, 21% versus 66% (P = .0002), stage T3c, 18% versus 64% (P = .001), and high-risk group (50% v 72%; P = .05). By continuous analysis PSA-N was an independent prognostic marker for BCR (P = .049) with a hazard ratio of 1.25 (95% CI, 1.001 to 1.57). By categorical analysis, PSA-N 100 or more was an independent variable (P = .021) with a relative risk of 1.98 (95% CI, 1.11 to 3.55) for BCR compared with PSA-N less than 100. CONCLUSION: PSA-N 100 or more is a new, independent molecular staging criterion for localized PC that identifies high-risk group patients with clinically relevant occult micrometastases in N0-PLN, who may benefit from additional therapy to prevent BCR.

Rat Sertoli cells express epithelial but also mesenchymal genes after immortalization with SV40.

A new immortal Sertoli cell line from pubertal rat testis was established and characterized. We have generated the clonal line SCIT-C8 expressing established markers for Sertoli cells (SC) like transferrin, clusterin and steel factor/stem cell factor (SCF). Additionally, the immortalized cells express afadin, a protein which is a member of tight and adherens junctions, therefore the cells may be useful for studies of the blood-testis barrier (BTB) in vitro. In contrast to primary SC, the immortalized cells lost expression of androgen receptor and responsiveness to androgens and follicle-stimulating hormone. Surprisingly, we found mRNA expression and protein secretion of the mesenchymal markers, fibronectin and entactin-1, which we also observed for the immortalized SC lines, ASC-17D and 93RS2. In comparison to primary SC, the immortalized cells demonstrated enhanced adhesion in vitro. This correlated with the expression of entactin-1 because adhesion was strongly reduced by antibody perturbation experiments. Additionally, we found the alternatively spliced and primarily muscle cell-specific long variant of TGF-beta2 not only in peritubular cells (PC), but also in the primary and immortalized SC. Furthermore, all immortalized cell lines secreted higher amounts of TGF-beta2 than primary SC. In conclusion, the immortalized SC lines from different developmental stages showed a similar pattern of epithelial and mesenchymal markers.

Prognostic value of combined "triple"-reverse transcription-PCR analysis for prostate-specific antigen, human kallikrein 2, and prostate-specific membrane antigen mRNA in peripheral blood and lymph nodes of prostate cancer patients.

PURPOSE: We present the largest study of both peripheral blood and lymph node samples examining the utility of reverse transcription-polymerase chain reaction (RT-PCR) for established molecular markers as a diagnostic tool in the molecular staging of prostate cancer patients undergoing radical prostatectomy. EXPERIMENTAL DESIGN: Peripheral blood from 358 patients was obtained before radical prostatectomy. Corresponding obturatory lymph node samples were collected from 153 of these patients. Nested RT-PCR for prostate-specific antigen (PSA), human kallikrein 2 (hK2), and prostate-specific membrane antigen (PSMA) were performed on cDNA from peripheral blood. The lymph node cDNA was analyzed for PSA und hK2 expression. RESULTS: RT-PCR in peripheral blood was positive in 124 (34.6%) of 358 samples for PSA, 215 (60.1%) of 358 for PSMA, and 97 (27.1%) of 358 for hK2. Comparison of positive RT-PCR rates of pT(2) and pT(3) tumors in corresponding peripheral blood for PSA, PSMA, and hK2 were 31.9 and 40.0%, 58.8 and 62.5%, and 26.9 and 27.5%, respectively. Histopathologically, cancer-free lymph node samples were positive in RT-PCR for PSA and hK2 in 70 (49.6%) of 141 and 89 (63.2%) of 141 of cases. All histologically positive lymph node samples (n = 12, pN+) were positive for PSA RT-PCR. PSA RT-PCR alone, as well as combined PSA/PSMA RT-PCR evaluation, in peripheral blood showed a significant association with grading. PSA RT-PCR lymph node-negative samples were significantly less likely positive in their corresponding peripheral blood RT-PCR sample. CONCLUSIONS: Although the preoperative PSA RT-PCR in peripheral blood correlated with the grading of prostate cancer, no combination of RT-PCR results using "triple" markers (PSA, hK2, PSMA) in peripheral blood and/or lymph nodes yielded additional preoperative staging information.

Quantitative PSA RT-PCR for preoperative staging of prostate cancer.

BACKGROUND: The clinical value of detecting prostate specific antigen (PSA) mRNA in the peripheral blood mononuclear cell fraction of patients (pts) by standard RT-PCR assays with localized prostate cancer remains controversial. We used a quantitative RT-PCR assay to measure the PSA mRNA copy number in addition to the qualitative PSA RT-PCR and correlated the results with clinical parameters. METHODS: Total RNA was extracted from the peripheral blood mononuclear cell fraction of 115 prostate cancer pts prior to radical retropubic prostatectomy (RP) who received 3 months of neoadjuvant androgen deprivation. For quantitative RT-PCR, a PSA-like internal standard (IS) was added to each sample prior to reverse transcription and the PCR products for PSA and IS were selectively detected with fluorescent europium chelates after hybridization. Corresponding qualitative PSA-RT-PCR was performed for all samples. RESULTS: The median PSA copy number was 126 (range: 0-37988). There were no significant correlations established between qualitative or quantitative RT-PCR results and given clinical parameters. Corresponding quantitative and qualitative RT-PCR results were significantly associated (P = 0.01). CONCLUSIONS: We were unable to show any additional value of quantitative as well as qualitative PSA RT-PCR for preoperative staging of prostate cancer so far. Nevertheless, the long-term follow up of the patients has to be awaited.

Macrophage migration inhibitory factor and development of type-1 diabetes in non-obese diabetic mice.

AIMS/HYPOTHESIS: T-cell activation by specific antigen has been found to increase macrophage migration inhibitory factor (MIF) expression, indicating its role as an important feature of T-cell activation in vitro and in vivo. To date, the potential role of MIF in the development of autoimmune-mediated diabetes mellitus has not been studied. METHODS: MIF-mRNA expression in splenic lymphocytes of spontaneously diabetic non-obese diabetic (NOD) mice (n=6), cyclophosphamide-treated NOD mice (n=6), 14-day-old non-diabetic NOD mice (n=7) and C57/Bl6 control mice (n=6) was monitored using an internally standardised competitive reverse transcription-polymerase chain reaction, and the MIF-protein levels were determined using Western blot analysis. In addition, the impact of intraperitoneally administered recombinant MIF-protein treatment on diabetes incidence in NOD mice was evaluated. RESULTS: MIF-mRNA expression was markedly increased in splenic lymphocytes of spontaneously diabetic NOD mice as well as in 8-week-old NOD mice treated with cyclophosphamide compared with 2-week-old non-diabetic NOD and healthy C57BL/6 control mice. Western blot analyses showed decreased lymphocytic MIF-protein content in diabetic as well as in cyclophosphamide-treated animals compared with 2-week-old non-diabetic NOD and healthy C57BL/6 mice, probably as a consequence of increased protein secretion. Furthermore, treatment of NOD mice with recombinant MIF-protein at 25 microg twice a week, from age 6 to 11 weeks, led to an increased diabetes incidence (86%; n=7) compared with untreated control groups (55%; n=20) at week 34. CONCLUSIONS/INTERPRETATION: In this study, we report for the first time that MIF-mRNA expression in splenic lymphocytes is up-regulated during development of cell-mediated diabetes in non-NOD mice. The data of our preliminary study suggest a possible role of MIF in autoimmune-inflammatory events, such as type-1 diabetes and also that anti-MIF therapeutic strategy might serve to attenuate autoimmune processes.

Human kallikrein 2 (hK2) mRNA in peripheral blood of patients with thyroid cancer: a novel molecular marker?

PURPOSE: To elucidate whether hK2 mRNA can be detected in peripheral blood of patients with thyroid disease using reverse transcription polymerase chain reaction (RT-PCR). METHODS: A nested RT-PCR protocol for the detection of hK2 mRNA was established, and blood samples of 72 patients with a history of thyroid cancer, 10 patients with current metastases of thyroid cancer, and 32 volunteers were tested. RESULTS: hK2-transcripts were significantly more often detected in patients with thyroid cancer (20/72=28%) than in the control group (2/32=6%, P = 0.03, chi-square analysis). CONCLUSIONS: This is the first study reporting on hK2 as a potential molecular marker for patients with thyroid cancer. We could demonstrate a correlation between diagnosis of thyroid cancer and the positive signal for hK2 in the RT-PCR assay. Future studies are necessary to prove the clinical value of hK2 as a molecular marker regarding recurrence and outcome.

Ribozyme-targeting of a secreted FGF-binding protein (FGF-BP) inhibits proliferation of prostate cancer cells in vitro and in vivo.

Prostate cancer is one of the most common malignant tumors with increasing incidence rates in the aging male. Since locally advanced or metastatic prostate tumors are essentially incurable, identification of new target molecules and treatment strategies is of critical importance. Fibroblast growth factor-2 (FGF-2) acts as potent mitogen which is upregulated in prostate cancers modulating cancer cell proliferation and development of an invasive phenotype. Normally it is tightly bound to the extracellular matrix that quenches its biological activity. The FGF-binding proteins (FGF-BP, HBp17) is a secreted protein which is able to mobilize and activate FGF-2 from the extracellular matrix. Here we show that FGF-BP is highly expressed in prostate tumor cells. To study the functional role of FGF-BP, we use a ribozyme-targeting approach to selectively deplete FGF-BP in prostate cancer cells achieving a more than 50% reduction of FGF-BP mRNA and protein levels in two mass-transfected cell lines. FGF-BP depletion reduces proliferation of the cells in vitro without changes in cell cycle distribution or apoptosis. Using cDNA microarrays, Northern blotting and RT-PCR, we show a complex pattern of changes in the gene expression profiles upon FGF-BP depletion. Most strikingly, ribozyme-mediated reduction of FGF-BP levels completely abolishes the ability of the highly metastatic PC-3 prostate carcinoma cells to grow tumors in an athymic nude mouse in vivo model which is far beyond the effects of FGF-BP ribozyme targeting observed previously in cells from other tumors in the same model. Taken together, our study identifies FGF-BP as a potential rate-limiting factor for prostate cancer growth and, due to its restricted expression pattern in adults, a potentially attractive target for prostate cancer therapy.

Quantification of Constitutive Endothelial and Inducible Nitric Oxide Synthase mRNA by Competitive Reverse Transcription-polymerase Chain Reaction.

Nitric oxide (NO) participates in the general homeostatic control of the vasculature, and it is involved in the process of vascular remodelling. NO in particular inhibits the proliferation of vascular smooth muscle cells and has been shown to possess antiatherogenic properties. Two important molecules control NO synthesis, namely constitutive endothelial (ecNOS) and inducible (iNOS) nitric oxide synthase. To investigate the regulation of the ecNOS and iNOS mRNA expression in various tissues, we describe the design and validation of a reliable and efficient competitive RT-PCR approach for quantification of ecNOS and iNOS mRNA in rat tissue. Prior to reverse transcription, the total RNA was supplemented with internal standard RNA-competitors, which were constructed by a modified site-directed mutagenesis followed by in vitro transcription using T7-polymerase. This technique allows the easy and fast (within a single day) construction of an internal, recombinant RNA-fragment without the use of cloning techniques. Only two additional "linker" primers containing the sequence of T7-promoter, the primers used for the wild type of ecNOS and iNOS mRNA and the primers of a spacer gene are needed. In addition, all steps of the procedure can be streamlined by convenient commercially available kits. We conclude that the described technique is a valid and reliable method for the absolute quantification of small amounts of specific mRNA.