Neuropilin-1 is upregulated in the adaptive response of prostate tumors to androgen-targeted therapies and is prognostic of metastatic progression and patient mortality.

Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.

Prostate Specific Membrane Antigen Positron Emission Tomography May Improve the Diagnostic Accuracy of Multiparametric Magnetic Resonance Imaging in Localized Prostate Cancer.

PURPOSE: Positron emission tomography using ligands targeting prostate specific membrane antigen has recently been introduced. Positron emission tomography imaging with (68)Ga-PSMA-HBED-CC has been shown to detect metastatic prostate cancer lesions at a high rate. In this study we compare multiparametric magnetic resonance imaging and prostate specific membrane antigen positron emission tomography of the prostate with whole mount ex vivo prostate histopathology to determine the true sensitivity and specificity of these imaging modalities for detecting and locating tumor foci within the prostate. MATERIALS AND METHODS: In a prospective clinical trial setting 20 patients with localized prostate cancer and a planned radical prostatectomy were recruited. All patients underwent multiparametric magnetic resonance imaging and positron emission tomography before surgery, and whole mount histopathology slides were directly compared to the images. European Society of Urogenital Radiology guidelines for reporting magnetic resonance imaging were used as a template for regional units of analysis. The uropathologist and radiologists were blinded to individual components of the study, and the final correlation was performed by visual and deformable registration analysis. RESULTS: A total of 50 clinically significant lesions were identified from the whole mount histopathological analysis. Based on regional analysis the sensitivity, specificity, positive predictive value and negative predictive value for multiparametric magnetic resonance imaging were 44%, 94%, 81% and 76%, respectively. With prostate specific membrane antigen positron emission tomography the sensitivity, specificity, positive predictive value and negative predictive value were 49%, 95%, 85% and 88%, respectively. Prostate specific membrane antigen positron emission tomography yielded a higher specificity and positive predictive value. CONCLUSIONS: A significant proportion of cancers are potentially missed and underestimated by both imaging modalities. Prostate specific membrane antigen positron emission tomography may be used in addition to multiparametric magnetic resonance imaging to help improve local staging in those patients undergoing retropubic radical prostatectomy.

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip.

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.

PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer.

Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.

Safety profile of Hoodia gordonii extract: mouse prenatal developmental toxicity study.

Hoodia gordonii extract (0, 5, 15 or 50mg/kg body weight/day, n=24 mice/group) was orally administered by gavage to female CD-1 mice from gestation days 5-17. On gestation day 18 the females were euthanized and examined. Treatment at 50mg/kg/day caused a marked reduction in feed intake and body weight gain. Feed consumption was sporadically reduced at 15 mg/kg/day. At 50 or 15 mg/kg/day fetal weights, ossification of some bones and full and empty uterus weights were reduced. There were no clear maternal or fetal effects at 5mg/kg/day. Reproductive indices were unaffected at all doses and there were no treatment-related malformations, anomalies or variations. The overall study no-observed-adverse-effect level was set at 5mg/kg/day. In summary, at doses that reduced maternal feed consumption, H. gordonii extract delayed fetal development. The fetal effects seen could be consequent to reduced maternal feed consumption, the desired biological activity of the test item.

Safety profile of Hoodia gordonii extract: rabbit prenatal developmental toxicity study.

Hoodia gordonii extract was orally administered by gavage to groups of 22 female New Zealand white rabbits from day 3-28 after mating at doses of 0 (control), 3, 6 or 12 mg/kg bodyweight/day. These doses were reached by a dose escalation phase between days 3 and 7 after mating. As well as a vehicle control group, a control group pair-fed to the high dose was also included. On day 29 after mating the females were euthanized and examined. Treatment at 6 or 12 mg/kg/day was associated with a dose-related reduction in feed intake and bodyweight gain. Feed consumption and bodyweight gain was unaffected at 3mg/kg/day. In spite of marked maternal effects at 12 mg/kg/day, reproductive indices were unaffected at all doses and there were no effects on fetal or placental weights and no morphological changes in the fetuses. The no-observed-effect level (NOEL) for developmental effects was therefore 12 mg/kg/day, and the maternal NOEL was 3mg/kg/day. At doses that caused marked maternal effects, H. gordonii extract did not affect embryonic or fetal development in a species that is considered predictive of developmental toxicity in man.

Chemical characterisation of Hoodia gordonii extract.

The chemical composition of a solvent extract of Hoodia gordonii termed 'H.gordonii extract' has been characterised by hyphenated chromatographic methods and traditional analytical techniques. The extract consists of a mixture of steroid glycosides, fatty acids, plant sterols and polar organic material. High performance liquid chromatography (HPLC) with ultra violet (UV) and mass spectrometric (MS) detection was used to quantify and confirm the identity of a number of steroid glycosides (73.7% w/w) present in the extract. Gas chromatography (GC) with MS and flame ionisation detection (FID) was applied to determine the fatty acid (3.12% w/w) sterol (0.39% w/w) and alcohol (0.03% w/w) content of a saponified sample of the extract. Polar organic material was quantified by gravimetric methodology using C(18) SPE separation and was determined to be a minimum of 3% w/w. Moisture content was measured by Karl Fischer coulometric titration (0.81% w/w). The protein content was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with SYPRO Ruby staining and a negative result was determined with a limit of detection of <0.001%w/w of protein per band. The chemical composition of the extract remained stable for 19 months when stored in re-sealable plastic bags at ambient (21-24°C) temperature and <60% relative humidity.

Targeting aurora kinases: a novel approach to curb the growth & chemoresistance of androgen refractory prostate cancer.

Aurora Kinase (AK) based therapy targeting AK-A & B is effective against some cancers. We have explored its potential against previously unreported incurable, metastatic androgen depletion independent Prostate Cancer (ADIPC). We used androgen sensitive (AS) and ADI lines derived from Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. The relevance of this model was unequivocally established through focussed array, quantitative PCR and western blotting studies; significantly greater alteration of genes (fold change and number) representing major cancer pathways was shown in ADI cells compared to AS lines. A marked enhancement of in vivo growth of the ADI subline showing the greatest degree of gene modulations [TRAMP C1 (TC1)-T5: TC1-T5] reflected this. In contrast to the parental AS TC1 line, TC1-T5 cells grew with 100% incidence in the prostate, as lung pseudometastases and migrated to the bone and other soft tissues. The potential involvement of AKs in this transition was indicated by the significant upregulation of AK-A/B and their downstream regulators, survivin and phosphorylated-histone H3 in TC1-T5 cells compared to TC1 cells. This led to enhanced sensitivity of TC1-T5 cells to the pan-AK inhibitor, VX680 and to significant reduction in in vivo tumour growth rates when AK-A and/or B were downregulated in TC1-T5 cells. This cell growth inhibition was markedly enhanced when both AKs were downregulated and also led to substantially greater sensitivity of these cells to docetaxel, the only chemotherapeutic with activity against ADI PC. Finally, use of VX680 with docetaxel led to impressive synergies suggesting promise for treating clinical ADI metastatic PC.

Co-expression of CD147 (EMMPRIN), CD44v3-10, MDR1 and monocarboxylate transporters is associated with prostate cancer drug resistance and progression.

BACKGROUND: The aim of this study is to seek an association between markers of metastatic potential, drug resistance-related protein and monocarboxylate transporters in prostate cancer (CaP). METHODS: We evaluated the expression of invasive markers (CD147, CD44v3-10), drug-resistance protein (MDR1) and monocarboxylate transporters (MCT1 and MCT4) in CaP metastatic cell lines and CaP tissue microarrays (n=140) by immunostaining. The co-expression of CD147 and CD44v3-10 with that of MDR1, MCT1 and MCT4 in CaP cell lines was evaluated using confocal microscopy. The relationship between the expression of CD147 and CD44v3-10 and the sensitivity (IC(50)) to docetaxel in CaP cell lines was assessed using MTT assay. The relationship between expression of CD44v3-10, MDR1 and MCT4 and various clinicopathological CaP progression parameters was examined. RESULTS: CD147 and CD44v3-10 were co-expressed with MDR1, MCT1 and MCT4 in primary and metastatic CaP cells. Both CD147 and CD44v3-10 expression levels were inversely related to docetaxel sensitivity (IC(50)) in metastatic CaP cell lines. Overexpression of CD44v3-10, MDR1 and MCT4 was found in most primary CaP tissues, and was significantly associated with CaP progression. CONCLUSIONS: Our results suggest that the overexpression of CD147, CD44v3-10, MDR1 and MCT4 is associated with CaP progression. Expression of both CD147 and CD44v3-10 is correlated with drug resistance during CaP metastasis and could be a useful potential therapeutic target in advanced disease.

Molecular and traditional chemotherapy: a united front against prostate cancer.

Castrate resistant prostate cancer (CRPC) is essentially incurable. Recently though, chemotherapy demonstrated a survival benefit ( approximately 2months) in the treatment of CRPC. While this was a landmark finding, suboptimal efficacy and systemic toxicities at the therapeutic doses warranted further development. Smart combination therapies, acting through multiple mechanisms to target the heterogeneous cell populations of PC and with potential for reduction in individual dosing, need to be developed. In that, targeted molecular chemotherapy has generated significant interest with the potential for localized treatment to generate systemic efficacy. This can be further enhanced through the use of oncolytic conditionally replicative adenoviruses (CRAds) to deliver molecular chemotherapy. The prospects of chemotherapy and molecular-chemotherapy as single and as components of combination therapies are discussed.

P53 expression is associated with malignant potential in xenograft tissues of a fibrosarcoma mouse model.

BACKGROUND: The expression of wild-type and mutant p53 was studied in two fibrosarcoma cell lines in a mouse xenograft model. MATERIALS AND METHODS: Human cell lines HT1080 and Hs913(D)T were implanted in athymic mice via intramuscular (i.m.) or subcutaneous (s.c.) routes. After eight weeks, liver, lung and primary inoculation sites were harvested. Sections were stained using two methods: a) haematoxylin and eosin to detect tumour at implantation site, liver and lung; b) immunohistochemistry using monoclonal antibodies to detect expression of wild-type (wt) and mutant p53. RESULTS: Both cell lines had similar implantation rates via either route but Hs913(D)T had a higher metastatic rate than HT1080. The Hs913(D)T cells exhibited greater expression of mutant and wild-type p53 than the HT1080 cells. CONCLUSION: The expression of wild-type and mutant p53 is associated with a cell line of greater malignant potential. The inoculation route does not affect primary tumour uptake or metastatic rate.

Androgen decreases osteoprotegerin expression in prostate cancer cells.

Osteoprotegerin (OPG), a key regulator of bone resorption, is hypothesized to have a role in prostate cancer (CaP) bone metastasis. As advanced CaP is treated by androgen ablation, we examined if androgen modulates OPG expression by CaP cell lines in vitro. Basal levels of secreted OPG protein were significantly greater in androgen-independent PC-3 cells compared with androgen-responsive LNCaP-FGC cells (P<0.001); OPG was not detected in the androgen-responsive CaP cell lines LAPC-4 or DuCaP. Treatment with 5alpha-dihydrotestosterone (5alpha-DHT) significantly decreased OPG protein levels in both PC-3 and LNCaP-FGC, with maximal suppression using 10(-9)-10(-7) M 5alpha-DHT in PC-3 (P<0.01; day 3), and using 10(-10)-10(-9) M 5alpha-DHT in LNCaP-FGC cells (P<0.01; day 6). OPG messenger RNA levels were not significantly altered by this 5alpha-DHT treatment. Co-treatment with 10(-6) M flutamide blocked 5alpha-DHT inhibition of OPG protein expression in LNCaP-FGC cells. These data suggest that androgen may modulate OPG protein levels in CaP cell lines in vitro using a post-transcriptional mechanism.

Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo.

This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous "take rate" in NOD-SCID mice, and increased production of PSA. Tumors expressing F134L and R273H grew slower than controls, and were associated with decreased necrosis and apoptosis, but not hypoxia. Interestingly, hypoxia levels were increased in tumors expressing M237L. There was less proliferation in F134L-bearing tumors compared to control, but this was not statistically significant. Angiogenesis was decreased in tumors expressing F134L and R273H compared with M237L, or controls. Conditioned medium from F134L tumors inhibited growth of normal human umbilical-vein endothelial cells but not telomerase-immortalized bone marrow endothelial cells. F134L tumor supernatants showed lower levels of VEGF and endostatin compared with supernatants from tumors expressing other mutants. Our results support the possibility that decreased angiogenesis might account for reduced growth rate of tumor cells expressing the F134L p53 mutation.

Expression of HER1/EGFR protein in human soft tissue sarcomas.

AIM: To measure epidermal growth factor receptor (HER1/EGFR) expression in a range of soft tissue sarcoma (STS) patient samples. METHOD: HER1/EGFR expression was examined by immunohistochemistry in archival tissues of 46 STS patients. RESULTS: HER1/EGFR was positively expressed in 36/46 of STS samples distributed among different histological types. The levels of HER1/EGFR in STS tumour tissues in positive samples were higher compared to those in nearby normal tissues. CONCLUSION: HER1/EGFR is significantly expressed in soft tissue sarcomas, which is a finding reflected in other series. The significance of this finding for targeted therapy is as yet unknown.

Knee muscular response strategies differ by developmental level but not gender during jump landing.

The purpose of this investigation was to determine differences between pre- and post-pubescent males and females in quadriceps (vastus medialis; VM) and hamstrings (medial hamstrings and biceps femoris; HAMS) muscular activation patterns via the root mean square of surface electromyography (SEMG) during self-initiated vertical jump landing. Fifty-eight subjects, divided into age and gender groupings, were compared on kinematic variables during pre-landing (100 msec preceeding initial ground contact), post-landing (100 msec following initial ground contact), and initial-contact-to-maximum-knee-flexion stages. Kinematic variables investigated were (1) SEMG values during each stage of the vertical-jump landing; (2) Co-contraction ratios (CCR), which represented the ratio of normalized hamstrings' activity to normalized quadriceps' activity; and, (3) knee angle at initial contact. Results indicated (1) no significant gender differences in variables measured; and, (2) significant developmental level differences. Post-pubescent subjects displayed greater HAMS acitivity and CCR values in the pre-landing stage relative to post-landing stages, indicating that post-pubescent subjects had a greater level of hamstrings co-contraction prior to landing than pre-pubescent subjects. Conversely, pre-pubescent subjects displayed greater post-landing and initial-contact-to-maximum-knee-flexion ratios, indicating a greater level of hamstrings' co-contraction during post-landing stages than post-pubescent subjects. There were no significant differences in knee angle at initial contact. The greater level of hamstrings' co-activation prior to landing by post-pubescent subjects indicated that they used a strategy of pre-tuning the hamstrings prior to landing (more CNS pre-activation) to control the ground reaction forces and anterior tibial displacement experienced by the knee during landing. On the other hand, pre-pubescent subjects controlled these forces by having a greater level of hamstrings' co-activation during landing, which represents more of a reflexive activation in response to ground impact.

Preclinical evaluation of a prostate-targeted gene-directed enzyme prodrug therapy delivered by ovine atadenovirus.

Gene-directed enzyme prodrug therapy (GDEPT) based on the Escherichia coli enzyme, purine nucleoside phosphorylase (PNP), provides a novel strategy for treating slowly growing tumors like prostate cancer (CaP). PNP converts systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine, that kills PNP-expressing and nearby cells by inhibiting DNA, RNA and protein synthesis. Reporter gene expression directed by a hybrid prostate-directed promoter and enhancer, PSMEPb, was assayed after plasmid transfection or viral transduction of prostate and non-CaP cell lines. Androgen-sensitive (AS) LNCaP-LN3 and androgen-independent (AI) PC3 human CaP xenografts in nude mice were injected intratumorally with an ovine atadenovirus vector, OAdV623, that carries the PNP gene under PSMEPb, formulated with cationic lipid for enhanced infectivity. Fludarabine phosphate was then given intraperitoneally for 5 days at 75 mg/m2/day. PNP expression was evaluated by enzymic conversion of its substrate using reverse phase HPLC. OAdV623 showed excellent in vitro transcriptional specificity for CaP cells. In vivo, expression of PNP persisted for > 6 days after OAdV623 injection and a single treatment provided 100% increase in tumor doubling time and > 50% inhibition of tumor growth for both LNCaP-LN3 and PC3 lines, with increased tumor necrosis and apoptosis and decreased tumor cell proliferation. OAdV623 significantly suppressed the growth of AS and AI human CaP xenografts in mice.

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer.

BACKGROUND: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. METHODS: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. RESULTS: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. CONCLUSIONS: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.

Regulation of epidermal growth factor receptor in human colon cancer cell lines by interferon alpha.

BACKGROUND AND AIM: The biology of growth factor receptor expression has implications for receptor specific cancer therapy. In this study, we examined: (a) regulation of epidermal growth factor receptor (EGFR) expression in a panel of 10 human colon cancer cell lines using interferon alpha (IFN-alpha); (b) ability of IFN-alpha to inhibit cell proliferation; and (c) sensitivity of IFN-alpha pretreated cells to EGF. METHODS: Cell proliferation was measured both by crystal violet colorimetric and clonogenic assays. Cell surface, intracellular, and/or total cell protein expression of EGFR was assessed by indirect immunofluorescence flow cytometry and/or fluorescein isothiocyanate (FITC)-EGF binding and internalisation flow cytometric assay. RESULTS: IFN-alpha treatment upregulated expression of cell surface EGFR in seven of 10 colon cancer cell lines within 16 hours, reaching a peak within 48-96 hours; this was accompanied by transient elevation of intracellular EGFR and marked growth inhibition. IFN-alpha treated cancer cells were still sensitive to EGF proliferative stimulation. CONCLUSIONS: Our results indicate that cytostatic concentrations of IFN-alpha can enhance cell surface and intracellular EGFR expression in a proportion of human colon cancer cells. The antiproliferative action of IFN-alpha could not block the signal transduction of the EGF-EGFR pathway. This may have clinical implications for improving treatment based on targeting of EGFR.

Downregulation of KAI1 mRNA in localised prostate cancer and its bony metastases does not correlate with p53 overexpression.

Recent data have proposed that transcription of the KAI1 metastasis suppressor gene is directly mediated by p53 and that loss of KAI1 expression in advanced prostate cancer is simply due to loss of p53 function after mutation. To investigate this possibility, we have examined KAI1 mRNA (by in situ hybridisation) and p53 protein expression (by immunohistochemistry) as an indicator of wildtype or mutant p53, in a series of 77 paraffin-embedded prostate tissue samples, including post-mortem normal prostates (2), benign prostatic hyperplasia (10), localised cancer (grades 4-6, 25; grades 7-9, 21) and prostate-derived bony metastases (19). Overall, we confirmed that expression of KAI1 mRNA decreased from normal tissue, through localised cancer to bony metastases (P=0.055, tending to significance), while levels of p53 staining significantly increased with cancer progression (P=0.046). These were consistent with the possibility that loss of p53 function might be responsible for loss of KAI1 mRNA. However, by close examination of KAI1 and p53 in adjacent tissue sections, we found no correlation between decreased levels of KAI1 mRNA and overexpression of p53 protein (P=0.497). In addition, high levels of KAI1 mRNA could be identified in samples irrespective of p53 staining. Our data suggest that mutation of p53 is independent of the loss of KAI1 mRNA, and do not support a role for p53 in regulating the expression of KAI1.

Characterization of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in prostate cancer cell lines.

Stromal expression of some matrix metalloproteinases (MMPs) has been associated with increasing tumour burden in prostate cancer. We investigated the expression of mRNA (by RT-PCR) and protein (by zymography and western blotting) of MMPs and endogenous inhibitors (tissue inhibitors of metalloproteinases, TIMPs) in two parent epithelial prostate cancer cell lines and sublines of increasing invasive/metastatic potential. Expression of membrane type MMPs, MT1-MMP and MT3-MMP mRNA was higher in PC3-derived than in LNCaP-derived lines, whereas MT2-MMP mRNA expression was higher in the LNCaPderived than in PC3-derived cell lines. Active MT1, MT2 and MT3-MMP protein levels were similar in all lines, but processed MT-MMPs, indicative of latent MMP activation, were increased in more aggressive sublines. Expression of MMP-1, MMP-13 and TIMP-1 was higher in the more aggressive sublines and may be implicated in invasive/metastatic ability. Regulation of MMP-1 and MMP-13 expression may offer important therapeutic options for treating patients with prostate cancer.