Abbreviated cell cycle progression induced by the serine/threonine protein phosphatase inhibitor okadaic acid at concentrations that promote neoplastic transformation.

We examined cell cycle-related effects of the phosphatase inhibitor okadaic acid (OA) in T51B rat liver epithelial cells under conditions chosen to mimic early stages of tumor promotion by this compound. Optimal transformation (colony formation in soft agar) was seen after prolonged culture of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated T51B cells in 7 nM OA. Paradoxically, T51B cells treated with 2-10 nM OA showed decreased, rather than increased, proliferation in response to epidermal growth factor (EGF), as measured by [3H]thymidine incorporation. Complete inhibition was observed within 24 h at 10 nM OA. This response paralleled a loss of EGF-stimulated cdk2 kinase activity and an increase in association of the inhibitors p21 (cip-1) and p27 (kip-1) with cdk2. An increase in p53 phosphorylated on serine 15 accompanied the rise in p21 (cip-1). Both phosphorylation of the retinoblastoma protein and induction of cyclin A by EGF were blocked in cells treated with OA, but there was an increase in cyclin E. Resting cells treated with OA alone also showed elevated cyclin E levels, together with reduced levels of the E2F regulator pRb2/p130. Taken together, these observations indicate transforming levels of okadaic acid elicit a G(1)-trapping effect by facilitating cell cycle progression to the G(1)/S checkpoint, where cells are trapped by mechanisms that include p21 (cip-1)-mediated inhibition of cdk2. They support the premise that disruption of cellular processes regulating the transitions from G(0) to G(1) to S-phase is an important early step in tumor promotion by low levels of okadaic acid.

Hydrogen peroxide promotes transformation of rat liver non-neoplastic epithelial cells through activation of epidermal growth factor receptor.

Previous studies demonstrated that hydrogen peroxide (H(2)O(2)) is a tumor promoter in the rat liver epithelial cell line T51B. We investigated the pathway linking H(2)O(2) to tumor promotion. H(2)O(2) can directly induce tyrosine phosphorylation of epidermal growth factor receptor (EGFR). H(2)O(2) and epidermal growth factor exerted similar effects on the induction of early growth response genes, disruption of gap junction communication, triggering of calcium inflow, and promotion of transformation. Furthermore, the effect of H(2)O(2) on tumor promotion was blocked by abrogation of EGFR activation. Our results suggested that tumor promotion by H(2)O(2) is mediated mainly through activation of EGFR in T51B cells.

Connexin 43 (cx43) enhances chemotherapy-induced apoptosis in human glioblastoma cells.

Stable re-expression of connexin 43 (cx43) in human glioblastoma suppresses transformation and tumorigenicity. The present study was designed to examine the role of cx43 in chemotherapy-induced apoptosis. Expression of cx43 in human glioblastoma cells significantly increased sensitivity to several common chemotherapeutic agents, including etoposide, paclitaxel (Taxol) and doxorubicin, compared with control-transfected cells. The increased sensitivity to chemotherapeutic agents resulted from apoptosis as evidenced by Hoechst dye staining, TUNEL assay and annexin V assay. These cx43-mediated effects were coupled with decreased expression of the specific apoptosis inhibitor bcl-2. Over-expression of bcl-2 in cx43-transfected cells partially confers the resistance to apoptosis induced by etoposide, suggesting that the cx43-mediated apoptosis to chemotherapeutic agents is regulated in part through the down-regulation of bcl-2 expression. Furthermore, the cx43-mediated apoptosis in response to chemotherapeutic drugs may not be linked to increased gap junctional communication in cx43-transfected cells. Our results demonstrate a new role of cx43 in the mediation of apoptosis during chemotherapy.

Prostate-specific membrane antigen (PSMA): current benefits and future value.

We will review the evolution, benefits, and limitations of PSMA testing in the past, as well as its current and future value. Prostate cancer has been the most frequently diagnosed cancer and the second leading cause of cancer death in men in the United States. It has a wide spectrum of biological behavior between latent (indolent) and progressive (aggressive). Further identification of prostate-specific membrane antigen (PSMA) as a prognostic proliferation marker may enhance our understanding of the types of prostate cancer. A review of PSMA testing in the past as well as currently was conducted. Studies were reviewed that deal with detection of PSMA in serum and seminal fluid, reverse transcriptase-polymerase chain reaction (RT-PCR), immunoscintigraphy, and immunohistochemical assays. PSMA is expressed primarily in benign and cancerous prostatic epithelial cells. It is up-regulated in hormone resistant states, and in metastatic situations or other clinical situations where there is tumor recurrence or extension. Based on current results, PSMA detected in the serum by western blotting can assist in the identification, staging, and monitoring of metastatic prostate cancer. In addition, PSMA shows a promising role in directed imaging and therapy of recurrent or metastatic disease.

Detection of extraprostatic prostate cells utilizing reverse transcription-polymerase chain reaction.

This article reviews the utility of reverse transcription-polymerase chain reaction (RT-PCR) in prostate cancer. RT-PCR aims to detect occult micrometastases in non-prostatic sites. Due to its exquisite analytical sensitivity, RT-PCR is able to amplify and detect even low-level, prostate-specific messages present at these extraprostatic sites. In recent years, a fair amount of data on the clinical utility of the technique had been reported. The target tissues under investigation are peripheral blood, bone marrow aspirate, and lymph nodes. Favorite markers of choice are prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and human glandular kallikrein-2 (hK2). False positives among negative controls are low. For the most part, RT-PCR is inadequate in detecting tumor cells in the peripheral blood from patients who are known to have metastatic prostate cancer. All studies showed that RT-PCR could detect PSA, PSMA or hK2 mRNAs in the circulation of patients who have organ-confined or extraprostatic disease. Most studies showed that RT-PCR utilizing current markers could not be used as a prospective test to diagnose prostate cancer. However, a few studies also showed that the detection rate could be predictive and sensitive enough to differentiate patients with organ-confined disease from those with extraprostatic disease. Data from PSA- or PSMA-RT-PCR using lymph nodes as the tissue source is more encouraging. RT-PCR was able to detect PSA and/or PSMA positive samples that have not been detected by conventional pathology.

Follow-up evaluation of a phase II prostate cancer vaccine trial.

BACKGROUND: A phase II trial, involving infusions of autologous dendritic cells (DC) and two human histocompatibility antigen (HLA-A2)-specific prostate-specific membrane antigen (PSMA) peptides, was recently completed. Thirty percent of the participants, including subjects with hormone-refractory metastastic disease, and those with suspected local recurrence of prostate cancer, were identified as clinical responders. This report describes the follow-up evaluation of 19 responders in the two study groups. METHODS: After conclusion of the study, study participants were subjected to follow-up evaluations at 6-8-week intervals. Each responder was reevaluated for response status, and duration of response was determined. RESULTS: Subjects were observed for an average of 291 days (metastastic group, group A-2) and 557 days (local recurrence group, group B), which included the treatment and follow-up periods. The average duration of response was 149 days for group A-2, and 187 days for group B. A majority of responders (11/19; 58%) were still responsive at the end of the current follow-up. CONCLUSIONS: The responses observed may be significant and relatively durable. This study suggests that DC-based cancer vaccines in the future may provide an additional therapy for advanced prostate cancer.

Expression and purification of prostate-specific membrane antigen in the baculovirus expression system and recognition by prostate-specific membrane antigen-specific T cells.

Antigen-specific immunotherapy of cancer depends on a consistent source of well-defined protein antigen. Production of recombinant protein offers the obvious solution to this problem but few comparisons of recombinant and native proteins in cellular immune assays have been reported. We report expression of a putative immunotherapy antigen, prostate-specific membrane antigen (PSMA), in insect cells using a baculovirus vector. T cells stimulated with recombinant PSMA or native PSMA derived from the LNCaP cell line recognized both native PSMA and recombinant, baculoviral PSMA. These data indicate that PSMA produced in Sf9 cells is immunologically cross-reactive with native PSMA and therefore suitable for immunotherapy as it is recognized by both cellular and humoral immune responses.

GM-CSF as a systemic adjuvant in a phase II prostate cancer vaccine trial.

BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; Leukine [sargramostim], Immunex Corp., Seattle, WA) was administered to a subgroup of 44 patients in a phase II clinical trial for prostate cancer using DC pulsed with HLA-A2-specific prostate-specific membrane antigen (PSMA) peptides. Our purpose was to determine if GM-CSF caused any enhancement of patients' immune responses, including enhancement of clinical response to the DC-peptide treatment. This report compares the clinical responses to DC-peptide infusions with and without systemic GM-CSF treatment. METHODS: GM-CSF was administered by subcutaneous injection at a dose of 75 microg/m2/day for 7 days with each of six infusion cycles. Prefilled syringes were supplied to the patients for self-administration. RESULTS: One complete and 8 partial responders were identified among 44 patients who received GM-CSF, as compared to 2 complete and 17 partial responders among 51 patients who did not receive GM-CSF. For patients who received GM-CSF and were tested by delayed-type hypersensitivity (DTH) skin test, 3 cases of improved immune response were identified, compared to 5 cases of improvement in patients who did not receive GM-CSF. The main GM-CSF side effects reported were local reactions at the site of injection, fatigue, pain, and fever. Most reported side effects were of mild severity, with some cases of moderate severity leading to discontinuation of GM-CSF. CONCLUSIONS: Our results suggest GM-CSF as employed in this trial did not detectably enhance clinical response to DC-peptide infusions, or significantly enhance the measured immune response.

Phase II prostate cancer vaccine trial: report of a study involving 37 patients with disease recurrence following primary treatment.

BACKGROUND: A phase II trial was conducted to assess the efficacy of infusions of dendritic cells (DC) and two HLA-A2-specific prostate-specific membrane antigen (PSMA) peptides (PSM-P1 and -P2). This report describes the evaluation of 37 subjects admitted with presumed local recurrence of prostate cancer after primary treatment failure. METHODS: All subjects received six infusions of DC pulsed with PSM-P1 and -P2 at 6-week intervals. Clinical monitoring was conducted pre-, during, and post-phase II study. Data included: complete blood count, bone and total alkaline phosphatase, prostate markers, physical examination, performance status, bone scan, ProstaScint scan, and chest X-ray, as well as other assays to monitor cellular and humoral immune responses. RESULTS: One complete and 10 partial responders were identified from this group based on National Prostate Cancer Project criteria, or on a 50% reduction of prostate-specific antigen (PSA), or on a significant resolution in lesions (biopsy-proven when possible) on ProstaScint scan. CONCLUSIONS: About 30% of study participants in this group showed a positive response at the conclusion of the trial. This study suggests that DC-based cancer vaccines may provide an alternative therapy for prostate cancer patients whose primary treatment failed.

Tumor promotion by hydrogen peroxide in rat liver epithelial cells.

Reactive oxygen species, including H2O2, play an important role in the tumor promotion process. Using an in vitro model of tumor promotion involving the rat liver epithelial oval cell line T51B, the tumor promoting activity of H2O2 in N-methyl-N'-nitro-N-nitrosoguanidine-initiated cells was studied. In this assay system, the promoting effect of H2O2 is evidenced by the formation of colonies in soft agar, appearance of foci in monolayer culture, disruption of gap junction communication (GJC) in foci areas and growth at higher saturation densities. H2O2 preferentially induced the expression of c-fos, c-jun, c-myc and egr-1, while JunB and JunD levels remained almost unchanged. H2O2 also induced hyperphosphorylation of Cx43 and disruption of GJC. The effects of H2O2 on tumor promotion, induction of immediate early (IE) genes and disruption of GJC are blocked by antioxidants. These results suggest that H2O2 acts as a tumor promoter in rat liver non-neoplastic epithelial cells and that the induction of IE genes and disruption of GJC are two possible targets of H2O2 during the tumor promotion process.

Disruption of gap junctional communication by the platelet-derived growth factor is mediated via multiple signaling pathways.

The platelet-derived growth factor (PDGF) mediates its cellular functions via activation of its receptor tyrosine kinase followed by the recruitment and activation of several signaling molecules. These signaling molecules then initiate specific signaling cascades, finally resulting in distinct physiological effects. To delineate the PDGF signaling pathway responsible for the disruption of gap junctional communication (GJC), wild-type PDGF receptor beta (PDGFRbeta) and a series of PDGFRbeta mutants were expressed in T51B rat liver epithelial cells. In cells expressing wild-type PDGFRbeta, PDGF induced disruption of GJC and phosphorylation of a gap junctional protein, connexin-43 (Cx43), which required activation of mitogen-activated protein kinase, although involvement of additional factors was also evident. In the F5 mutant lacking binding sites for phosphatidylinositol 3-kinase, GTPase-activating protein, SHP-2, and phospholipase Cgamma1 (PLCgamma1), PDGF induced mitogen-activated protein kinase, but failed to affect GJC or Cx43, indicating involvement of additional signals presumably initiated by one or more of the mutated binding sites. Examination of the single-site mutants revealed that PDGF effects were not mediated via a single signaling component. This was confirmed by the "add-back" mutants, which showed that restoration of either SHP-2 or PLCgamma1 binding was sufficient to propagate the GJC inhibitory actions of PDGF. Further analysis showed that activation of PLCgamma1 is involved in Cx43 phosphorylation, which surprisingly failed to correlate with GJC blockade. The results of our study demonstrate that PDGF-induced disruption of GJC can be mediated by multiple signaling pathways and requires participation of multiple components.

Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate.

Disruption of gap junctional communication (GJC) by various compounds, including growth factors and tumor promoters, is believed to be modulated by the phosphorylation of a gap junctional protein, connexin43 (Cx43). We have previously demonstrated a platelet-derived growth factor (PDGF)-induced blockade of GJC and phosphorylation of Cx43 in T51B rat liver epithelial cells expressing wild-type PDGF receptor beta (PDGFr beta). Both of these actions of PDGF required participation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Similar requirements of MAPK were suggested in the modulation of GJC by other agents, including epidermal growth factor (EGF) and lysophosphatidic acid (LPA). Since many of these agents activate additional protein kinases, our present study examined whether activation of MAPK was sufficient for Cx43 phosphorylation and GJC blockade. By utilizing a variety of MAPK activators, we now show that activation of MAPK is not always associated with either Cx43 phosphorylation or disruption of GJC, which suggests a requirement for additional factors. Furthermore, pretreatment with hydrogen peroxide (H2O2), a potent MAPK activator but inefficient GJC/Cx43 modulator, abrogated PDGF- or TPA-induced disruption of GJC. While a 5 min H2O2 pretreatment abolished both PDGF- and TPA-induced Cx43 phosphorylation and GJC blockade, a simultaneous H2O2 treatment interfered only with GJC closure but not with the phosphorylation of Cx43 induced by PDGF and TPA. This finding indicates that, in addition to the Cx43 phosphorylation step, inhibition of GJC requires interaction with other components. H2O2-mediated abrogation of PDGF/TPA signaling can be neutralized by the antioxidant N-acetylcysteine (NAC) or by the tyrosine kinase inhibitor genistein. Taken together, our results suggest that disruption of GJC is not solely mediated by either activated MAPK or Cx43 phosphorylation but requires the participation of additional kinases and regulatory components. This complex mode of regulation is perhaps essential for the proposed functional role of GJC.

Impaired expression and posttranslational processing of connexin43 and downregulation of gap junctional communication in neoplastic human prostate cells.

BACKGROUND: Gap junctional communication (GJC) has been implicated in the control of cell proliferation. Numerous cancer cells show a decrease or loss of GJC compared to their normal counterparts. Lack of adequate information on the status of gap junctions during prostate neoplasia prompted us to examine this form of cancer, which comprises about 14% of male cancer deaths in America. METHODS: Cultured normal human prostate epithelial cells and several different human prostate tumor lines were used in this study. GJC was assayed by dye transfer, whereas Western blot and immunofluorescence methods were used to examine connexin43 (Cx43) levels and the presence of gap junctions, respectively. RESULTS: Normal human prostate cultures exhibited extensive cell-communication which was completely absent in all the examined tumor cells. This disrupted communication was associated with a decreased expression and an impaired posttranslational modification of Cx43 in these cells. Abundant immunostaining of gap junctional channels by a Cx43-antibody was observed in normal prostate cells but not in tumor cells. CONCLUSIONS: Our data provide further support for the hypothesis that loss of junctional communication is a critical step in progression to human prostate neoplasia.

Infusion of dendritic cells pulsed with HLA-A2-specific prostate-specific membrane antigen peptides: a phase II prostate cancer vaccine trial involving patients with hormone-refractory metastatic disease.

BACKGROUND: A phase II trial was conducted to assess the efficacy of infusions of dendritic cells (DC) and two HLA-A2-specific PSMA peptides (PSM-P1 and -P2). This report describes thirty three subjects with hormone-refractory metastatic prostate cancer without prior vaccine therapy history who were evaluated and reported as a group. METHODS: All subjects received six infusions of DC pulsed with PSM-P1 and -P2 at six week intervals. Clinical monitoring was conducted pre-, during, and post- phase II study. Data collected include: complete blood count, bone and total alkaline phosphatase, prostate markers, physical examination, performance status, bone scan, ProstaScint scan, chest x-ray, as well as assays to monitor cellular immune responses. RESULTS: Six partial and two complete responders were identified in the phase II study based on NPCP criteria, plus 50% reduction of prostate-specific antigen (PSA), or resolution in previously measurable lesions on ProstaScint scan. CONCLUSIONS: Over 30% of study participants in this group showed a positive response at the conclusion of the trial. This study suggested that DC-based cancer vaccines may provide an alternative therapy for prostate cancer patients whose disease no longer responds to hormone therapy.

Reduced connexin43 expression in high-grade human brain glioma cells.

BACKGROUND AND OBJECTIVES: Connexin43 (cx43), a gap junction protein, is implicated in the suppression of tumor cell growth. Numerous cancer cells show a reduction or loss of cx43 expression compared to their normal counterparts. Our previous studies suggest that cx43 expression is decreased in a variety of human brain tumor cell lines. To further investigate the role of cx43 in the development of human gliomas, we performed the present study on human glioma grades I-IV. METHODS: Immunohistochemistry was performed on paraffin-embedded tissue sections of 18 human gliomas to analyze the expression levels of cx43 in different stages of human gliomas. RESULTS: High levels of cx43 were observed in all normal brain tissue and in glioma grades I and II. In contrast, the expression of cx43 was very weak in grade III gliomas and almost undetectable in grade IV gliomas. CONCLUSIONS: Our data support the hypothesis that reduction of cx43 is involved in the progression of human gliomas.

Dendritic cell-based immunotherapy for prostate cancer.

Dendritic cells are unique in their ability to stimulate naive T cells. These investigators have developed a prostate cancer vaccine using autologous dendritic cells as a vehicle to present prostate antigens to T cells in vivo.

Antisense human neuroglia related cell adhesion molecule hNr-CAM, reduces the tumorigenic properties of human glioblastoma cells.

BACKGROUND: Human Nr-CAM (Neuroglia related Cell Adhesion Molecule) is over expressed in glioblastoma multiforme tissue (GMT) as compared to normal brain tissue (NBT). MATERIALS AND METHODS: We transfected a human glioblastoma cell line (2020-CRL) with a vector that overexpresses antisense hNr-CAM using a CMVpromoter. RESULTS: Antisense hNr- CAM caused reduction in the native hNr-CAM expression, changed cell morphology, reduced the cell proliferation rate and lengthening of the cell cycle. Furthermore, antisense hNr-CAM overexpression in these cells caused extensive reduction in the number of soft agar colonies and invasion through extra cellular matrix (ECM) gel in vitro. Subcutaneous injection of antisense hNr-CAM overexpressing glioblastoma cells into nude mice caused complete inhibition of tumor formation as compared to vector only transfected cells. Intra-tumoral inoculation of antisense hNr-CAM expressing plasmid also caused slow tumor growth in nude mice in vivo. CONCLUSION: On the basis of these results, we conclude that hNr-CAM is a valid target for potential gene therapy of glioblastoma tumors.

Isolation and characterization of monoclonal antibodies specific for the extracellular domain of prostate specific membrane antigen.

PURPOSE: Monoclonal antibodies specific for protein epitopes of prostate specific membrane antigen (PSMA) expressed on the external surface of prostatic epithelial cells were prepared to provide material for use in the diagnosis or treatment of prostatic cancer. MATERIALS AND METHODS: Mice were immunized with LNCaP cell membranes followed by purified PSMA before fusion. Hybridomas were screened by reactivity with purified PSMA. Resulting antibodies were characterized by enzyme-linked immunosorbent assay, Western blot and fluorescence-activated cell sorter analyses. RESULTS: Monoclonal antibody producing hybridomas designated 3E11, 3C2, 4E10-1.14, 3C9 and 1G3 were obtained which displayed specificities for differing regions of the extracellular domain of the PSMA protein. These antibodies reacted strongly with PSMA from multiple sources and specifically stained unfixed PSMA expressing cells by flow cytometric analysis. CONCLUSIONS: The antibodies obtained displayed strong reactivity and specificity for extracellular epitopes of PSMA. These antibodies will have value in future diagnostic and therapeutic applications focusing on PSMA as a target antigen.

CXCR-4, a chemokine receptor, is overexpressed in and required for proliferation of glioblastoma tumor cells.

BACKGROUND AND OBJECTIVES: Using the technique of differential hybridization of Atlas Human cDNA expression arrays, we previously reported the isolation of a G protein coupled receptor, CXCR-4, which is overexpressed in glioblastoma multiforme tumor tissue (GMTT) compared to normal brain tissue (NBT). METHODS: Using gene specific reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization, we studied its expression in a variety of brain and breast tumor tissue samples. To demonstrate the requirement of CXCR-4 in glioblastoma cell proliferation an antisense construct was overexpressed. Glioblastoma cells were also treated with antibodies against CXCR-4 and its ligand, SDFbeta-1. RESULTS: Expression analysis indicated that CXCR-4 is overexpressed in 57% of the primary glioblastoma tissues and in 88% of the glioblastoma cell lines analyzed. Overexpression of CXCR-4 in glioblastoma cell lines enhanced their soft agar colony-forming capability. Expression of anti-sense CXCR-4 in glioblastoma cell lines caused neurite outgrowth and cellular differentiation. Treatment of glioblastoma cell lines with CXCR-4 and SDFbeta-1 specific antibodies caused inhibition of glioblastoma cell proliferation. CONCLUSIONS: On the basis of these results, we conclude that CXCR-4 gene is required for the proliferation of human glioblastoma tumors.