Shrinkage of experimental benign prostatic hyperplasia and reduction of prostatic cell volume by a gastrin-releasing peptide antagonist.

Gastrin releasing-peptide (GRP) is a potent growth factor in many malignancies. Benign prostatic hyperplasia (BPH) is a progressive age-related proliferation of glandular and stromal tissues; various growth factors and inflammatory processes are involved in its pathogenesis. We have demonstrated that potent antagonists of GRP inhibit growth of experimental human tumors including prostate cancer, but their effect on models of BPH has not been studied. Here, we evaluated the effects of GRP antagonist RC-3940-II on viability and cell volume of BPH-1 human prostate epithelial cells and WPMY-1 prostate stromal cells in vitro, and in testosterone-induced BPH in Wistar rats in vivo. RC-3940-II inhibited the proliferation of BPH-1 and WPMY-1 cells in a dose-dependent manner and reduced prostatic cell volume in vitro. Shrinkage of prostates was observed after 6 wk of treatment with RC-3940-II: a 15.9% decline with 25 µg/d; and a 18.4% reduction with 50 µg/d (P < 0.05 for all). Significant reduction in levels of proliferating cell nuclear antigen, NF-κß/p50, cyclooxygenase-2, and androgen receptor was also seen. Analysis of transcript levels of genes related to growth, inflammatory processes, and signal transduction showed significant changes in the expression of more than 90 genes (P < 0.05). In conclusion, GRP antagonists reduce volume of human prostatic cells and lower prostate weight in experimental BPH through direct inhibitory effects on prostatic GRP receptors. GRP antagonists should be considered for further development as therapy for BPH.

Mechanisms of synergism between antagonists of growth hormone-releasing hormone and antagonists of luteinizing hormone-releasing hormone in shrinking experimental benign prostatic hyperplasia.

BACKGROUND: Benign prostatic hyperplasia (BPH) affects aging men. Combined therapy with antagonists of growth hormone-releasing hormone (GHRH) and of luteinizing hormone-releasing hormone (LHRH or GnRH) induces prostate shrinkage in rat models. We investigated the mechanisms of action of this combination on cell cycle traverse and expression of prostatic genes. METHODS: Effects of GHRH antagonist, JMR-132 (40 µg/day), the LHRH antagonist, cetrorelix (0.625 mg/kg), and their combination were evaluated on testosterone-induced benign prostatic hyperplasia in male Wistar rats. Influence of JMR-132, cetrorelix, and their combinations on cell viability was assessed by MTS assay in BPH-1 human prostate epithelial cells and WPMY-1 normal prostate stromal cells. Cell cycle was analyzed by laser flow cytometry. Real-time PCR arrays were performed. RESULTS: The combination of antagonists caused marked shrinkage of rat prostate (29.5%). In vitro, JMR-132 plus cetrorelix (both 5µM) produced synergistic (57.4%) inhibition of growth of BPH-1 cells, but a lesser inhibition (46%) of WPMY-1 cells. Co-treatment of with JMR-132 plus cetrorelix induced a significant increase of BPH-1 cells blocked in S-phase plus cells with lower G0 /G1 and G2 /M DNA content. Significant changes in expression of >40 gene transcripts related to growth factors, inflammatory cytokines, and signal transduction were identified. CONCLUSIONS: GHRH antagonist and LHRH antagonist combination potentiates rat prostate weight reduction and synergistically inhibits of growth of BPH-1 leading to cell cycle arrest in S-phase. These effects were lesser in normal stromal prostate cell line, WPMY-1. Our findings suggest that GHRH antagonists could be useful for BPH therapy, possibly in combination with LHRH antagonists.

Flow immunocytochemistry of marker expression in cells from body cavity fluids.

Diagnostic cytology based on the examination of cells from body cavity fluids misses approximately 50% of patients with a proven malignancy. In an earlier study, we used immunohistochemical detection of epithelial membrane antigen expression with flow cytometric detection of DNA aneuploidy to reduce the number of false negatives. In the present study, we have combined DNA flow cytometry with flow cytometric detection of marker expression to analyze cells from body cavity fluids. Seventy-nine specimens of ascites and pleural fluids were analyzed by diagnostic cytology, DNA flow cytometry, and for the expression of the following markers: Ber-EP4, progesterone (PR), MUC4, and thyroid transcription factor-1 (TTF-1). DNA index of equal to or greater than 1.2 was seen in 33/79 (41.7%) of the samples. Statistical analysis of 79 samples in which data from cytology, DNA aneuploidy, and expression of at least one of the markers was available showed that by combining data from positive marker expression with that of aneuploidy, the sensitivity was increased from 58.5 to 100%. In contrast, out of the 38 samples designated as non-malignant by diagnostic cytology, nine had aneuploid DNA content and 16 of the diploid samples had a positive marker expression. Specificity was reduced from 74.7 to 31.6% due to the presence of aneuploidy and marker expression in these samples. ALDH1(pos)/CD44(pos)/CD24(neg) expression has been reported to be associated with human breast tumor stem cells. Some of our samples had cells with this phenotype. Flow cytometry offers the advantage of rapid multiparametric analysis of DNA aneuploidy and marker expression in cells from body cavity fluids based on the analysis of a large number of cells without observer bias. By further developing the use of specific markers and aneuploidy, it may be possible to refine flow cytometric analysis for rapid detection of malignant cells in body cavity fluids.

Click-iT assay with improved DNA distribution histograms.

The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.

Are increased tumor aneuploidy and heightened cell proliferation along with heterogeneity associated with patient outcome for carcinomas of the uterine cervix? A combined analysis of subjects treated in RTOG 9001 and a single-institution trial.

PURPOSE: To look for possible associations between measurements of DNA index (DI), S-phase fraction (SPF), and tumor heterogeneity (TH) using flow cytometry and overall survival for patients with invasive cervical carcinoma treated with definitive irradiation. METHODS AND MATERIALS: A total of 57 patients with International Federation of Obstetrics and Gynecology Stages IB(2) through IVB cervical carcinomas treated with definitive radiotherapy with or without concurrent chemotherapy were enrolled into this registry study that involved flow cytometric analysis of fresh tissue from each cervical cancer obtained by pretreatment biopsy. These specimens were evaluated for DNA aneuploidy (DI 1.5), SPF (15%), and TH (uniploid vs. multiploid). RESULTS: In these analyses 27 of the patients were treated in Radiation Therapy Oncology Group protocol 9001, and an additional 30 were offered chemoradiation at a single institution. Forty-one patients had DI 1.5. Twenty-nine patients had SPF 15%, and 2 had no determinable SPF. Forty-three patients had uniploid and 14 multiploid tumors. The 4-year estimated overall survival rate for the entire study cohort was 62% (95% confidence interval 48%-74%). With a median follow-up of 3.7 years, there were no observable associations by univariate analysis for DI, SPF, or TH concerning patient survival. CONCLUSIONS: There were no statistically significant associations among DI, SPF, or TH and patient outcome. Additional studies are indicated to identify tumor biomarkers that could predict patients at risk for disseminated disease.

Cellular volume and marker expression in human peripheral blood apheresis stem cells.

Coulter volume is far more accurate measure of cell volume than forward angle light scatter. In this report, we have used Coulter volume to determine the mean cell volume and diameter of normal human peripheral blood cells and hematopoietic progenitor cells obtained by apheresis (HPC-A) from patients with hematological malignancies. Fresh peripheral blood samples (treated with Beckman Coulter IMMUNOPrep erythrocyte lysis solution), HPC-A samples (treated with BD Biosciences FACSLysing solution), or processed by Ficoll Hypaque sedimentation method were stained with CD45-FITC and PE-labeled CD34, CD90, CD117, and CD133 antibodies and analyzed for electronic volume and two color fluorescence. The mean electronic volume and diameter of mononuclear cells from fresh peripheral blood samples prepared with IMMUNOPrep were lymphocytes (191 microm(3), 7.16 microm), monocytes (370 microm(3), 9.91 microm), and granulocytes (328 microm(3), 8.56 microm). In mononuclear cells of HPC-A samples prepared by Histopaque-1077 sedimentation, the lymphocytes had volume and diameter of 311 microm(3), 8.4 microm, monocytes were 486 microm(3), 9.76 microm, and granulocytes were 515 microm(3), 9.95 microm. In contrast, HPC-A samples prepared after lysis with FACSLysing solution had mean electronic volume and diameter of lymphocytes (414 microm(3), 9.25 microm), monocytes (797 microm(3), 11.5 microm), and granulocytes (670 microm(3), 10.85 microm). Cell volume of mononuclear cells in the HPC-A samples prepared by Histopaque-1077 sedimentation method was correlated with the expression of stem cell markers CD34, CD90, CD117, and CD133. CD90 positive cells had the smallest mean electronic volume of 299.93 microm(3) when compared with cells with positive expression of CD133 (322 microm(3)), CD117 (349 microm(3)), CD34 (407 microm(3)), and CD45 (453 microm(3)). Correlation of cell volume with stem cell marker expression may allow for the identification of small stem cells, which may not express the conventional markers used for the identification of stem cells in HPC-A samples.

Efficacy of 2-halogen substituted D-glucose analogs in blocking glycolysis and killing "hypoxic tumor cells".

PURPOSE: Since 2-deoxy-D-glucose (2-DG) is currently in phase I clinical trials to selectively target slow-growing hypoxic tumor cells, 2-halogenated D-glucose analogs were synthesized for improved activity. Given the fact that 2-DG competes with D-glucose for binding to hexokinase, in silico modeling of molecular interactions between hexokinase I and these new analogs was used to determine whether binding energies correlate with biological effects, i.e. inhibition of glycolysis and subsequent toxicity in hypoxic tumor cells. METHODS AND RESULTS: Using a QSAR-like approach along with a flexible docking strategy, it was determined that the binding affinities of the analogs to hexokinase I decrease as a function of increasing halogen size as follows: 2-fluoro-2-deoxy-D-glucose (2-FG) > 2-chloro-2-deoxy-D-glucose (2-CG) > 2-bromo-2-deoxy-D-glucose (2-BG). Furthermore, D-glucose was found to have the highest affinity followed by 2-FG and 2-DG, respectively. Similarly, flow cytometry and trypan blue exclusion assays showed that the efficacy of the halogenated analogs in preferentially inhibiting growth and killing hypoxic vs. aerobic cells increases as a function of their relative binding affinities. These results correlate with the inhibition of glycolysis as measured by lactate inhibition, i.e. ID50 1 mM for 2-FG, 6 mM for 2-CG and > 6 mM for 2-BG. Moreover, 2-FG was found to be more potent than 2-DG for both glycolytic inhibition and cytotoxicity. CONCLUSIONS: Overall, our in vitro results suggest that 2-FG is more potent than 2-DG in killing hypoxic tumor cells, and therefore may be more clinically effective when combined with standard chemotherapeutic protocols.

Androgen & vitamin D nuclear receptor expression in archival breast tumour samples.

BACKGROUND & OBJECTIVE: Breast tumour cells have receptors for androgen and vitamin D and their clinical significance is not completely understood. Therefore, the present study was undertaken to analyze androgen and vitamin D receptor levels in human primary infiltrating ductal breast carcinomas (IDC) and benign breast tumour archival samples and to find out their correlation, if any, with the clinical findings. METHODS: Paraffin blocks of benign and malignant breast tumours were sectioned, deparaffinized, and nuclei released by pepsin digestion. After antigen retrieval, nuclei were stained with primary antibodies for androgen or vitamin D receptors and secondary fluorescein isothiocyanate (FITC) labeled antibodies and propidium iodide respectively, to quantitative receptor expression and DNA content by flow cytometry. RESULTS: Androgen receptor positive nuclei ranged from 16-66 per cent in the IDC tumours as compared to 36-67 per cent in the benign tumours. Based on flow cytometric comparison of AR expression in AR positive and negative cell lines established earlier, 24 of 28 tumours from postmenopausal women were AR positive compared to all benign tumours and 32 of 33 tumours from pre-menopausal patients. Vitamin D receptor positive nuclei ranged from 14-89 and 2-75 per cent in IDC and benign tumours, respectively. All pre- or post-menopausal tumours were VDR positive as compared to 10 of 15 benign tumours that were VDR positive. No correlation was seen between nuclear androgen and vitamin D receptor expression of the IDC or benign tumours. There was a positive correlation between per cent of receptor positive nuclei and antigen density as measured by ratio of the mean log fluorescence channel value (MFC). No statistically significant correlation was found between nuclear receptor expression (per cent positive nuclei or antigen density) with that of tumour stage, lymph node status, tumour grade, patient age or menopausal status. INTERPRETATION & CONCLUSION: There was no significant correlation between androgen or vitamin D receptor expression and clinical findings. The expression of AR and VDR and the antigen density in the nuclei of the archival breast tumour samples were highly variable because of the tumour heterogeneity. Future studies with fresh biopsy samples of tumour on AR and VDR levels and their up- or down-regulation may be useful while stratifying the patients for hormonal therapy.

Detection of tumor cells in body cavity fluids by flow cytometric and immunocytochemical analysis.

Measurement of electronic volume versus DNA content of nuclei can be used to discriminate between normal and malignant cells. Epithelial membrane antigen immunocytochemistry (EMA-ICC), a helpful ancillary test in body cavity fluids, is not universally accurate for detecting malignancy in effusions. The current study was undertaken to determine if multiparametric flow cytometry (based on simultaneous analysis of light scatter, nuclear volume, DNA, and nuclear protein content) in combination with (EMA-ICC) could be used for the detection of malignant cells in peritoneal and pleural fluids. We studied 130 body cavity fluids (68 peritoneal and 62 pleural fluids) by conventional cytology and multiparametric laser flow cytometry. EMA-ICC was performed using EMA antibodies and L-SAB detection system (DakoCytomation, Carpinteria, CA). EMA-ICC had significantly higher sensitivity than conventional cytology (79% versus 59%, P = 0.016) and ploidy (79% versus 38%, P = 0.001). Cytology had significantly higher specificity than ploidy (97% versus 82%, P = 0.012). The differences in specificity between EMA-ICC and ploidy (87% versus 82%, P= 0.607) or EMA-ICC and cytology (87% versus 97%, P = 0.109) were not statistically significant. However, assuming serial testing, sensitivity increased significantly for the combinations of cytology and EMA-ICC (79.4%, P = 0.016) and cytology and ploidy (73.5%, P = 0.004) as compared to cytology alone (58.8%). Also, the combination of cytology and ploidy had a higher sensitivity than ploidy alone (73% versus 38%, P < 0.0001). However, the sensitivity associated with the three tests used in serial (85.3%) was not significantly different from the sensitivities corresponding to the combination of cytology and EMA-ICC (79%) or cytology and ploidy (73%). Multiparametric flow cytometry utilizing high resolution DNA, nuclear volume, protein measurement, and ICC, in combination with cytomorphology, may be a valuable tool for rapid identification of malignant cells in body cavity fluids.

DAPI fluorescence in nuclei isolated from tumors.

In DNA histograms of some human solid tumors stained with nuclear isolation medium--4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI), the coefficient of variation (CV) of the G0/G1 peak was broad, and in nuclear volume vs DNA scattergrams, a prominent slope was seen. To determine the cause for this, nuclei from frozen breast tumors were stained with NIM-DAPI and analyzed after dilution or resuspension in PBS. In two-color (blue vs red) analysis, most of the slope and broad CV was due to red fluorescence of nuclei stained with NIM-DAPI, which was reduced on dilution or resuspension in PBS, resulting in elimination of the slope and tightening of the CV.

Synergistic cytotoxicity of pyrazoloacridine with doxorubicin, etoposide, and topotecan in drug-resistant tumor cells.

Pyrazoloacridine (NSC 366140, PD115934, PZA) is a new class of acridine anticancer agents under investigation in Phase II clinical trials in patients with advanced cancers. Although poor responses in patients to the treatment with PZA alone have been observed, this class of agents remains of interest because of its distinct mechanism of action from other topoisomerase poisons. Therefore, the combination of PZA with conventional anticancer agents presents an attractive approach to treat drug-resistant human tumors. In the present study, the cytotoxic effects of PZA combined with doxorubicin, topotecan, and etoposide were determined using paired parental and doxorubicin-resistant human colon carcinoma (SW-620 and SW620/AD-300) and breast cancer cell lines (MCF-7 and MCF-7/TH). Cytotoxicity was measured by soft agar clonogenic assays. Dose effect and combination effects were analyzed by the method of Chou and Talalay. The combination of PZA with doxorubicin, topotecan, and etoposide in fixed ratios demonstrated synergistic cytotoxicity on both SW-620 and SW620/AD-300 cell lines. The combination of PZA with doxorubicin also exhibited synergistic cytotoxicity against both MCF-7 and MCF-7/TH cell lines. The mechanism of synergism appeared independent of topoisomerase I and II inhibition, and interference with protein-DNA complexes. Strategies to define optimal drug combinations are proving to be of significant value when considering potential clinical applications of new and established agents.

Flow cytometric monitoring of fluorescent drug retention and efflux.

Laser flow cytometry has been used for monitoring cellular retention of fluorescent drugs such as fluorescent anticancer antibiotics (e.g., doxorubicin) and fluorochromes used for the detection of cellular drug efflux and resistance (e.g., rhodamine 123, Hoechst 33342). Multiparametric flow cytometry can be used for identification of tumor cell subpopulations based on their drug retention profiles with or without the presence of an efflux blocker. This rapid procedure can be used for identification of tumor cells with the drug-resistance phenotype based on drug efflux as well as for efflux blockers that may block efflux of a chemotherapeutic agent and thus increase cellular retention and sensitivity. It has been reported recently that some of the bone marrow stem cells (SP cells) efflux the Hoechst 33342 fluorochrome and thus can be rapidly identified by comparing red vs blue fluorescence in the presence or absence of an efflux blocker such as verapamil. The present chapter discusses some of the flow cytometric methods used for the study of cellular drug retention and the artifacts that may arise in such analysis.

Flow cytometric analysis of electronic nuclear volume and DNA content in normal mouse tissues.

Light scatter is used in flow cytometry for identification of cells based on their size and/or granularity. However, forward light scatter is not an accurate measure of cell size. The measurement of Electronic Volume (EV) by Coulter principle is more accurate. However, EV cannot be measured on most of the commercially available flow cytometers. We have described the development and applications of a flow cytometer that can simultaneously measure Electronic Nuclear Volume (ENV) and DNA content. In the present study we have used a commercially available NPE Quanta for measuring EV and DNA content of different normal mice tissues. Fresh/frozen or formalin fixed-paraffin embedded tissues from mice were processed for isolation of nuclei, which were then analyzed for EV versus DNA content. By using these two parameters, distinct sub-populations were identified in liver, thymus, small intestine and bone marrow. Dual parametric analysis of EV versus DNA content can be a valuable technique for identification of sub-populations in heterogeneous cell mixtures such as those of complex tissues like bone marrow, intestine and tumors. The methods established are rapid and can provide valuable data for identification and characterization of sub-populations for cell cycle analysis by flow cytometry.

Microplate screening for apoptosis with antibody to single-stranded DNA distinguishes anticancer drugs from toxic chemicals.

The effect of anticancer drugs and toxic compounds on cultures of human leukemic cells was evaluated by an enzyme-linked immunosorbent assay (Apoptosis ELISA) that uses a monoclonal antibody against single-stranded DNA to quantitate the apoptotic cells. The concentrations of 13 anticancer drugs, which increased Apoptosis ELISA absorbance, were close to the cytotoxic concentrations determined by the long-term cell survival assay. Short-term tetrazolium-based microculture tetrazolium (MTT) assay was significantly less sensitive than the Apoptosis ELISA and the cell survival assay for all anticancer drugs. For 6 drugs, cytotoxic concentrations measured by the MTT assay were at least 1 log higher than the concentrations inducing apoptosis. Importantly, in contrast to the anticancer drugs, 14 toxic chemicals did not increase the Apoptosis ELISA absorbance at cytotoxic concentrations. The difference in apoptosis induction by the anticancer drugs and the toxic chemicals was especially large in cultures treated with drug concentrations 2-fold higher than the IC(50) dose. Although all of the anticancer drugs tested induced intense ELISA reaction (mean absorbance 2.0), all toxic chemicals tested did not induce apoptosis. The Apoptosis ELISA assay could have useful applications in drug development as it can distinguish between clinically useful anticancer drugs and toxic compounds, has sensitivity similar to that of the long-term cell survival assay, and provides insight into the mechanism of drug cytotoxicity by differentiating between compounds killing cells by apoptosis and necrosis.

Greater cell cycle inhibition and cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under hypoxic vs aerobic conditions.

PURPOSE: In order to investigate the hypothesis that cells found in hypoxic areas of solid tumors are more sensitive to glycolytic inhibitors than cells growing aerobically, we have previously characterized three distinct in vitro models (A, B and C) that simulate this condition. In all of the models it was shown that cells growing under hypoxic conditions are hypersensitive to the glycolytic inhibitor 2-deoxy- d-glucose (2-DG). However, in those studies cytostatic and cytotoxic effects were not distinguished from one another. Since successful treatment of cancer includes not only slowing down but also actually killing tumor cells, studies were undertaken to assess the effects of 2-DG on cell cycle progression and cell death. METHODS AND RESULTS: Using flow cytometry and cell viability assays, it was found that 2-DG caused significantly greater cell cycle inhibition and cell death in all three hypoxic models as compared to aerobically growing control cells. In model A (a chemically induced model of hypoxia in which rhodamine-123 is used to block oxidative phosphorylation), 1200 microg/ml of 2-DG was shown to induce more cell cycle arrest in late S/G(2) and more cell death than in the aerobic cell counterpart treated with 3600 microg/ml 2-DG. In rho(0) cells which are genetically constructed to be unable to perform oxidative phosphorylation (model B), an even greater window of selectivity (more than tenfold) between hypoxic and aerobic cells was found when considering 2-DG's effects on cell cycle arrest and cell death. In the environmental model (model C), where cells were grown under reduced amounts of external oxygen (0.1%), hypersensitivity to the effects of 2-DG with respect to cell cycle arrest and cell death were also observed. CONCLUSIONS: Overall, these results indicate that cells growing under anaerobic conditions respond with greater sensitivity to the effects of 2-DG on cell cycle inhibition and cell death than those growing under aerobic conditions. This supports our contention that glycolytic inhibitors added to standard chemotherapeutic protocols should increase treatment efficacy by selectively killing the slow-growing cells, which are found in the hypoxic portions of solid tumors, while sparing most of the normal cells that are also slow-growing but are living under aerobic conditions.

Monitoring of cellular resistance to cancer chemotherapy.

Cellular resistance to a broad spectrum of natural products used as antitumor drugs is believed to be a major cause for the failure of chemotherapy. Flow cytometry has been used for monitoring the expression of drug resistance markers, determining accumulation of fluorescent drugs, and for screening of drugs that enhance chemosensitivity by blocking efflux and enhancing drug retention. This article reviews recent developments in our understanding of the multiple drug resistance phenotype and the use of flow cytometry for the study of drug efflux and its modulation in human tumor cells.

Androgen and vitamin D receptor expression in archival human breast tumors.

BACKGROUND: The present study was undertaken for quantitation of androgen (AR) and vitamin D (VDR) receptor expression in human male and female breast tumors by flow cytometry. METHODS: Nuclei isolated from sections of paraffin-embedded tumors by pepsin digestion were treated for antigen unmasking and incubated with antibodies to AR and VDR. Flow cytometric analysis was used to determine the percentage of receptor-positive nuclei with fluorescence greater than 95% of the isotype nuclei. Mean log fluorescence channel values were used for comparing antigen density of the isotype and the antibody-treated nuclei. RESULTS: Six of 23 female breast tumors had aneuploid DNA content. Nineteen of 20 estrogen receptor-positive female tumors by immunohistochemical analysis (IHC) were also AR positive by flow analysis. Aneuploid subpopulations had higher percentages of AR-positive nuclei than did diploid populations. Eight of 33 male breast tumors had aneuploid DNA content. Twenty-three of 33 male breast tumors were AR positive by flow analysis compared with six that were AR positive by IHC. Six AR-positive (IHC) male tumors were also AR positive by flow analysis. VDR expression was higher in diploid female tumors than in aneuploid tumors. CONCLUSIONS: Lack of a strong correlation between IHC and flow analysis may be due to differences in criteria used for identification of receptor-positive and -negative tumors by the two methods.

Apoptosis enzyme-linked immunosorbent assay distinguishes anticancer drugs from toxic chemicals and predicts drug synergism.

The effects of anticancer drugs and toxic compounds on leukemic cells in culture were evaluated by enzyme-linked-immunosorbent assay (ELISA) based on the detection of apoptotic cells by a monoclonal antibody against single-stranded DNA. The concentrations of 13 anticancer drugs, which increased apoptosis ELISA absorbance, were similar to the concentrations decreasing long-term cell survival. Short-term metabolic tetrazolium-based 3-(4,5-dimethylthiazol-yl)-2,5-diphenyformazan bromide (MTT) assay was significantly less sensitive than apoptosis ELISA and the cell survival assay. In contrast to anticancer drugs, 12 toxic chemicals did not increase apoptosis ELISA absorbance at cytotoxic concentrations. The difference between two groups of compounds by apoptosis ELISA was especially large in cultures treated with twofold of concentrations producing 50% inhibition of cell growth: all anticancer drugs induced intense reaction (mean absorbance 2.0), while none of the toxic chemicals induced apoptosis. The application of apoptosis ELISA to chemosensitivity testing was evaluated by its ability to detect synergism of anticancer drug combinations. Among 66 drug combinations tested, only combination of nitrogen mustard with mithramycin was highly synergistic by the apoptosis ELISA, as defined by apoptosis induction with the combination containing each drug at 50% of effective concentration. This combination was also synergistic in the cell survival assay, producing significant cell kill while each drug alone had no effect on cell survival. This synergism was not detected by MTT assay. We conclude that apoptosis ELISA could be useful for drug development and chemosensitivity assessment as it can distinguish clinically useful anticancer drugs from toxic compounds, is as sensitive as the long-term cell survival assay and can detect anticancer drug synergism by rapid evaluation of apoptosis induction.

Novel GHRH antagonists suppress the growth of human malignant melanoma by restoring nuclear p27 function.

Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent forms remains a challenge. It has recently been reported that growth hormone-releasing hormone (GHRH) receptor is involved in the pathogenesis of melanoma. Therefore, we investigated the effects of our new GHRH antagonists on a human melanoma cancer cell line. Antiproliferative effects of GHRH antagonists, MIA-602, MIA-606 and MIA-690, on the human melanoma cell line, A-375, were studied in vitro using the MTS assay. The effect of MIA-690 (5 µg/day 28 d) was further evaluated in vivo in nude mice bearing xenografts of A-375. Subcellular localization of p27 was detected with Western blot and immunofluorescent staining. MIA-690 inhibited the proliferation of A-375 cells in a dose-dependent manner (33% at 10 µM, and 19.2% at 5 µM, P < 0 .05 vs. control), and suppressed the growth of xenografted tumors by 70.45% (P < 0.05). Flow cytometric analysis of cell cycle effects following the administration of MIA-690 revealed a decrease in the number of cells in G2/M phase (from 19.7% to 12.9%, P < 0.001). Additionally, Western blot and immunofluorescent studies showed that exposure of A-375 cells to MIA-690 triggered the nuclear accumulation of p27. MIA-690 inhibited tumor growth in vitro and in vivo, and increased the translocation of p27 into the nucleus thus inhibiting progression of the cell cycle. Our findings indicate that patients with malignant melanoma could benefit from treatment regimens, which combine existing chemotherapy agents and novel GHRH-antagonists.