Androgen inhibits the growth of carcinoma cell lines established from prostate cancer xenografts that escape androgen treatment.

Most prostate cancers escape endocrine therapy by diverse mechanisms. One of them might be growth repression by androgen. We reported that androgen represses the growth in culture of MOP cells (a sub-line of LNCaP cells) and that of MOP cell xenografts, although tumor growth becomes androgen-independent (AI). Here we explore whether AI tumors contain androgen-responsive cells. ME carcinoma cells were established from AI tumors. The responses to androgen were examined by cell counting, DAPI labeling, flow cytometry, PSA immunoassay and tumor size follow-up. Androgen receptors (AR) were analyzed by western blotting and DNA sequencing. The pattern of responses of these cells to androgen was compared to that of MOP cells and that of JAC cells established from LNCaP-like MOP cells. R1881, a synthetic androgen: (1) repressed the growth of all the six ME cell lines obtained, MOP and JAC cells, (2) augmented the secretion of PSA, (3) induced spectacular cell bubbling/fragmentation and (4) blocked the cell cycle and induced a modest increase of apoptosis. All the androgen-repressed cells expressed the same level of mutated AR as LNCaP cells. In nude mice, the growth of ME-2 cell xenografts displayed transient androgen repression similar to that of MOP cells. In culture neither fibroblasts nor extra-cellular matrix altered the effects of R1881 on cell proliferation. These results demonstrate that androgen-independent tumors contain androgen-responsive cells. The apparent discrepancy between the responses to androgen of tumors and those of carcinoma cells in culture suggests that microenvironmental factors contribute to the androgen responsiveness of tumor cells in vivo. These modifications, albeit unspecified, could be suitable targets for restoring the androgen responsiveness of AI tumors.

Identification and relative quantification of adenosine to inosine editing in serotonin 2c receptor mRNA by CE.

A new method has been developed allowing the identification and relative quantification of different forms of mRNA after RNA editing. This method was applied to the serotonin 2c receptor mRNA that potentially exhibits 32 different forms after adenosine to inosine editing at five different sites located in a row of 13 nucleotides. CE was used to characterize fluorescently labeled ssDNA molecules on the basis of their conformational polymorphism. The relative amount of these 32 mRNA forms has been estimated by measuring the fluorescence intensity of each individual DNA strand. Accuracy of quantification was established by diluting one form into another or into a mixture of cDNA, showing linear and precise proportion of each form (0.06

Production and characterization of neutralizing monoclonal antibodies that recognize an epitope in domain 2 of Bacillus anthracis protective antigen.

Antibodies against the protective antigen (PA) of Bacillus anthracis play a key role in response to infection by this important pathogen. The aim of this study was to produce and characterize monoclonal antibodies (mAbs) specific for PA and to identify novel neutralizing epitopes. Three murine mAbs with high specificity and nanomolar affinity for B. anthracis recombinant protective antigen (rPA) were produced and characterized. Western immunoblot analysis, coupled with epitope mapping using overlapping synthetic peptides, revealed that these mAbs recognize a linear epitope within domain 2 of rPA. Neutralization assays demonstrate that these mAbs effectively neutralize lethal toxin in vitro.

Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus.

Nipah virus, a zoonotic paramyxovirus which emerged recently was chemically inactivated using binary ethylenimine (BEI). The inactivated virus was concentrated and purified by sucrose gradient centrifugation. The gradient fractions were examined by electron microscopy and Western immunoblot, and gradient fraction containing mainly Nipah matrix (M) and nucleocapsid (N) proteins was used for immunizing BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones identified five strongly positive clones producing IgG monoclonal antibodies (mAbs) reactive to the Nipah virus antigen. The protein specificity of these mAbs was determined by Western immunoblot using Nipah virus and recombinant Nipah virus proteins expressed in mammalian cells. Four mAbs reacted with Nipah N protein and one reacted with Nipah M protein. None of the mAbs neutralized Nipah virus infectivity in vitro. However, all mAbs recognized Nipah virus in ELISA and immunofluorescence assay. F45G2 mAb was most suitable for immunohistochemistry on long term formalin-fixed Nipah virus infected swine tissues. Three of the anti-nucleocapsid mAbs (F45G2, F45G3 and F45G6) showed cross-reactivity with closely related Hendra virus N protein in both immunofluorescence and Western Immunoblot assays. Two of the mAbs were specific for the Nipah virus only, F45G4 (anti-N) and F45G5 (anti-M), and could be used in the primary identification of Nipah virus. The use of these immunoreagents to develop new diagnostic assays is discussed.

Divergent biological effects of estradiol and diethylstilbestrol in the prostate cancer cell line MOP.

The involvement of mutated androgen receptors (mut-AR) in the actions of estrogens in prostate cancer cells is controversial. This work was designed to determine the role of such receptors in the growth inhibition by estradiol (E2) and androgens of the MOP cell line, a derivative of the LNCaP cell line. Diethylstilbestrol (DES) was used as a "tool". E2 like DHT and R1881 inhibits MOP cell proliferation while DES does not. E2 and R1881 down regulate mut-AR mRNA, DES does not. E2 enhances mut-AR transcriptional activity less efficiently than R1881 while DES does not. E2 and R1881 up regulate PSA secretion in a dose-dependent manner, DES does it marginally at 10(-6)M. MOP cells express low amounts of ERalpha and ERbeta mRNA but neither DES nor E2 and R1881 do enhance ER transcriptional activity. DES and E2 bind to mut-AR with relative binding affinities which are respectively 1/175 and 1/10 that of DHT. The E2 and androgen-repressed proliferation is prevented by DES and by the anti-androgen bicalutamide. In LNCaP cells, DES prevents the androgen-enhanced proliferation. These results strongly suggest that: (a) the putative endogenous ERs are biologically inactive in MOP cells, (b) the E2-repressed proliferation results from hormone binding to mut-AR and, (c) DES is an anti-androgen in mut-AR expressing cell line.

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus.

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.

Disseminated neuroblastoma in children older than one year at diagnosis: comparable results with three consecutive high-dose protocols adopted by the Italian Co-Operative Group for Neuroblastoma.

PURPOSE: To compare the outcomes associated with modifications in three consecutive protocols employed by the Italian Co-Operative Group for Neuroblastoma (ICGNB) in disseminated neuroblastoma. PATIENTS AND METHODS: Between January 1985 and November 1997, a total of 359 children aged 1 to 15 years with newly diagnosed stage 4 neuroblastoma were enrolled in three consecutive protocols. Compared with ICGNB-85, the ICGNB-89 protocol contained two more chemotherapy cycles, and some drugs were given at greater doses, whereas in the ICGNB-92 protocol, the induction phase included a chelating agent, and individual cycles contained four drugs instead of two. RESULTS: A total of 330 of 359 evaluable children were included in this analysis; 106 children were treated with ICGNB-85, 65 children were treated with ICGNB-89, and 159 children were treated with ICGNB-92 protocols. Radical resection of primary tumor was carried out in 59.4%, 50.8%, and 57.9% of the patients, respectively. Major tumor response after induction therapy was achieved in 66.7%, 69.2%, and 68.6% of the patients, respectively. A total of 218 of 232 patients received consolidation therapy consisting of conventional chemotherapy in 65 patients and of high-dose chemotherapy in 153 patients. Disease recurrence or progression occurred in 82.1%, 69.2%, and 74.8% of the patients, respectively. Therapy-related deaths occurred in 1.9%, 12.3%, and 6.9% of the patients, respectively. Five-year overall survival (OS) for the three studies was 26%, 23%, and 28%, and event-free survival (EFS) was 19%, 17%, and 17%, respectively. CONCLUSION: The therapeutic modifications adopted in the ICGNB-89 and ICGNB-92 protocols were not associated with a significant improvement in response rate or in the 5-year OS and EFS as compared with the ICGNB-85 protocol. Attempts at intensifying chemotherapy were associated with greater toxicity.