Peptidylglycine alpha-amidating monooxygenase- and proadrenomedullin-derived peptide-associated neuroendocrine differentiation are induced by androgen deprivation in the neoplastic prostate.

Most PCs show NE differentiation. Several studies have tried to correlate NE expression with disease status, but the reported findings have been contradictory. Prostatic NE cells synthesize peptides with a wide spectrum of potential functions. Some of these active peptides, such as PAMP, are amidated. PAM is the only carboxy-terminal peptide-amidating enzyme identified. We studied expression of PAMP and PAM in normal prostate and prostatic tumors (clinical specimens and human xenograft models) with or without prior androgen-deprivation therapy and found a wide distribution of both molecules in NE subpopulations of all kinds. Although the correlation of either marker to tumor grade, clinical progression or disease prognosis did not reach statistical significance, PAMP- or PAM-immunoreactive cells were induced after androgen-blockade therapy. In the PC-310 and PC-295 androgen-dependent models, PAMP or PAM NE differentiation was induced after castration in different ways, being higher in PC-310, which might explain its long-term survival after androgen deprivation. We show induction of expression of 2 new NE markers in clinical specimens and xenografted PC after endocrine therapy.

Androgen deprivation of the PC-310 [correction of prohormone convertase-310] human prostate cancer model system induces neuroendocrine differentiation.

Neuroendocrine (NE) cells are androgen-independent cells and secrete growth-modulating neuropeptides via a regulated secretory pathway (RSP). We studied NE differentiation after androgen withdrawal in the androgen-dependent prostate cancer xenograft PC-310. Expression patterns of chromogranin A, secretogranin III, and prohormone convertase-1 were analyzed at both protein and mRNA level to mark the kinetics of NE differentiation both in vivo and in vitro. PC-310 tumor-bearing nude mice were killed at 0, 2, 5, 7, 14, and 21 days postcastration. PC-310C cultures initiated from collagenase-treated tumor tissue could be maintained up to four passages, and androgen-deprivation experiments were performed similarly. PC-310 tumor volumes decreased by 50% in 10 days postcastration. Proliferative activity and prostate-specific antigen (PSA) serum levels decreased to zero postcastration, whereas PSA levels in PC-310C culture media first decreased and subsequently increased after 5 days. In vivo, androgen receptor (AR) expression decreased initially but returned to control level from 5 days postcastration on. CgA, secretogranin III, and secretogranin V expression increased in vivo from 5 days postcastration on. Subsequently, prohormone convertase-1 and peptidyl alpha-amidating monooxygenase as well as the vascular endothelial growth factor were expressed from 7 days postcastration on, and, finally, growth factors such as gastrin-releasing peptide and serotonin were expressed in a small part of the NE cells 21 days postcastration. The PC-310 tumors did not show colocalization of the AR on the NE cells in the tumor residues after 21 days. As in the PC-310 xenograft, NE differentiation was induced and AR expression relapsed after prolonged androgen suppression in PC-310C. For PC-310C cells, this relapse was associated with the secretion of PSA. PC-310C is the first culture of human prostatic cancer cells having the NE phenotype. The PC-310 model system is a potential androgen-dependent model for studying the role of NE cells in the progression of clinical prostate cancer. Androgen deprivation of NE-differentiated prostate cancer may induce the formation of both NE- and AR-positive dormant tumor residues, capable of actively producing NE growth factors via a RSP, possibly leading to hormone refractory disease.

Androgen-independent growth is induced by neuropeptides in human prostate cancer cell lines.

BACKGROUND: Androgen-independent growth leads to progressive prostate cancer after androgen-ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand-independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells. METHODS: We established and characterized the androgen-independent FGC-DCC from the androgen-dependent LNCaP fast growing colony (FGC) cell line. The androgen-independent DU-145, FGC-DCC, and PC-3, and the androgen-dependent LNCaP and PC-346C cell lines were used to study growth modulation of gastrin-releasing peptide (GRP), calcitonin (CT), serotonin (5-HT), and vasoactive intestinal peptide (VIP) by (3)H-thymidine incorporation. Specificity of the growth-modulating effects was tested with the anti-GRP monoclonal antibody 2A11 and induction of cAMP by neuropeptides. RESULTS: Androgen-independent growth stimulation by neuropeptides was shown in DU-145 and PC-346C. 2A11 inhibited GRP-induced (3)H-thymidine incorporation in DU-145 and PC-346C and inhibited proliferation of the FGC-DCC and PC-3 cell lines. With some exceptions, cAMP induction paralleled growth stimulation. Dideoxyadenosine (DDA) inhibited the GRP-induced growth effect in DU-145 and PC-346C, whereas oxadiazoloquinoxaline-1-one (ODQ) had no effect on (3)H-thymidine incorporation. None of the neuropeptides stimulated growth of LNCaP, FGC-DCC, or PC-3. CONCLUSIONS: GRP-induced growth of DU-145 and PC-346C was specific and cAMP-mediated. Androgen-independent growth of FGC-DCC cells was mainly due to an induction of Bcl-2 expression and possibly through the activation of an autocrine and NE-like pathway, as has been shown also for the PC-3 cell line. Growth induction of non-NE cells by neuropeptides could be a possible role for NE cells in clinical prostate cancer.

Kinetics of neuroendocrine differentiation in an androgen-dependent human prostate xenograft model.

It was previously shown in the PC-295 xenograft that the number of chromogranin A (CgA)-positive neuroendocrine (NE) cells increased after androgen withdrawal. NE cells did not proliferate and differentiated from G0-phase-arrested cells. Here we further characterized NE differentiation, androgen receptor status, and apoptosis-associated Bcl-2 expression in the PC-295 model after androgen withdrawal to assess the origin of NE cells. PC-295 tumor volumes decreased by 50% in 4 days. Intraperitoneal bromodeoxyuridine (BrdU) incorporation and MIB-1 labeling decreased to 0%, and the apoptosis was maximal at day 4. Androgen receptor expression and prostate-specific antigen (PSA) serum levels decreased rapidly within 2 days. The number of NE cells increased 6-fold at day 4 and 30-fold at day 7. Five and ten percent of the CgA-positive cells were BrdU positive after continuous BrdU labeling for 2 and 4 days, respectively. However, no MIB-1 expression was observed in CgA-positive cells. NE cells expressed the regulated secretory pathway marker secretogranin III but were negative for androgen receptor and Bcl-2. Bcl-2 expression did increase in the non-NE tumor cells. In conclusion, androgen withdrawal leads to a rapid PC-295 tumor regression and a proliferation-independent induction of NE differentiation. The strictly androgen-independent NE cells that were still present after 21 days differentiated mainly from G0-phase-arrested cells.

Immunofluorescence for detection of antibodies against human cytomegalovirus-induced membrane antigens.

This paper describes an improved method for the in vitro detection of antibodies specifically directed against human cytomegalovirus (CMV)-induced membrane antigens present on the surface of CMV-infected fibroblasts (CMV-MA). Viable cells were found to be essential for specific visualization of CMV-MA staining. The addition of divalent cations (2.6 mM Ca2+ and 2.2 mM Mg2+) and glucose (180 mM) to the incubation and washing buffers improved the viability and morphology of the cells and increased the cell yield at the end of the assay. Clustering of antigen-antibody complexes on the surface of viable CMV-infected cells was prevented by low-temperature incubation (0 to 4 degrees C) rather than by the addition of agents which act on the metabolism of the cell. No interaction with the CMV-induced Fc receptor was observed at 0 degrees C with either human sera or murine monoclonal antibodies. The specificity of the CMV-MA reaction was confirmed by using monoclonal antibodies to CMV nuclear, cytoplasmic, and membrane-associated antigens. Furthermore, a microplate modification of the membrane fluorescence test is described which is suitable for multisample screening purposes. This method can be applied to the determination of anti-CMV-MA antibody titers in human sera and to the screening of hybridoma supernatants for the presence of antibodies with specificity for CMV-MA.

Killing of human cytomegalovirus-infected fibroblasts by antiviral antibody and complement.

Complement-dependent cytolytic antibodies (CyAb) to cytomegalovirus (CMV)-infected fibroblasts were detectable in acute- and convalescent-phase sera from renal allograft recipients (n = 44) and nonimmunocompromised patients (n = 14) with symptomatic CMV infection but not in sera from control donors (n = 75; P less than .001 by Wilcoxon rank sum test). Renal allograft recipients with secondary CMV infection had the highest levels of CyAb activity. Activity closely correlated with the serum antibody titer to CMV membrane antigens (r = .9106 by linear regression analysis) and was present in both the IgM and IgG fractions of human sera. IgG F(ab)2 fragments were inactive, thus implicating the classical pathway of complement activation. Maximal CMV-specific lysis was obtained with target cells expressing CMV late membrane antigens (greater than or equal to 72 hr after inoculation) irrespective of the CMV strain used. Adsorption and cold target inhibition studies indicated that the target antigens for the CyAb response are specific for the plasma membrane of CMV-infected cells and may only partly be shared by the virion envelope.

Cytomegalovirus early and late membrane antigens detected by antibodies in human convalescent sera.

Using indirect immunofluorescence and a panel of human convalescent-phase sera, we identified cytomegalovirus (CMV) early and late membrane antigens (CMV-EMA and CMV-LMA, respectively) as separate entities on the surfaces of viable CMV-infected fibroblasts starting at 6 to 12 and 36 to 48 h postinoculation, respectively. For expression of CMV-EMA and CMV-LMA, infectious virus and active protein synthesis were required, whereas the expression of CMV-LMA, in addition, required viral DNA synthesis. Our data suggest that CMV-EMA and CMV-LMA form an individual set of CMV antigens that are different from intracellular CMV antigens and possibly (partly) different from the viral envelope.

Detection of immunoglobulin M and G antibodies against cytomegalovirus early and late antigens by enzyme-linked immunosorbent assay.

A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) is described for the detection of immunoglobulin M and antibodies with specifity for human cytomegalovirus (CMV) early (CMV-EA) and late (CMV-LA) antigens. The emphasis is on the production of high-quality CMV antigens, CMV-EA and CMV-LA separately, and conditions for their application in the ELISA. The induction of CMV-EA and -LA in infected cell extracts was studied in detail by using human sera with defined antibody specificity for CMV-EA and CMV-LA. This resulted in the development of a simple whole cell extraction procedure that provided a high yield of CMV antigens with reproducible antigen quality. The antigens were specific for the detection of anti-CMV antibodies. The influence of autoantibodies on the determination of CMV-specific antibodies was investigated. Parallel analysis of 322 human sera by indirect immunofluorescence and ELISA showed a high correlation between both assays (r = 0.9674 for CMV-EA and 0.9362 for CMV-LA). Antibody titers determined by ELISA were equal to (for CMV-EA) or slightly higher (for CMV-LA) that those determined by immunofluorescence but significantly higher (20- to 5,120-fold) than those determined by complement fixation. From 191 sera positive by ELISA (titer greater than or equal to 40) 4 (2.1%) were negative by immunofluorescence (titer less than 40), and from 61 ELISA-positive sera 12 (19.6%) were negative (titer less than 8) when tested by complement fixation. Consequently, ELISA for CMV may prove to be more reliable for the selection of CMV-seronegative blood donors than these other methods. The use of high-quality antigens allows more economic handling of large-scale serum determinations. Possibilities for further automation are discussed.