The relationship of androgen receptor levels to androgen responsiveness in the Dunning R3327 rat prostate tumor sublines

The objective of this study was to determine whether androgen receptor levels in a transplantable animal model of prostatic adenocarcinoma correlated with androgen responsiveness of the tumor. This is the first comparative study of androgen receptor levels in 3 subcellular compartments (cytosol, nuclear salt-extractable and nuclear salt-resistant fractions) of 4 Dunning R3327 rat prostatic adenocarcinoma sublines that vary in their response to androgen ablation. Tumors were harvested from intact adult male rats in order to best approximate the human clinical setting in which receptor levels are quantitated prior to androgen ablative therapy. Only the nuclear salt-resistant (nuclear matrix) and total nuclear androgen receptor contents were significantly different among all tumor sublines. The properties of the tumors studied and their nuclear salt-resistant androgen receptor levels were as follows: H tumor--well-differentiated, slow growing, androgen-dependent, 63 +/- 11 fmol./mg. DNA; HI tumor--well-differentiated, slow growing, androgen-insensitive, 19 +/- 8 fmol./mg. DNA; G tumor--poorly-differentiated, fast growing, androgen-sensitive, 195 +/- 42 fmol./mg. DNA; and AT-2 tumor--anaplastic, fast growing, androgen-insensitive, no detectable receptors. There was no apparent quantitative relationship between androgen receptor content and tumor growth rates, which varied considerably irrespective of the androgen responsiveness of the tumor. However, there was a qualitative relationship between nuclear salt-resistant or total nuclear receptor content and androgen responsiveness. Higher levels of receptor (H and G tumor sublines) were associated with responsiveness to androgen ablation (cessation or slowing of growth, respectively), whereas lower levels of receptor (HI and AT-2 sublines) were associated with androgen insensitivity. These observations, based on relatively homogeneous tumors, may have important implications for human prostatic cancers which appear to be composed of heterogeneous cell populations.     

A human monoclonal antibody reactive with human prostate

A human immunoglobulin M monoclonal antibody secreting hybridoma, termed MHG7, has been isolated and characterized for its reactivity against human prostate cells. Lymphocytes isolated from a regional draining lymph node of a patient with prostate carcinoma were fused with murine P3-NS1-Ag4-1 myeloma cells. Supernatants from the generated mouse-human somatic cell hybrids were first screened for human immunoglobulin production by an enzyme immunoassay. The identified human immunoglobulin-secreting hybridomas were expanded for further analysis and their supernatants screened by enzyme immunoassay against a panel of prostate cell lines. The human immunoglobulin M monoclonal antibody MHG7, in addition to reacting with prostate cell lines, also reacted with prostate carcinoma cells and benign prostatic hypertrophy cells on both frozen and paraffin embedded tissue sections. These data suggest that regional draining lymph nodes of prostate carcinoma patients can be used as a source of human lymphocytes for generating human immunoglobulin-secreting hybridomas reactive with human prostate cells.     

Proliferation of Dunning R-3327 rat prostatic adenocarcinoma and of its sublines (CUA and CUB)

Proliferating cells in tumors of the androgen-dependent R-3327 and its partially dependent and autonomous sublines, CUA and CUB, were examined. Two types of cells, myoepithelial cells and large stromal cells, in the R-3327 have a high proliferating ability, as estimated by thymidine uptake. These cells were also observed in the normal dorsolateral prostate of rats, the lobes from which R-3327 originated. CUA is a squamous carcinoma, which proliferates at the border of the epithelial layer and large stromal cells. The tumor cells of CUB appear as clusters of large stromal cells which have a strong potential for dividing. Therefore, R-3327 contains two types of dividing cells, and their uncoordinated proliferation may be responsible for causing the different features seen in the sublines.     

Differences in expression of oligosaccharide determinants by phenotypically distinct sublines of the Dunning 3327 rat prostate cancer

Oligosaccharides expressed by the 3327-H and 3327-MAT LyLu sublines of the Dunning rat prostate cancer model have been compared in formalin-fixed and routinely paraffin-embedded tumour tissues. Binding by lectins of defined specificity has been employed to identify expression of seven oligosaccharide structures by primary and metastatic prostatic carcinoma cells. Neuraminidase digestion was employed to reveal determinants masked by sialic acid. The presence of core Man alpha 1----3(Man alpha 1----6)Man beta 1----4GlcNAc beta 1----4 determinants recognised by Con-A (Canavalia ensiformis) confirmed expression of complex-type glycoconjugates by plasma membrane and cytoplasmic components of the 3327-H tumour but only by cytoplasmic determinants within 3327 MAT LyLu variant tumour-cells. The only other oligosaccharide freely expressed by either tumour-subline was (GlcNAc beta 1----4GlcNAc beta 1----4-)n, recognised by WGA (Triticum vulgaris). Prior to neuraminidase digestion, PNA (Arachis hypogaea) (which identifies Type I oligosaccharides: Gal beta 1----3GalNAc-) bound to pseudoluminal membranes of the 3327-H tumour. However, ECG (Erythrina cristagalli) (which identifies type II oligosaccharides: Gal beta 1----4GlcNAc-) did not bind to this tumour. Unmasked Type I (Gal beta 1----3GalNAc-) and Type II (Gal beta 1----4GlcNAc-) oligosaccharides were not identified in the MAT-LyLu variant. After neuraminidase digestion, PNA-binding was identified along pseudoluminal plasma membranes within 3327-H tumours but only within the cytoplasm of 3327-MAT LyLu primary and metastatic tumour cells. Following neuraminidase digestion, ECG-binding was observed along pseudoluminal plasma membranes of 3327-H tumours and heterogeneously within the cytoplasm of primary, but not metastatic 3327-MAT LyLu tumours. Terminal alpha/beta GalNAc- residues recognised by SBA (Glycine max) were not freely expressed by either subline. These structures were readily detected along luminal membranes of 3327-H cells and weakly detected within the cytoplasm of primary but not metastatic MAT 3327-LyLu tumour cells following neuraminidase digestion. Fucosylated Type II structures Fuc alpha 1----2Gal(GalNAc)-), recognised by UEA-1 (Ulex europaeus-1) and GalNAc alpha 1----3GalNAc- structures recognised by DBF (Dolichos biflorus) were not identified as a component of either tumour subline. The different patterns of oligosaccharide expression, identified by lectin-binding, clearly differentiated between the two tumour sublines and distinguished them from normal prostatic epithelium. The Dunning 3327 rat prostatic cancer sublines offer a useful model with which to examine the relationship between cell-surface oligosaccharide structures and phenotypic variants within a defined tumour-cell population.(ABSTRACT TRUNCATED AT 400 WORDS)     

Time lapse videomicroscopic identification of Dunning R-3327 adenocarcinoma and normal rat prostate cells

A method for accurate prediction of prognosis in human prostatic cancer does not exist. The limitations of pathologic grading systems may result from the failure of standard pathological examination of fixed dead tissue to accurately assess the biological behavior of live tumor cells. Many of the sublines of the Dunning R-3327 rat adenocarcinoma are histologically similar yet differ widely in their metastatic potential. The nonmetastatic G, occasionally metastatic AT-1 and AT-2, and highly metastatic AT-3 and MAT-Lu Dunning sublines, and normal dorsal prostate were grown in culture and filmed by time-lapse videomicroscopy. Cell membrane ruffling, undulation and pseudopodal extension, vectoral translation, irregularity of pathway, and overall subjective motility (gestalt) were visually graded. Intra-assay, intra-observer, and inter-observer reproducibility were 75, 80 and 75% respectively. The combination of ruffling, pseudopodal extension and vectoral translation was most successful in identifying the six sublines. To validate this technique prospectively, five tumor sublines and two normal prostates were graded by 10 observers unfamiliar with the technique. Fifty-nine percent of unknowns were correctly identified when motility profiles were compared to previously developed standards by least sum of squares analysis. We devised a new technique for characterizing the motility of living prostate cells which was more accurate in identifying normal rat prostate and the Dunning sublines than standard pathological examination. Prostatic cancer cell motility may reflect biological behavior and metastatic potential and thus contribute to the assessment of an individual patient's prognosis.     

Estramustine in combination with vinblastine and mitomycin-C for patients with progressive androgen independent adenocarcinoma of the prostate

A phase II trial was performed to assess the antitumor activity and toxicity of estramustine in combination with vinblastine and mitomycin-C in 46 consecutive patients with androgen independent prostate cancer. All patients presented with disease progression following castrate serum testosterone levels and were treated for six consecutive weeks with three daily 140 mg doses of oral estramustine, vinblastine at 5 mg/m(2) weekly by intravenous bolus and mitomycin-C at 15 mg/m(2) every six weeks by intravenous bolus. Prostate specific antigen levels decreased by greater than 50% from baseline in 16 (41%; 95% CI 25-58%) and normalized in 11 (28%; 95% CI 14-45%) of 39 evaluable patients. Patients who demonstrated a greater than 50% reduction in PSA had a longer delay in time to disease progression. Non-hematologic toxicity was mild, predominately gastrointestinal. Hematologic toxicity was apparent in five patients with Grade III granulocytopenia and in 21 patients with Grade IV granulocytopenia of 43 evaluable patients for toxicity. Three patients were admitted to the hospital for neutropenic fever. Eight patients had Grade III thrombocytopenia, four patients had Grade IV thrombocytopenia, no bleeding occurred. Estramustine in combination with vinblastine and mitomycin-C is an active regimen. The non-hematologic toxicity was tolerable, while the hematologic toxicity required individual dosage reduction. The combination and the clinical significance of the decline in the PSA warrants further investigation.     

Expression of a transfected v-Harvey-ras oncogene in a Dunning rat prostate adenocarcinoma and the development of high metastatic ability

To investigate the role of oncogenes in the development of metastatic ability by prostatic cancer, the viral-Harvey-ras (v-H-ras) oncogene was introduced into the Dunning rat prostate adenocarcinoma cell line, AT2.1 by means of DNA transfection. The AT2.1 cell line is a cloned cell line that is anaplastic, rapidly growing, and has low metastatic potential; after subcutaneous (s.c.) inoculation in syngeneic rats, fewer than 10% of inoculated rats develop distant metastases. Calcium phosphate mediated DNA transfections of AT2.1 cells were performed with the v-H-ras oncogene or with control DNA. The in vitro growth rate of cloned transfectants, which contain and express the v-H-ras oncogene is similar to that of untransfected AT2.1 cells and of control transfectants. After s.c. inoculation in syngeneic rats, all transfectants produced rapidly growing tumors with similar growth rates. While control transfectants had low metastatic ability comparable to untransfected AT2.1 cells, the H-ras expressing transfectants metastasized in over 80% of inoculated rats. While the mechanism by which nonmetastatic Dunning tumor sublines spontaneously develop high metastatic ability in vivo during serial s.c. passage has not been addressed in the present studies, these studies do demonstrate that expression of an activated H-ras oncogene can reproducibly convert a tumorigenic nonmetastatic prostatic cell line to a highly metastatic state.     

Prostate-specific antigenic domain of human prostate specific antigen identified with monoclonal antibodies

With the use of five murine monoclonal antibodies (1A5, 2A4, 3F1, F5 and 3A12) and an antigen-affinity purified goat polyclonal IgG antibody, the presence of a prostate-specific antigenic domain in human prostate-specific antigen molecule was identified. The results were based upon a series of quantitative competitive inhibition assays of each 125I-labeled monoclonal antibody and polyclonal antibody binding to prostate-specific antigen by unlabeled monoclonal antibodies as inhibitors, and immunohistochemical examination of an extensive panel of human tissue specimens. A cluster of two epitopes that are spatially related or in close topographical proximity and represent a prostate-specific antigenic domain are defined by the monoclonal antibodies 1A5, 2A4, 3F1, and F5, 3A12, respectively.     

Tumor lysis syndrome in a patient with metastatic, androgen independent prostate cancer

Tumor lysis syndrome (TLS) is an uncommon, but well described, clinical entity that typically occurs following chemotherapy in patients with rapidly growing hematological malignancies. It is rarely described in patients with solid tumors. We report a case of TLS in a patient with metastatic adenocarcinoma of the prostate after treatment with paclitaxel chemotherapy.     

Antibodies reactive with autologous prostatic tissue in adenocarcinoma of prostate

Circulating autoantibodies reactive with interepithelial constituents of secretory epithelial cells of autologous human prostatic tissue in a patient with metastatic adenocarcinoma of the prostate have been observed by fluorescent microscopy. Identification of autoantibodies in prostatic cancer provides further evidence of the participation of immunologic factors, perhaps of an autoimmune nature, in diseases of the prostate.     

Role of prolactin in modulating the effects of tamoxifen on growth of the Dunning R3327 rat prostate adenocarcinoma

Tamoxifen (TAM) has previously been shown to inhibit growth of the Dunning R3327 rat prostate adenocarcinoma and to elevate serum prolactin levels. The purpose of this study was to determine the role of prolactin in modulating the effects of tamoxifen on growth of the R3327 prostatic adenocarcinoma. Intact and castrated Copenhagen-Fischer male rats bearing the Dunning R3327 rat prostatic tumor were divided into groups and injected sc five times per week for 16 weeks as follows: vehicle; TAM (0.5 mg/kg); haloperidol (HALO; 0.5 mg/kg); bromocriptine (CB-154; 5 mg/kg); TAM plus HALO; or TAM plus CB-154. In both intact and castrated rats, agents that either raised (HALO) or lowered (CB-154) serum prolactin had little effect on prostatic tumor growth when administered singly. In intact rats, average tumor diameter in vehicle-treated controls increased 421% 16 weeks after the start of the experiment, and treatment with TAM or TAM plus HALO reduced this tumor growth by approximately one-half. Interestingly, CB-154 administered in combination with TAM completely blocked TAM inhibition of tumor growth in intact rats. In contrast to these results in intact rats, average tumor diameter increased 129% in TAM- and 118% in TAM plus HALO-treated castrated rats and was significantly greater than the characteristic retardation of tumor growth (49% increase) that occurred in the vehicle-treated castrate controls. In addition, combined treatment of TAM plus CB-154 in castrate rats resulted in an even greater increase (188%) in average tumor diameter. The inhibitory effect of TAM on R3327 prostatic tumor growth in intact rats appears to be an indirect effect resulting from its ability to reduce serum testosterone levels. In contrast, the stimulatory effect of TAM in castrate rats appears to result directly from an estrogen-like action, which can directly enhance prostatic tumor growth in the presence of low levels of circulating androgens; this stimulatory effect of TAM is more pronounced when prolactin levels are suppressed by CB-154. Clearly, castration alone is more effective than TAM therapy alone or in combination with castration in the retardation of the growth of the androgen-dependent R3327 prostatic tumor in rats.     

Microarray analysis of differential gene expression in androgen independent prostate cancer using a metastatic human prostate cancer cell line model

Progression to androgen independence (AI) leading to uncontrolled cell growth is the main cause of death in prostate cancer. While almost all patients with metastatic prostate cancer will initially respond to anti-androgen treatments, the majority will fail hormonal treatments in less than 2 yrs. Both genetic and epigenetic alterations in gene expression contribute significantly to the development of AI. To investigate this we have used an in vitro cell line model of AI prostate cancer from which we have identified a number of differentially expressed genes associated with progression to AI in prostate cancer. We used an in vitro cell line model of AI prostate cancer, to study differential gene expression using cDNA microarray analysis and corroborated the microarray results with Ribonuclease Protection Assay (RPA). Approximately 4480 out of 7075 (63.3%) cDNA cloned genes were differentially expressed, of which, 6 genes were differentially expressed by at least fivefold. RPA was used to corroborate the microarray results for the five most highly differentially expressed genes. Using an in vitro cell line model and microarray analysis we have identified a number of candidate genes for further investigation in AI prostate cancer.     

抗人前列腺干细胞抗原单克隆抗体的制备及鉴定

目的制备并鉴定抗人前列腺干细胞抗原(PSCA)的单克隆抗体.方法以纯化的PSCA为抗原免疫Balb/c小鼠,经细胞融合、克隆化培养建立抗人PSCA的杂交瘤细胞株,采用免疫组化、Western blot等方法鉴定其特异性.结果利用小鼠杂交瘤技术,建立了1株分泌抗PSCA抗体的杂交瘤细胞系D3,经鉴定抗体类别为IgG1,能特异性识别PSCA,与其他因子无交叉反应.PSCA在5例正常前列腺组织、10例良性前列腺增生组织、38例前列腺癌组织中有不同程度的阳性染色,95%(38/40例)的前列腺癌PSCA染色呈阳性、强阳性染色,50%～100%的肿瘤细胞着色,与良性前列腺增生及正常前列腺组织相比,其染色强度及范围的差异有统计学意义(P＜0.01).结论成功制备了组织特异性的抗人PSCA单克隆抗体,为前列腺癌的诊断和免疫治疗提供了新的手段.     

Production of monoclonal antibodies specific to human lung cancer--squamous cell carcinoma and adenocarcinoma

Hybridomas secreting monoclonal antibodies specific for human lung cancer were produced by fusing immunized mouse spleen cells with mouse myeloma line 653. Methods: BALB/c mice were hyperimmunized with two different histological types of human lung cancer (squamous cell carcinoma and adenocarcinoma) obtained from surgery. An immunocytoadherence test was used to select hybridomas secreting antibodies that bound to the patient's lung tumor but did not bind to a B-lymphoblastoid cell line derived from the same patient. Five stable antibody-producing hybrids (2C6, 5D7 and 5E8 derived from squamous cell carcinoma, 5C7 and 2B7 derived from adenocarcinoma) have been established and cloned. Results: The antibodies bound to other lung tumors and established lung tumor cell lines of the same histological type. Also, some significant reactivity was observed with large cell carcinoma, but the antibodies did not react with small cell carcinoma of the lung, bronchiolo-alveolar cell carcinoma, cancer of the stomach and esophagus, melanoma, several types of leukemias, normal human lung tissue, fibroblasts, or erythrocytes of type A, B or O. Two of the five antibodies, 5C7 and 5E8 cross-reacted with one breast cancer obtained from surgery and 5C7 also cross-reacted with one melanoma biopsy specimen. Moreover, the antibodies have been characterized according to their light and heavy chain isotypes by ouchterlony and radioimmunoassay.     

Pre-treatment nomogram for disease-specific survival of patients with chemotherapy-naive androgen independent prostate cancer

OBJECTIVE: Our objective was to develop a nomogram that predicts the probability of cancer-specific survival in men with untreated androgen-independent prostate cancer (AIPC). METHODS: AIPC was diagnosed in 129 consecutive patients between 1989 and 2002. No patient received cytotoxic chemotherapy. Univariate and multivariate Cox regression models were used to test the association between prostate-specific antigen (PSA) level at initiation of androgen deprivation, PSA doubling time (PSADT), PSA nadir on androgen deprivation therapy (ADT), time from ADT to AIPC, and AIPC-specific mortality. Multivariate regression coefficients were then used to develop a nomogram predicting AIPC-specific survival at 12-60 mo after AIPC diagnosis. Two-hundred bootstrap resamples were used to internally validate the nomogram. RESULTS: AIPC-specific mortality was recorded in 74 of 129 patients (57.4%). Other-cause mortality was recorded in 7 men (5.4%). Median overall survival was 52.0 mo (mean, 36.0 mo) and median AIPC-specific survival was 54.0 mo (mean, 35.0 mo). In univariate regression models, all variables were significant predictors of AIPC-specific survival (p < or = 0.02). In multivariate models, PSADT and time from androgen deprivation to AIPC remained statistically significant (p < or = 0.004). Bootstrap-corrected predictive accuracy of the nomogram was 80.9% versus 74.9% for our previous model. CONCLUSIONS: A nomogram predicting AIPC-specific survival is between 13% and 14% more accurate than previous nomograms and 6% more accurate than tree regression-based predictions obtained from the same data. Moreover, a nomogram approach combines several advantages, such as user-friendly interface and precise estimation of individual recurrence probability at several time points after AIPC diagnosis, which all patients deserve to know and all treating physicians need to know.     

Immunocytochemical demonstration of S phase cells by anti-bromodeoxyuridine monoclonal antibody in human prostate adenocarcinoma

Using a monoclonal antibody to bromodeoxyuridine and immunohistochemistry, we measured the incorporation of this thymidine analogue into the deoxyribonucleic acid of human prostate adenocarcinoma cells exposed in situ. Fifteen patients with prostate cancer were given an intravenous infusion of 500 mg. bromodeoxyuridine at needle biopsy to label tumor cells in the deoxyribonucleic acid synthesis phase (S phase). The tumor specimens were fixed with 70 per cent ethanol, embedded in paraffin, sectioned and stained by an indirect immunoperoxidase method using anti-bromodeoxyuridine monoclonal antibody as the first antibody. The results showed that this method demonstrated bromodeoxyuridine-labeled nuclei satisfactorily in tissue section. The bromodeoxyuridine labeling index, S phase fraction, was determined by counting the number of bromodeoxyuridine-labeled cells in the tissue sections. Grade 3 tumors averaged 4.37 +/- 0.48 per cent labeling versus 2.41 +/- 0.49 per cent in grade 2 tumors, and grade 1 tumor in the series had an S phase fraction of 1.36 +/- 0.39 per cent. The average S phase fractions for single gland, cribriform, fused and medullary were 1.16, 2.30, 3.74 and 4.95 per cent, respectively. The results obtained with S phase fraction measured with bromodeoxyuridine labeling proved to be comparable to the results of histological grade and growth pattern. Thus, the higher S phase fraction may indicate biological malignancy. Moreover, the degree of heterogeneity concerning S phase fraction distribution within prostate cancer tissue could be compared to the morphological appearance. Our preliminary results suggest that the measurement of bromodeoxyuridine labeling index in prostate cancer may prove to be a new objective and quantitative assay of biological potential of individual tumor.     

Broadening of transgenic adenocarcinoma of the mouse prostate (TRAMP) model to represent late stage androgen depletion independent cancer

BACKGROUND: The transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mimics PC-progression as it occurs in humans. However, the timing of disease incidence and progression (especially late stage) makes it logistically difficult to conduct experiments synchronously and economically. The development and characterization of androgen depletion independent (ADI) TRAMP sublines are reported. METHODS: Sublines were derived from androgen-sensitive TRAMP-C1 and TRAMP-C2 cell lines by androgen deprivation in vitro and in vivo. Epithelial origin (cytokeratin) and expression of late stage biomarkers (E-cadherin and KAI-1) were evaluated using immunohistochemistry. Androgen receptor (AR) status was assessed through quantitative real time PCR, Western blotting, and immunohistochemistry. Coexpression of AR and E-cadherin was also evaluated. Clonogenicity and invasive potential were measured by soft agar and matrigel invasion assays. Proliferation/survival of sublines in response to androgen was assessed by WST-1 assay. In vivo growth of subcutaneous tumors was assessed in castrated and sham-castrated C57BL/6 mice. RESULTS: The sublines were epithelial and displayed ADI in vitro and in vivo. Compared to the parental lines, these showed (1) significantly faster growth rates in vitro and in vivo independent of androgen depletion, (2) greater tumorigenic, and invasive potential in vitro. All showed substantial downregulation in expression levels of tumor suppressor, E-cadherin, and metastatis suppressor, KAI-1. Interestingly, the percentage of cells expressing AR with downregulated E-cadherin was higher in ADI cells, suggesting a possible interaction between the two pathways. CONCLUSIONS: The TRAMP model now encompasses ADI sublines potentially representing different phenotypes with increased tumorigenicity and invasiveness.