Molecular cytogenetic analysis of the bladder carcinoma cell line BK-10 by spectral karyotyping.

The bladder cancer cell line BK-10 was established from a grade III-IV transitional cell carcinoma (TCC). BK-10 is near-tetraploid (+/-4n) and consists of two subclones with 20-25 structural aberrations. Here we report the cytogenetic analysis of BK-10 by G-banding, spectral karyotyping (SKY), and FISH. SKY refers to the hybridization of 24 differentially labeled chromosome painting probes and the simultaneous visualization of all human chromosomes using spectral imaging. SKY enabled us to confirm 12 markers in BK-10 previously described by G-banding, redefine 11 aberrations, and detect 4 hidden chromosomal rearrangements, 2 of which had been identified as normal or deleted copies of chromosome 20 and 1 as a normal chromosome 3. Twenty out of 21 translocations identified were unbalanced. FISH analysis of BK-10 using chromosome arm-specific paints, centromere probes, and oncogene/tumor suppressor gene-specific probes revealed a deletion of CDKN2A (p16) in all copies of chromosome 9, a low-level amplification of MYC (five copies), and loss of one copy of TP53; detected the presence of the Y chromosome in a hidden translocation; and detected four copies of ERBB-2. A probe set for BCR and ABL verified breakpoints for all translocations involving chromosomes 9 and 22. A new karyotype presentation, "SKY-gram," is introduced by combining data from G-banding, SKY, and FISH analysis. This study demonstrates the approach of combining molecular cytogenetic techniques to characterize fully the multiple complex chromosomal rearrangements found in the bladder cancer cell line BK-10, and to refine the chromosomal breakpoints for all translocations.

Peptidyl membrane-interactive molecules are cytotoxic to prostatic cancer cells in vitro.

Cytotoxic membrane disruption via lytic peptides is a well-recognized mechanism of immune surveillance for antifungal and antibacterial host protection. Naturally occurring lytic peptides were shown to exhibit antitumor activity as well. Peptidyl membrane-interactive molecules (MIMs) are synthetic lytic peptides specifically designed to maximize antitumor activity. We tested nine novel Peptidyl MIMs for activity against four androgen-insensitive prostate-cancer cell lines using a standard microculture tetrazolium (MTT) assay. Five Peptidyl MIMs known to form alpha-helical secondary structures were active against prostate carcinoma and were chosen for further study. Three peptides configured in beta-pleated sheets were noticeably less effective. Concentrations lethal to 50% of the prostate-cancer cell lines treated (D50 values) with the five chosen Peptidyl MIMs ranged from 0.6 to 1.8 microM. For comparison, two alpha-helically structured peptides, D2A21 and DP1E, were tested on several other cancer types: breast (n = 2), colon (n = 2). bladder, cervical and lung carcinomas (n = 1 each). Resulting LD50 values obtained in breast carcinoma cells were significantly higher (P < 0.05) than those observed in prostate cancer cells. LD50 values recorded for D2A21 and DP1E in cervical, colon, bladder, and lung cancer lines were similar to those obtained in prostate cancer cells. As compared with cisplatin, a standard chemotherapeutic drug, the LD50 values recorded for D2A21 were significantly lower (P < 0.04) in prostate-cancer cell lines, suggesting the therapeutic efficacy of Peptidyl MIMs. These data demonstrate for the first time the cytotoxic potential of Peptidyl MIMs against prostate cancer cells and suggest a dependence on a specific secondary alpha-helical structure of the peptide.

Isolation and characterization of a novel human bladder cancer cell line: BK10.

Molecular studies of bladder carcinomas have aided in determining causative genetic events and the prognosis of cancers endowed with certain abnormalities. In vitro bladder cancer characterization of key cytogenetic alterations is useful for study of molecular changes that may promote oncogenic events. In our laboratory, a novel human bladder cancer cell line, BK10, has been established in vitro and passaged for more than 20 mo. This new bladder cancer cell line (BK10) was derived from bladder tissue containing grade III-IV/IV transitional cell carcinoma. Bladder cancer tissue was obtained at the time of radical cystoprostatectomy extirpation. Cell cultures derived from this surgical sample exhibited an epithelial morphology and expressed epithelial cytokeratins. Immunostains of BK10 were negative for prostate specific antigen (PSA), fibronectin, smooth muscle actin alpha, and desmin. Karyotypic analysis revealed an aneuploid chromosomal content <4n> with many numerical and structural abnormalities previously linked to bladder oncogenesis. Translocations occurred in chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 16, 17, 19, 20, 21, 22, X and Y. G-banding analysis revealed rearrangements involving chromosomes 9q and 17p, and the location of the ab11 oncogene and the p53 gene, respectively. The availability of this bladder cancer cell line will provide a useful tool for the further study of bladder carcinoma oncogenesis and gene therapy.

Fenretinide: induction of apoptosis and endogenous transforming growth factor beta in PC-3 prostate cancer cells.

N-(4-Hydroxyphenyl)retinamide (4-HPR, Fenretinide) is a retinoid derivative with antineoplastic activity in various tumor types including prostate carcinoma. The mechanism of action of 4-HPR toxicity is unknown. 4-HPR induces apoptosis in leukemia- and lymphoma-derived cells, neuroblastoma, and small cell lung cancers. The present study was designed to investigate: (a) the mechanism of 4-HPR cytotoxicity in prostate cancer cells; and (b) correlate increased expression of transforming growth factor beta 1 (TGF beta 1) with induction of apoptosis. 4-HPR exposure to PC-3 cells in vitro was associated with apoptosis as evidenced by increased incidence of hypodiploid nuclei in propidium iodide fluorescence histograms and DNA fragmentation. An increase in the percentage of nuclei in the G1 phase of the cell cycle preceded induction of apoptosis. TGF beta 1-increased expression was noted in mRNA levels and in secretion of active TGF beta 1 into culture media. TGF beta 1 and TGF-beta receptor type II detected immunohistochemically were increased in 4-HPR-treated PC-3 cells. Furthermore, 4-HPR-induced cytotoxicity in PC-3 cells was abrogated by the addition of anti-TGF beta 1 antibody. In BT-20 cells, a 4-HPR-resistant breast carcinoma cell line, apoptosis was not observed after exposure to 4-HPR nor was TGF beta 1 expression enhanced in stained cells or in conditioned media. It is concluded that 4-HPR induces the expression of TGF beta 1 in association with the induction of apoptosis.

Induction of apoptosis by diethylstilbestrol in hormone-insensitive prostate cancer cells.

BACKGROUND: Diethylstillbestrol (DES) and diethylstilbestrol diphosphate (DESdP) are effective agents for the treatment of advanced prostate cancers. Tumor-inhibiting effects of DES and DESdP are presumed secondary to suppression of androgen production in vivo. Little is known, however, about the direct cellular mechanisms of the tumor inhibition. Estrogens have been reported not only to stimulate growth but also to disrupt microtubule formation in prostate cancer cells. PURPOSE: The study was designed to examine and compare mechanisms of in vitro growth inhibition of DES and DESdP in human androgen-insensitive prostate cancer cells (DU145, 1-LN, and PC-3) and human androgen-sensitive prostate cancer cells (LNCaP) and to examine estrogen receptor modulation of such effects. METHODS: The cytotoxic effects of DES and DESdP were examined in vitro by use of a standard microculture tetrazolium assay to quantitate numbers of viable cells. Immunofluorescence microscopy, DNA fragmentation analysis, and fluorescence flow cytometry were used to investigate microtubules, the induction of apoptosis, and changes in cell cycle distribution. The degree of estrogen receptor positivity of untreated and treated cells was determined by immunohistochemistry and quantitative image analysis. RESULTS: LD50 levels (the dose at which 50% of cells are no longer viable) in the concentration range of 19-25 microM were observed for both DES and DESdP in all cell lines examined. DESdP-induced growth inhibition was found to be dependent on heat-labile phosphatases present in fetal calf serum. DES-induced cytotoxicity was not affected by the presence of 17 beta-estradiol, and it was not dependent on the presence of estrogen receptor. Estrogen receptor-positive cells and estrogen receptor-negative cells were equally responsive to DES. PC-3 cells stained with fluorescent anti-tubulin, phalloidin (actin stain), and 4',6-diamidino-2-phenylindole (DNA stain) showed no inhibition of microtubules or actin filaments but revealed the presence of apoptotic bodies in the nuclei. Fluorescence flow cytometry of nuclear DNA content of propidium iodide-stained nuclei from androgen-insensitive prostate cancer cells treated with 15 or 30 microM DES or DESdP revealed an increase in relative numbers of hypodiploid (apoptotic) nuclei, a depletion of G1- and S-phase cells, and an accumulation of cells in G2/M phase. Conversely, androgen-sensitive cells contained a lower percentage of hypodiploid nuclei but no accumulation of cells in G2/M phase. CONCLUSIONS: Direct cytotoxic effects of DES in prostate cancer cells are estrogen receptor independent and do not involve disruption of microtubule architecture but do involve the promotion of cell cycle arrest and apoptosis. These are the first data confirming direct cytotoxic effects of DES and DESdP in prostate cancer cells via an apoptotic mechanism. IMPLICATIONS. These results suggest that DES and DESdP have potential value as agents against androgen-insensitive prostate neoplasms through induction of an apoptotic cascade.

Glutathione and glutathione S-transferase in benign and malignant prostate cell lines and prostate tissues.

Metastatic prostate adenocarcinoma is unresponsive to alkylator chemotherapy with virtually no prolonged remissions. Glutathione (GSH) and glutathione S-transferase (GST) have been reported to play a role in tumor resistance to alkylator therapy; however, there are no baseline studies that have investigated and compared GSH and GST in human prostate cell lines and tissues. Thus, we determined the GSH content and GST activity in benign prostate, in primary and metastatic prostate adenocarcinoma tissues, in immortal adenocarcinoma cell lines, and in primary cell cultures derived from both benign prostate and primary prostatic carcinoma tissue. The GSH content was higher in the immortal cell lines than in the fresh tissues and primary cultures. Conversely, the GST activity was significantly higher in the tissues and primary cultures than in the cell lines. The GSH content and GST activity of the primary cultured prostatic cells were similar to those of the prostate tissues. The differences between the immortal prostate cancer cell lines and prostate tissue are of sufficient magnitude to suggest that in vitro results with cell lines may not extrapolate to prostate cancer in vivo. The GSH content and GST activity in a prostate specific antigen-secreting human prostate tumor xenograft, LuCaP23, maintained in nude mice were similar to those of human prostate tissue and primary cultures. Both the xenograft and primary cultures from patients with prostate cancer may be more appropriate models than established cell lines for investigating techniques to increase the effectiveness of alkylators in prostate cancer.

Isolation and characterization of a novel human prostatic stromal cell culture: DuK50.

A novel human prostatic stromal cell culture, designated DuK50, has been passed in vitro > 12 mo. Tissue cultures were obtained from material harvested within a normal region of a radical prostatectomy specimen. These monolayers exhibited normal fibroblastic characteristics with each cell having a flattened, elongated appearance. Karyotypic analysis revealed a normal, male 46, XY chromosomal content with no numerical or structural abnormalities. DNA analysis using a Cell Analysis Systems Image Analyzer confirmed a euploid DNA content (7.9 pg DNA). Cellular markers for verification of stromal cell type were performed by immunohistochemical techniques. DuK50 stained positive for vimentin and fibronectin. Immunostains for epithelial cytokeratins and prostate-specific antigen were negative, which ruled out contamination with prostatic epithelial cells. Negative immunostaining with desmin monoclonal antibody and light staining with smooth muscle actin alpha is consistent with the staining pattern of myofibroblasts. Response to various androgens, measured by a microculture tetrazolium assay technique, revealed a significant growth stimulation of DuK50. Soft agar invasiveness assays and tumorigenicity studies in nude mice were negative. DuK50 exhibits a rapid doubling time with excellent plating efficiency, thrives in a readily available media supplemented with fetal bovine serum, and passes with routine trypsin protocols. The availability of this prostatic stromal cell culture may facilitate studies on this cell type's role in growth factor modulation, drug and steroid metabolism, and stromal-epithelial interactions in the prostate.

The alpha 1C-adrenoceptor in human prostate: cloning, functional expression, and localization to specific prostatic cell types.

1. Benign prostatic hyperplasia (BPH) causes urinary obstruction in aging men that frequently requires surgery to relieve the symptoms of urinary retention, nocturia, and micturition. Smooth muscle tone which contributes to the urethral constriction in the enlarged gland appears to be mediated by the alpha 1-adrenoceptors. In this paper, molecular and pharmacological approaches are used to establish the role played by the alpha 1C-adrenoceptor subtype in the prostate. 2. The alpha 1-adrenoceptor subtype(s) expressed in human prostate were investigated by use of polymerase chain reaction (PCR), Northern blot, and in situ hybridization. The alpha 1C subtype was found in both prostate stromal and glandular cells while alpha 1B and alpha 1D subtypes were expressed in glandular cells. High expression levels for alpha 1C were observed in prostate cancer tissues in both stroma and glandular cells. 3. Full length alpha 1C-adrenoceptor cDNA was cloned from human prostate. Stable mammalian cell lines expressing human alpha 1B-, alpha 1C-, and alpha 1D-adrenoceptors were made. Membranes prepared from these cell lines and human prostate were used to evaluate the pharmacological profiles of human alpha 1B-, alpha 1C- and alpha 1D-adrenoceptors in comparison to human prostate. Leverage plot analysis of compound affinities determined by competition for [125I]-I-HEAT binding demonstrated that the alpha 1C subtype is the predominant alpha 1-adrenoceptor in human prostate. 4. The alpha 1-adrenoceptors cause smooth muscle constriction by coupling to IP3 turnover and intracellular Ca2+ release. Using stable cell lines to measure IP3 production in response to noradrenaline, alpha 1C stimulated IP3 production most efficiently, with alpha 1B at an intermediate level, while little IP3 above background could be detected with alpha 1D. These results supported a functional role of the alpha 1C-adrenoceptor on prostate smooth muscle constriction by noradrenaline stimulation.

Efficient gene transfer with adeno-associated virus-based plasmids complexed to cationic liposomes for gene therapy of human prostate cancer.

We have shown previously that treatment of rats bearing the Dunning R3327 MatLyLu prostatic tumor with human interleukin 2 (IL-2) gene-modified tumor cell preparations induces potent antitumor immunity in the animal. To test the clinical feasibility of using genetically modified tumor vaccines for the treatment of prostate cancer, we have explored the use of a simplified gene delivery system based on liposomes to introduce and express the IL-2 gene in the Dunning rat R3327 MatLyLu prostatic tumor cell line (MatLyLu) and in short-term cultures of primary human prostatic tumor cells. Liposome-DNA complexes containing the adeno-associated virus inverted terminal repeats exhibited 3-10-fold higher levels of gene transfer and IL-2 expression than did liposome complexes with non-adeno-associated virus containing plasmids. Single transfections resulted in IL-2 expression for an extended period of time that exceeded severalfold the amount of IL-2 secreted from retrovirally transduced MatLyLu cells. X-irradiation of cells (4000 rads) prior to transfection did not affect cytokine secretion, indicating that liposome-mediated gene transfer does not depend on cell proliferation. High levels of gene transfer and IL-2 expression were also achieved in short-term cultures of primary human prostatic tumor cells established from tumor specimens obtained following radical prostatectomy of cancer patients. Depending on the type of liposome used, IL-2 levels secreted from the human prostatic tumor cells were comparable to or exceeded the levels of IL-2 secreted from retrovirally transduced MatLyLu cells, which induced antitumor immunity in the rat model. The ability to culture and expand ex vivo human prostatic tumor cells, and the use of a simple and highly efficient gene transfer method to generate genetically modified tumor vaccines, set the stage for clinical exploration of gene-based immunotherapy of prostate cancer.

6-Methylene progesterone is cytotoxic to human cancer cell lines independent of its 5-alpha-reductase activity.

This investigation examined the effects of 6-methylene progesterone (6MP), an irreversible inhibitor of 5-alpha-reductase, on prostatic cancer (PC) cell lines. Dose titration microculture tetrazolium assays were used to evaluate cytotoxicity in cultures treated for 72 hr with 6MP (0-20 micrograms/ml). An androgen-sensitive cell line, LNCaP, was drug-sensitive with a mean 50% lethal dose value (LD50) of 2.632 +/- 0.103. Hormone-resistant PC cell lines 1-LN, DU 145, and PC3 also demonstrated sensitivity with LD50 values between 0.8579-1.110 micrograms/ml with a group average of 1.023 +/- 0.082 micrograms/ml. Increasing dosages of dihydrotestosterone in the growth media did not alter 6MP cytotoxicity in androgen-insensitive prostatic cancer cell lines. No correlation between androgen-responsiveness and 6MP-induced cytotoxicity was observed. In nonprostatic malignancies, 6MP inhibited adenocarcinoma cell lines with a mean group LD50 value of 0.7772 micrograms/ml +/- 0.110. J82, a transitional cell carcinoma cell line of bladder origin, exhibited an average LD50 value of 1.041 +/- 0.260. In an epidermoid cervical cancer cell line, ME180, an LD50 value of 0.5356 micrograms/ml +/- 0.010 was noted. In a melanoma cell line, Du Mel 6, a mean LD50 of 0.7428 +/- 0.023 micrograms/ml was achieved with 6MP. We conclude that 6MP, a novel 5-alpha-reductase inhibitor, has potential as a cytotoxic agent in prostatic carcinoma and additional human malignancies. Further study is justified.

Differential immunoreactivity of transforming growth factor alpha in benign, dysplastic and malignant prostatic tissues.

Immunohistochemical examination of radical prostatectomy specimens from 57 patients was performed to determine the differential expression of transforming growth factor alpha in the human prostate. In addition, epidermal growth factor receptor (EGFr) immunoreactivity was assessed in each case. Stromal versus epithelial staining was determined for each histological subtype: benign prostatic hypertrophy (BPH), prostatic intra-epithelial neoplasia (PIN), and prostatic cancer (CaP) by a single pathologist reviewer. TGFa staining was predominant in stroma while EGFr was localized to the epithelial basal cell layer. Immunoreactivity of both TGFa (P = 0.002) and EGFr (P < 0.001) revealed a significant reduction in CaP compared to BPH or PIN. Autocrine stimulation of EGFr by TGFa or other unrecognized factors may be present in CaP. Conversely, altered stromal influence of CaP via TGFa may be present. These observations could form the basis for future cancer therapeutic strategies using antagonist factors.

Melanoma patient antibody responses to melanoma tumor-associated antigens defined by murine monoclonal antibodies.

Sera from melanoma patients undergoing immunization with three distinct immunogens were evaluated for development of IgG antibody specific for the melanoma tumor-associated antigens (TAAs) GD3 and 250-kd glycoprotein (high molecular weight; HMW). Of 99 patients receiving either irradiated allogeneic melanoma cells admixed with bacillus Calmette-Guérin, vaccinia viral oncolysate melanoma cell membranes, or cholesterol hemisuccinate-modified melanoma cells, 12 were determined to have responded against GD3 and 17 against the HMW TAA. This reactivity was measured using both direct binding to purified TAA and specific inhibition of binding of murine monoclonal antibodies R24 and Me-1-14 for GD3 and HMW TAA, respectively. Preimmune sera from these patients did not react with these TAAs, nor did preimmune sera or follow-up sera from melanoma patients electing not to receive immunotherapy. These results suggest that melanoma patients can be immunized against these TAAs as presented on the melanoma cell membrane in an immunotherapy setting and that immunization using purified TAAs might likewise be feasible.

Serological response of non-human primates to human melanoma disialoganglioside GD3.

The immunogenicity of the disialoganglioside, GD3, a melanoma-tumor-associated antigen, has been evaluated in non-human primates. Sera from four chimpanzees and two monkeys were evaluated for anti-GD3 antibody activity by solid-phase radioimmunoassay using GD3 and control gangliosides as targets. Serum from one monkey, immunized with cells from a melanoma cell line, was strongly reactive with GD3, having a titer of greater than 2500. In contrast, serum from this animal was non-reactive with several other gangliosides including the structurally similar GM3. Anti-GD3 reactivity was also demonstrable, albeit in low titer, in the sera of an additional monkey and a chimpanzee. Each of these animals had likewise been immunized using cells from melanoma cell lines. On the basis of these observations, suggestive of a primate anti-GD3 antibody response, we initiated a series of immunizations of chimpanzee using purified GD3 bound to Salmonella minnesota, R595. IgG reactive with melanoma cells in the cell-binding assay was first detected in sera collected after 4 immunizations and increased in titer against each reactive melanoma cell line during the immunizations. Reactivity of this serum with melanoma cell lines demonstrated a direct correlation with the expression of GD3 by the respective cell line. Anti-GD3 reactivity was evident in solid-phase radioimmunoassay against purified GD3 beginning with serum collected after 11 immunizations. By comparison with its binding to the control ganglioside panel, this serum demonstrated strong specificity for GD3 (titer = 640) while having only marginal reactivity with GM3 (titer = 40). Immune serum from this animal was also able specifically to block subsequent binding of a murine IgM anti-GD3 antibody (DMab7) to target GD3 in solid-phase radioimmunoassay. Together, these observations suggest that GD3, in the form of a purified molecule bound to a bacterial matrix or as part of the intact melanoma cell membrane, can be immunogenic in non-human primates, and is able to elicit an antibody response of appropriate specificity.