Kathryn M. Roberson

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. Genes Chromosomes Cancer 25:53–59, 1999 Published 1999 Wiley‐Liss, Inc.

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.

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

SummaryA novel human prostatic stromal cell culture, designated DuK50, has been passedin 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.

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

Abstract 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 μM. 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.

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 BLADDER CANCER CELL LINE :BK10

SummaryMolecular 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 too for the further study of bladder carcinoma oncogenesis and gene therapy.

Isolation and growth of human primary prostate epithelial cultures

Human primary prostate epithelial cultures derived from radical prostatectomy specimens were isolated and propagated using serum-free medium and culture technique developed in this laboratory. In this report, growth and characterization data from the first 95 radical prostatectomies is presented. Detailed methodology for the isolation and growth of prostate epithelial cultures is described.

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

SummaryThe 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 >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.