Loss of heterozygosity occurs centromeric to RB without associated abnormalities in the retinoblastoma gene in tumors from patients with metastatic renal cell carcinoma.

Tumor suppressor genes have been found to have loss of function in a number of malignancies. This loss of function is believed to contribute to malignant transformation or metastatic spread. In the present study, expression of the retinoblastoma (RB) tumor suppressor gene was examined in cell lines and tumor tissue obtained from primary renal and metastatic sites in patients with metastatic renal cell carcinoma. Three of fifteen (20%) of informative renal carcinoma cell lines had loss of heterozygosity (LOH) in the RB gene (intron 20) detected by polymerase chain reaction analysis. Using restriction fragment length polymorphism (RFLP) analysis, 7 of 22 (32%) informative cell lines had LOH centromeric to the RB gene at the D13S1 locus. No LOH (0 of 7) was seen telomeric to the RB gene at the D13S2 locus. None of the 28 cell lines examined had decreased RB mRNA expression compared with short-term cultures of proximal renal tubular cells. Western blotting demonstrated phosphorylated and unphosphorylated forms of RB protein of expected molecular weight in all 41 cell lines (33 primary and 8 metastatic) examined. Twenty-nine primary cell lines and 6 metastatic cell lines all demonstrated normal immunohistochemical staining. Loss of RB immunohistochemical staining in paraffin-embedded tissue was detected in none of the primary tumors (0 of 30) or metastatic tumors (0 of 12). The absence of abnormalities of RB expression detected in these renal cell carcinomas suggests that abnormalities of the RB gene are not central to malignant transformation or progression in this tumor type; however, another tumor suppressor gene centromeric to the RB locus may be important in renal cell carcinogenesis.

Suramin inhibits proliferation and DNA synthesis in transitional carcinoma cell lines.

Suramin is polysulfonated naphthylurea which has a broad range of antitumor activity. The mechanism of action of suramin is not completely understood, although it is known to inhibit enzymes in all cellular compartments, inhibit steroidogenesis and interfere with ligand-receptor binding. Suramin's large molecular size and negative charge should make it poorly absorbed through the bladder mucosa, a desired characteristic for an intravesical chemotherapeutic agent. We examined the ability of suramin to inhibit thymidine uptake and decrease cellular proliferation in 4 transitional cell carcinoma cell lines grown in vitro to determine if suramin might be a new candidate drug of treatment for patients with superficial bladder cancer. Suramin inhibited cellular proliferation of all cell lines tested (MBT2, T24, RT4 and TCCSUP) in a dose-dependent fashion. Fifty per cent inhibition of cellular proliferation compared with controls was seen with suramin concentrations of 250 to 400 micrograms/ml. by, at most, 5 to 9 days of exposure. The cell line RT4 was the cell line most sensitive to the growth inhibitory effect of suramin, with 50% growth inhibition compared with controls achieved after 3 days' exposure to a suramin concentration of 100 micrograms/ml. Suramin inhibited DNA synthesis in a dose-dependent fashion, as measured by thymidine uptake, in 3 of the 4 cell lines tested (MBT2, T24, and RT4). Suramin inhibited thymidine uptake by TCCSUP in a dose-dependent fashion, but did not achieve statistical significance.

Suramin inhibits bone resorption and reduces osteoblast number in a neonatal mouse calvarial bone resorption assay.

The antineoplastic properties of suramin, a polyanionic agent with demonstrated antigrowth factor activity, are under evaluation in vitro, in vivo, and in clinical trials. Suramin has been shown to have antitumor activity in patients with advanced, hormone refractory prostate cancer. During these trials, significant resolution of osseous pain was observed in nearly three quarters of the patients treated with suramin. To evaluate the effect of suramin on bone cells, we studied the effect of suramin on bone resorption in a neonatal mouse calvarial assay. Suramin inhibited bone-resorbing activity in a dose-related fashion and had an additive effect with calcitonin. Calvaria pretreated with suramin had less bone-resorbing activity, fewer attached osteoblasts, and less medium alkaline phosphatase activity than control calvaria. Suramin also inhibited osteoclastic release of tritiated proline from labeled bone in a dose-dependent fashion. The effect of metastatic prostate carcinoma on bone is incompletely understood, but may be moderated by tumor-produced factors and/or cytokines. The effects of several such agents, therefore, were examined in combination with suramin. Bone resorption induced by PTH, epidermal growth factor, tumor necrosis factor, and a tumor-produced factor, PTH related-protein, was blocked by suramin. The ability of suramin to inhibit the bone-resorbing effects of several cytokines suggests that its mechanism may involve direct action on bone metabolism. Autoradiography performed on calvaria treated with labeled suramin demonstrated heavy deposition of suramin on the outer surface of the matrix, adjacent to osteoblasts and osteoclasts lining the outer table, suggesting that bone cells may be subject to high local concentrations of the drug, in keeping with this hypothesis.

Molecular and cellular characterization of human renal cell carcinoma cell lines.

Recent studies have suggested that a tumor suppressor gene located in the region 3p21-26 of chromosome 3 is essential to the genesis of sporadic renal cell carcinoma (RCC) and that other tumor suppressor genes located on other chromosomes may be involved with progression of this malignancy. The cellular heterogeneity of solid tumors complicates their analysis. In order to analyze a homogeneous population of tumor cells and identify genetic changes associated with histology in renal cortical tumors, we have established and characterized 35 RCC lines derived from tumor tissue from 31 patients with renal cell carcinomas. The overall success rate in establishing these cell lines from fresh or frozen specimens was 75% (18 of 24) and 35% (17 of 48), respectively. These lines differed in their morphology, growth rates, and tumorigenicity in athymic nude mice. Molecular characterization utilizing DNA fingerprinting and restriction fragment length polymorphism deletion analysis was performed to detect somatic mutations and loss of heterozygosity on the short arm of chromosome 3. Analysis revealed loss of heterozygosity on chromosome 3p in 25 cell lines derived from 28 informative nonpapillary forms of RCC (89%). Deletion-mapping analysis showed the retention of the distal locus, D3S18, in one of the RCC cell lines, which further localized the position of the putative tumor suppressor gene to the region proximal to D3S18. Although deletions on chromosome 3 have been recently suggested to be specific to the clear cell-type phenotype, our results revealed no correlation between loss of heterozygosity and clear or granular cell types.

The effect of suramin, tumor necrosis factor and interferon gamma on human prostate carcinoma.

Suramin, an antiparasitic agent which has been shown to block the stimulatory effect of growth factors on certain cancers, is currently being evaluated in clinical trials and as an antineoplastic agent in patients with advanced prostate carcinoma. Preliminary studies suggested that suramin inhibits the growth in vitro of human prostate carcinoma. The present study was performed in order to evaluate the effect of suramin, recombinant human tumor necrosis factor (TNF), interferon gamma and the combination of suramin plus TNF or interferon gamma on proliferation of PC-3, a human, hormone unresponsive prostate carcinoma cell line. In medium containing 2% fetal calf serum (FCS) suramin, at doses of 10 microM, 30 microM and 100 microM (levels readily achievable in humans) inhibited proliferation of PC-3. TNF, at a concentration of 500 units/ml., induced an approximately 50% inhibition of growth of PC-3. The combination of suramin plus TNF induced a greater inhibition of growth than did either agent alone, even at the low dose of 10 microM suramin. Interferon gamma, 500 units/ml., inhibited PC-3 growth. However, the combination of interferon gamma plus suramin (30 microM) induced less inhibition of proliferation than did interferon gamma alone. These results may serve as a rationale for clinical trials employing the combination of TNF plus suramin in patients with advanced prostate carcinoma.

Identification of features of metaplastic cells in sputum for the detection of squamous-cell carcinoma of the lung.

Digitized images of 1,556 squamous metaplastic cells from the sputum of 15 patients with confirmed squamous-cell carcinoma of the lung and 13 subjects without apparent neoplasia were analyzed to determine if features existed within these relatively normal cells that could be used to differentiate between the two populations. Results indicate that such marker features do exist. A cross-validation study using an additional 465 cells confirmed the conclusion that squamous metaplastic cells from patients with squamous-cell carcinoma of the lung do differ from those of subjects without cancer. While such marker features alone are not reliable enough to assist pathologists in the diagnosis of squamous-cell carcinoma of the lung, they might, in sufficient numbers, be used to identify patients for whom follow-up testing would be advised.

The reliability of cytopathologists' classifications of bronchial epithelial atypias from Kodachrome slides.

The accuracy of computerized cell image analysis techniques for classification of atypical cells is determined by comparing computer-generated classifications with those made by cytopathologists. This measure of accuracy depends not only on the reliability of the computer classifications but also on the reliability of the cytopathologists' classifications. This study reports on the observed reliability of cytopathologists' classifications of squamous epithelial atypias in sputum across cytopathologists and two different classification times. Results indicated the percentage agreement among cytopathologists and computerized cell image analysis techniques. It is recommended that, in the future, all analyses of computerized classification schemes by interpreted in light of the consistency of the cytopathologists' classifications.