Immunotherapeutic alternatives in superficial bladder cancer. Interferon, interleukin-2, and keyhole-limpet hemocyanin.

Interleukin-2, IFNs, and TNF are biologic response modifiers that are part of an intricate network of interacting cytokines released during an immune response. Lack of sufficient endogenous cytokine activity secondary to the immunosuppressive effects of tumor growth may be overcome by direct, local application of biologic response modifiers or immunostimulants such as BCG or KLH. Bacillus Calmette-Guérin remains the most effective immunotherapeutic agent for superficial transitional-cell carcinoma. Although the mechanism of action is unknown, the weight of evidence suggests that local cytokine release is involved in the effector pathway. Recent data have shown that the local application of new single-agent immunotherapies can have an effect on superficial transitional-cell carcinoma and CIS similar to that of chemotherapeutic agents and nearly identical to that of BCG. But, unlike the situation with chemotherapy or BCG, these effects are attended by minimal or no toxicity. Chemotherapy and BCG failures have also shown responses to direct instillation of cytokines. Further understanding of the exact mechanism of action of these agents and of their interaction should lead to the optimal antitumor regimen with the least toxicity. Determining the degree of host immunoresponsiveness and which combination of cytokines or immunotherapeutic or chemotherapeutic agents is most effective for a specific tumor type is the challenge for the future.

Suramin interference with transforming growth factor-beta inhibition of human renal cell carcinoma in culture.

Suramin is a polyanionic compound used clinically for the treatment of trypanosomiasis, which is known to inhibit the action of many protein factors in vitro. Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory protein which inhibits the growth of renal cell carcinoma in culture. While suramin at 50-500 micrograms/ml had no significant effect on the growth of renal cell carcinoma in culture in our experiments, it did partially reverse the growth inhibition induced by TGF-beta in the two cell lines tested. This effect apparently is caused by suramin's direct interference with 125I-labeled TGF-beta's ability to bind to the cell, and not by any effect of suramin on the TGF-beta receptor. Furthermore, suramin dissociates TGF-beta bound to the cell with a t1/2 of less than 30 min. These results are consistent with those previously reported regarding suramin's interaction with other protein growth factors, and suggest that suramin may interact with the TGF-beta protein itself to inactivate it.

Epidermal growth factor receptor gene analysis in renal cell carcinoma.

The epidermal growth factor receptor binds the mitogens epidermal growth factor and transforming growth factor-alpha. Increased expression of the epidermal growth factor receptor has been noted in many types of tumors and is associated with gene amplification in several including epidermoid carcinoma, lung carcinoma, breast carcinoma and glioblastoma. We have recently observed increased expression of the epidermal growth factor receptor messenger RNA in neoplastic tissue relative to normal kidney tissue from patients with renal cell carcinoma. To determine if epidermal growth factor receptor gene amplification was present in renal cell carcinoma, DNA was extracted from renal cell carcinoma cell lines and from normal kidney and renal cell carcinoma tissues derived from radical nephrectomy specimens from thirty patients. DNA was analyzed by Southern blot hybridization. There was no epidermal growth factor receptor gene amplification detected in the renal cell carcinoma samples studied, indicating the increased epidermal growth factor gene expression observed in renal cell carcinoma does not occur through gene amplification. Unlike other tumors with enhanced epidermal growth factor receptor gene expression, amplification of this gene does not appear to be a common feature of renal cell carcinoma.

Expression of transforming growth factor alpha in normal human adult kidney and enhanced expression of transforming growth factors alpha and beta 1 in renal cell carcinoma.

Normal kidney and renal cell carcinoma tissues from ten patients were studied for mRNA and DNA for both transforming growth factors alpha and beta 1. Northern and Southern hybridizations were conducted on samples extracted from the solid tumor and surrounding normal tissues and two tumor-derived cell lines. Low levels of constitutive expression of TGF-alpha mRNA were detected in all normal kidney tissues; six of the ten patients, however, demonstrated an increased (2- to 8-fold) expression of TGF-alpha in the tumor versus normal kidney as determined by densitometry of RNA blots. All ten patients had elevated mRNA levels for TGF-beta 1 in the tumor (2.5-to 22-fold increase) relative to normal kidney. Two tumor-derived cell lines also expressed TGF-alpha and TGF-beta 1 mRNA. Southern blot hybridization of the DNA extracted from the normal tumor pairs revealed no gene amplification or gross rearrangement for either the TGF-alpha or TGF-beta 1 genes. These results demonstrate the expected constitutive expression of TGF-beta 1 by normal kidney; however, the constitutive expression of TGF-alpha by Northern blot analysis in normal adult human kidney is previously unreported. Enhanced expression of TGF-alpha and TGF-beta 1 mRNA in solid tumor may be related to the development of renal cell carcinoma.

Epidermal growth factor receptor gene expression in normal human kidney and renal cell carcinoma.

The epidermal growth factor receptor (EGFr) is a transmembrane glycoprotein detected on many cells and tissues including neoplastic and normal kidney. EGFr binds the mitogenic polypeptide hormone epidermal growth factor (EGF) as well as EGF-related transforming growth factor-alpha (TGF-alpha). Increases in EGFr gene expression and protein production have been implicated in the development of the malignant phenotype for certain cancers. To determine if alterations in EGFr gene expression are present in human renal cell carcinoma, paired samples of normal and neoplastic renal tissue from ten patients with advanced renal cell carcinoma were analyzed for EGFr mRNA content by Northern blot hybridization. The EGFr gene was constitutively expressed in 90% of normal kidney samples. In seven out of nine evaluable patients, tumors expressed 1.7 to 8.4 times more EGFr mRNA than corresponding normal tissue. Two patients showed no elevation of tumor EGFr mRNA and one patient had no identifiable EGFr transcripts in either neoplastic or normal kidney. Expression of EGFr mRNA was also detected in three tumor-derived and two established renal cell carcinoma cell lines. EGFr transcripts were not found in tumor infiltrating lymphocytes (TIL). These findings suggest that overexpression of EGFr mRNA may be associated with malignant transformation in renal cell carcinomas.

Transforming growth factor-beta inhibits the growth of renal cell carcinoma in vitro.

Transforming growth factor-beta (TGF-beta) is a bifunctional growth regulatory hormone which inhibits the growth of many normal and neoplastic epithelial cell lines in monolayer culture. Endogenous and exogenous TGF-beta may influence cell proliferation through autocrine and paracrine binding to specific TGF-beta receptors. Growth effects of TGF-beta on human renal cell carcinoma cell lines have not been thus far described. We have studied the effects of TGF-beta on one renal tumor-derived (UOK-39) and one established (SKRC-7) renal cell carcinoma cell line. Exogenous addition of biologically active TGF-beta to cell cultures at concentrations between two and five ng./ml. inhibited the anchorage-dependent growth of UOK-39 by 75% and SKRC-7 by 44%, relative to controls. Low numbers of high affinity TGF-beta receptors were identified on both cell lines in 125I-TGF-beta binding assays. UOK-39 cells bound radiolabeled TGF-beta with higher affinity than SKRC-7 cells, but had fewer receptor sites, by Scatchard analysis of binding data. These results suggest that TGF-beta inhibits proliferation of renal carcinoma cells in vitro which may be mediated through binding of exogenous TGF-beta to functional TGF-beta receptors on the cell surface.

Expression of mRNA for transforming growth factors-alpha and -beta and secretion of transforming growth factor-beta by renal cell carcinoma cell lines.

Transforming growth factors (TGFs)-alpha and -beta are regulatory polypeptides that reversibly confer a transformed phenotype upon normal cultured fibroblasts. TGF-alpha is synthesized primarily by malignant cells and shares many properties with the tissue mitogen, epidermal growth factor (EGF). The expression of TGF-beta mRNA has been demonstrated in a variety of normal and malignant cell types, some of which secrete the mature protein in an inactive form. To investigate the role of TGFs in human renal cell carcinoma (RCC), we used two renal tumor-derived cell lines and one established RCC cell line for analysis of TGF-alpha and TGF-beta mRNA production and for evaluation of TGF-beta protein secretion. By northern blot hybridization, all three RCC cell lines expressed TGF-alpha and -beta mRNA. In addition, TGF-beta activity was found in the conditioned medium from these cells. The secreted TGF-beta protein, however, displayed biological activity only after activation by acid-treatment. These data demonstrate the constitutive expression of TGF-alpha and -beta mRNA by RCC cell lines and, also, the secretion by this tumor of endogenous TGF-beta protein in a latent form.