Human Renal Carcinoma Cell Line Research Articles

Effects of low concentration of vinblastine on the anchorage-independent growth and in vitro invasion of human renal carcinoma cell lines

A study was conducted on the effect of vinblastine (VBL), an anti-mitotic drug that is commonly employed in the treatment of human renal cell carcinoma. When VBL was added to serum-free cultures of the ACHN and NT cell lines (both lines are of human renal carcinoma origin), a concentration of 1 μg/ml resulted in death of most of the cells of both cell types. However, at a concentration of 10 ng/ml or less, although the cells detached from the culture dish, many viable cells were observed. In addition, in an in vitro invasion assay, the invasiveness of these detached cells was demonstrated to be accelerated in comparison with the parent monolayed cells. This increase of invasion was observed in the treatment of TN-16 which is known to have a metaphasearresting effect, not to have an anti-cancer effect. When detached cells by VBL were inoculated into soft agar, their colony-forming ability was clearly increased in comparison with the parent cells or TN-16 treated cells. These results indicate that low concentration of VBL appears to increase the malignant potential of human renal carcinoma cells in culture.

AMPLIFICATION OF THE EXPRESSION OF MAJOR HISTOCOMPATIBILITY CLASS-I ANTIGENS BY INDUCERS OF DIFFERENTIATION AND GAMMA INTERFERON IN MURINE AND HUMAN SOLID TUMOR-CELL LINES

Treatment of B16 F10-9 mouse melanoma cell line and RC-29 human renal carcinoma cell line with chemical inducers of differentiation such as sodium butyrate (SB) hexamethylene bisacetamide (HMBA) and L-histidinol significantly increased the expression of major histocompatability complex (MHC) class I antigens. This effect depends on the continued exposure of the cells to these materials. Combined treatment of the B16 F10-9 melanoma cells with recombinant murine gamma interferon and SB increased the expression of MHC class I antigens in a synergistic manner. This effect of chemical inducers of differentiation might be of importance in rendering tumor cells more sensitive to the immune defense mechanisms.

Role of protein kinase C during interferon-gamma- and phorbol ester-stimulated immunocytochemical expression of ICAM-1 in human renal carcinoma cells.

Incubation of the human renal carcinoma cell line CaKi-1 with interferon-gamma (IFN-gamma) or the phorbol ester, phorbol-12-myristate 13-acetate (PMA) strongly stimulated the immunocytochemical expression of the intercellular adhesion molecule-1 (ICAM-1) in a dose-dependent manner. Since PMA is capable of activating the Ca2+/phospholipid-dependent protein kinase C (PKC), we investigated the role of this kinase during IFN-gamma signal transduction. Calcium ionophore A23187 significantly enhanced IFN-gamma- and PMA-induced ICAM-1 staining. While staurosporine, H7 and sphingosine, three known PKC inhibitors, blocked the PMA effect, only staurosporine abrogated the action of IFN-gamma. Finally, 24 h of PMA pretreatment with subsequent IFN-gamma stimulation enhanced ICAM-1 staining above values from cultures where IFN-gamma was omitted. This occurred despite the fact that 24 h of PMA pretreatment abolished the effect of IFN-gamma on PKC activation, as determined by acetylated myelin basic protein 4-14 phosphorylation. In conclusion, these results suggest that additional events other than PKC activation are required for complete regulation of ICAM-1 antigen by IFN-gamma in the whole cell population. Hence, other Ca(2+)-dependent signalling pathway(s) mediated by IFN-gamma receptors must act. Further studies are needed to elucidate these specific pathway(s) activated during IFN-gamma stimulation in our model.

Interleukin-6 in Renal Cell Carcinoma

We studied interleukin-6 production in 4 human renal cell carcinoma cell lines and measured the serum level in 71 patients with renal cell carcinoma, thus, clarifying a relationship between interleukin-6 secretion and an occurrence of the paraneoplastic syndrome in the carcinoma. Interleukin-6 was produced by 3 cell lines and detected in 25% of the patients. The level of interleukin-6 did not directly correlate with tumor volume and the differentiation grade of the carcinoma. However, the positive rate increased with progression of the stage. The serum level affected the 5-year survival of patients without distant metastasis. When serum interleukin-6 was elevated patients had a significantly higher frequency of unexplained fever and an elevation of acute phase proteins. These results suggest that some renal cell carcinomas can produce interleukin-6 and this cytokine is responsible for several paraneoplastic syndromes in the carcinoma.

Truncated fibronectin. An autologous growth-promoting substance secreted by renal carcinoma cells.

The human renal carcinoma cell (RCC) line ACHN proliferates in the absence of exogenous growth factors and secretes a 178-kilodalton growth-promoting substance (GPS). Complementary DNA (cDNA) was isolated that coded for polypeptides antigenically cross-reactive with GPS. Nucleotide sequencing of the cDNA showed strong homology with human fibronectin (FN). The deletion of an adenine in human FN codon 1482 caused a reading-frame shift that predicted early termination of translation after 1518 amino acid residues. Western immunoblotting human FN and GPS with anti-human FN antibodies showed that GPS was a truncated FN. Previous work found that malignant cells synthesized, bound, and deposited into the extracellular matrix decreased amounts of FN. Addition of this substance to transformed cells changed their morphology but not their rate of growth. By contrast, partial proteolysis of FN resulted in a prominent 180-kilodalton fragment that stimulated DNA synthesis. Similar to this finding, the authors showed that truncation of fibronectin during synthesis appeared to unmask latent DNA synthetic stimulating activity. Polymerase chain reaction methods using genomic DNA from normal kidneys and RCC and primers specific for the GPS-human FN gene showed two products of identical size, indicating that genomic amplification did not cause activation of the human FN gene in RCC to produce GPS. Restriction-fragment length analysis demonstrated identical patterns in DNA extracted from both normal kidneys and RCC, suggesting that chromosomal rearrangements did not activate the GPS-human FN gene. This study showed that genetic changes detectable only by DNA sequencing can explain the activation of the normal human FN gene to produce GPS, a product important for autologous growth stimulation of RCC.

Inhibitory Effects of Suramin on a Human Renal Cell Carcinoma Line, Causing Nephrogenic Hepatic Dysfunction

A sarcomatoid renal cell carcinoma has been isolated from a patient with Stauffer’s syndrome. The tumor, designated BA1119, has been established in tissue culture over 80 passages. Subcutaneous deposition of BA1119 in athymic mice induced splenomegaly and hepatic dysfunction which became fatal within four weeks without metastasis. Suramin is a synthetic polyanionic compound which is capable of altering the function of a number of biologic systems and inhibiting the activity of a variety of protein and growth factors. In this study we attempted to study the effect of suramin on growth of BA1119 in culture and in nude mice. Suramin, at 300μg/ml., had a profound inhibitory effect on cell growth during a six-day culture period. Suramin given i.p. weekly to nude mice at clinically relevant doses (200mg./kg.) caused significant shrinkage of subcapsular tumor deposits. Splenic hypertrophy secondary to BAlll9-induced Stauffer’s syndrome was inhibited by suramin. Synergistic effect with enhanced cytotoxicity on BA1119 cells was observed when suramin (100μg./ml.) was used in combination with lymphokines, such as γ interferon (500 units/ml.) and α tumor necrosis factor (300ng./ml.). These results may suggest a therapeutic efficacy of suramin in renal cell carcinoma patients with Stauffer’s syndrome.

Downregulation of the c-Fes protein-tyrosine kinase inhibits the proliferation of human renal carcinoma cells

The c-Fes protein-tyrosine kinase is associated with growth and differentiation of hematopoietic, neuronal, vascular endothelial and epithelial cell types. In this study, we investigated whether small interfering RNA (siRNA)-mediated knockdown of c-Fes expression affected proliferation of the human renal carcinoma cell lines, ACHN and VMRC-RCW. Immunofluorescence microscopy showed that c-Fes was expressed in both the cytosol and nuclei of these cells, and siRNA treatment preferentially downregulated c-Fes expression in the cytosol. Knock-down of c-Fes inhibited cellular proliferation in a dose-dependent manner with minimal increase in cell death. c-Fes siRNA treatment also downregulated the phosphorylation of Akt1 on S473 and IKKalpha on T23, and cyclin D1 expression, enhanced the expression of IkappaBalpha, and prevented the nuclear localization of NFkappaB. Treatment with an NFkappaB inhibitory peptide (SN50) also blocked the proliferation and nuclear localization of NFkappaB in these cells. The effect of SN50 treatment was not enhanced by c-Fes siRNA, suggesting that downregulation of c-Fes expression inhibited cell cycle progression through the Akt1/NFkappaB pathway. In contrast to siRNA-mediated knockdown, ectopic expression of either wild-type or kinase-inactive c-Fes in renal carcinoma cells failed to alter their proliferation in vitro and in vivo. Thus, suppression of proliferation resulting from siRNA-mediated knockdown may depend upon an expression of c-Fes protein rather than its kinase activity. Taken together, our results indicate that downregulation of c-Fes expression may be a potential therapeutic strategy for advanced human renal cell carcinoma and inhibition of its kinase activity as an antiangiogenic therapy does not seem to induce the growth of human renal carcinoma cells.

GTI-2040 displays cooperative anti-tumor activity when combined with interferon α against human renal carcinoma xenografts

GTI-2040, an antisense oligonucleotide targeting the small subunit of ribonucleotide reductase, acts as an anti-tumor agent in animal models of human cancer. In the present study, the anti-tumor activity of GTI-2040, in combination with interferon alpha (IFNalpha) was investigated against human renal cell carcinoma tumors xenografted into mice. The human renal cell carcinoma cell lines, Caki-1 and A498 were sensitive to IFNalpha both in vitro and when implanted into mice. In combination with GTI-2040 there were cooperative effects at intermediate doses of the two agents and complete tumor regression at higher combination doses. A control oligonucleotide was not effective as a monotherapy and did not improve the efficacy of IFNalpha. The effect of combination treatment on apoptosis and proliferation of tumor cells, isolated from xenografted tumors, was examined by histochemistry. GTI-2040 increased the percentage of cells undergoing apoptosis with a concomitant decrease in proliferation. IFNalpha alone had no effect but in combination with GTI-2040 resulted in increased apoptosis and decreased proliferation compared to GTI-2040 alone. Taken together these results expand the potential clinical applications of GTI-2040 to include combination therapy with IFNalpha.

Generation and Characterization of the Murine Monoclonal Antibody M-KID 2 to VLA-3 Integrin

Through the analysis of the antigenic phenotype of a recently established human renal carcinoma cell line (KJ29), we have demonstrated that alpha 3 subunit of the integrin family is selectively expressed by the plastic adherent cell subpopulation. Because of the scanty availability of monoclonal antibodies to this adhesion molecule, we have used KJ29 cell line as immunogen to raise novel murine monoclonal antibodies. We isolated an hybridoma secreting the mAb M-KID 2 of the IGg1k isotype that immunoprecipitates from intrinsically [35S]-Methionine labeled KJ29 cells, an heterodimer of 130/130 and 110/150 Kd, in reducing and nonreducing conditions respectively. This reactivity was completely abolished by immunodepletion of the cell extract with a polyclonal anti alpha 3 chain antiserum. Treatment of M-KID 2 immunoprecipitates with various solutions of pH ranging from 2 to 10.5, to dissociate alpha 3 from beta 1 chains, showed a retention of both alpha 3 beta 1 chains thus indicating that the epitope identified by mAb M-Kid 2 is likely to be constituted by the alpha 3 beta 1 heterodimer. Furthermore immunohistochemical studies on selected frozen and paraffin embedded tissues with mAb M-Kid 2 have provided staining pattern indicating the recognition of Vla-3. These findings demonstrate that mAb M-KID 2 can represent a valuable reagent for the study of Vla-3 integrin in normal and pathologic conditions.

Combined Effect of Tumor Necrosis Factor α and Anticancer Chemotherapeutic Agents against Human Renal Carcinoma Cell Lines

Antitumor effect of recombinant human tumor necrosis factor alpha (TNF) alone or in combination with various anticancer chemotherapeutic agents was tested using an in vitro antiproliferative assay on four human renal carcinoma cell lines (VMRC-RCW, VMRC-RCZ, Caki-1 and A-498). When the dose-dependent effect of TNF was studied, none of the four cell lines showed a 50% or more inhibition even at a concentration of 10,000 U/ml of TNF, so that the susceptibility to TNF was low. An enhancing effect of TNF on the cytotoxic effect of almost all the chemotherapeutic agents tested was observed against VMRC-RCZ and A-498. Against VMRC-RCW and Caki-1, the combination of TNF with the chemotherapeutic agents did not result in an enhancing effect. Mitomycin C showed an enhancing effect of TNF against all four cell lines. These results suggested that the combined treatment of TNF with chemotherapeutic agents may have favorable results in clinical trials.

Correlations between natural resistance to doxorubicin, proliferative activity, and expression of P-glycoprotein 170 in human kidney tumor cell lines

The natural resistance to doxorubicin of 15 human renal carcinoma cell lines was analyzed and compared to proliferative activity and expression of P-glycoprotein. We found a significant negative correlation between proliferative activity and natural resistance to doxorubicin, as well as between proliferative activity and the expression of P-glycoprotein. A positive correlation between resistance and expression of P-glycoprotein was found.

Parathyroid hormone related peptide can function as an autocrine growth factor in human renal cell carcinoma

Parathyroid hormone related peptide (PTHrP) has been implicated in the cause of the hypercalcemia associated with a number of malignant tumours. The data presented here suggests that PTHrP (in addition to its known role of mediating hypercalcemia) may be involved in the autocrine regulation of growth of some tumours. Polyclonal PTHrP antiserum almost totally inhibited the growth of a human renal cell carcinoma cell line, known to secrete PTHrP, in vitro and growth was significantly inhibited by the competitive PTH antagonist PTH (3–34)NH2.

A possible role for epidermal growth factor and its receptor in the growth of human prostatic and renal cell carcinoma cell lines in athymic nude mice

A possible role for epidermal growth factor and its receptor in the growth of human prostatic and renal cell carcinoma cell lines in athymic nude mice

Combination therapy for renal cell carcinoma using IFN-.GAMMA. and OK-432: In vitro study.

Synergistic effect of recombinant IFN-gamma (IFN-gamma) and OK-432, a streptococcal preparation, using chromium release assay was studied in vitro on killer cell induction. The target cells utilized for assay were a human leukemia cell line K562, a human renal carcinoma cell line KU-2, autologous normal kidney tissues and autologous renal cell carcinomas. Culture supernatant of peripheral blood lymphocytes (PBL) and OK-432 (designated as OK conditioned medium or OK-CM) demonstrated enhanced cytotoxicity of fresh PBL against these target cells. Killer cell activity against autologous cancer cells could be also induced from PBL of renal cell carcinoma patients. Pretreatment of PBL with IFN-gamma revealed synergistic effect of OK-CM on killer cell induction. OK-CM derived from patients was shown to contain IL-2 activity as well as high titer of interferon. Neutralizing monoclonal antibody against IFN-gamma and IL-2 receptor demonstrated reduction of cytotoxicity. These results suggested potential benefit of sequential use of IFN-gamma and OK-432 for the treatment of metastatic renal cell carcinoma.

Biomechanical Effects of Extracorporeal Shock Wave Lithotripsy: Relation of Applied Voltage and Shock Wave Number to Cell Viability and Stone Fragmentation*

High-energy shock waves (HESW) are harmful to both benign and malignant cells in culture. However, the extent to which the applied voltage (kV) and shock wave number interact to induce both cellular injury and stone fragmentation remain unclear. To investigate these interactions, single-cell suspensions of a human renal carcinoma cell line (TK-10) were subjected to HESW using a Dornier HM3 lithotripter at 15,18, 21, and 24 kV each at 100, 1200, and 2400 shocks. Cell viability was measured by trypan-blue exclusion. Similarly, the relation of kV and shock wave number to stone fragmentation was elucidated by exposing standardized plaster of Paris blocks in three trials to HESW at 16,18,20,22, and 24 kV at 750 shocks and to 250, 500, 750,1000,1500, and 2000 shocks at 18 kV. Stone fragmentation was recorded as the per cent weight loss after passage of the fragments through a 1-mm pore-diameter sieve. Ceil viability decreased as a linear function of kV and as an inverse exponential function of shock wave number. Stone fragmentation, on the other hand, related linearly to both kV and shock wave number. As the total number of delivered shock waves increases, kV apparently becomes the primary determinant of cell viability, whereas kV and shock wave number retain approximately equivalent effects on stone fragmentation over a wide range of power settings.

Effect of Tumor Necrosis Factor and Interferon Gamma on Human Renal Carcinoma Cell Line Growth

Four human renal carcinoma cell lines (786-0, CaKi-1, TK-10 and TK-164) were studied in vitro for susceptibility to human recombinant interferon-gamma (IFNγ) and/or human recombinant tumor necrosis factor (TNF). All four cell lines tested demonstrated a dose dependent sensitivity to the cytotoxic effects of TNF alone. The degree of sensitivity varied for each cell line. IFNγ alone also mediated a dose dependent antiproliferative effect in three of the four cell lines. Combinations of IFNγ and TNF produced diverse inhibitory effects. Preincubation of the cells for various time intervals with noninhibitory concentrations of IFNγ prior to the addition of TNF resulted in distinct effects for each cell line. Pretreatment enhanced the cytotoxic effect of TNF for some cell lines; however, others became less susceptible and some demonstrated enhanced proliferation in the presence of both agents. Optimal pretreatment times also varied from cell line to cell line. These results demonstrate variability in responsiveness by renal carcinoma cell lines to combination IFNγ and TNF treatments and suggest that timing as well as concentration may be important in in vivo therapy. (J. Urol, 142: 875–878, 1989)

Establishment of three human renal cell carcinoma cell lines (SMKT-R-1, SMKT-R-2, and SMKT-R-3) and their characters.

We have established three new cell lines of human renal cell carcinoma (RCC), designated as SMKT-R-1, SMKT-R-2 and SMKT-R-3. These cell lines were derived from a primary lesion of the tumor or a tumor initially xenotransplanted in nude mice. These cell lines have maintained a stable growth in vitro for more than a year. They also exhibited characteristics showing a lack of contact inhibition of cells, colony formation in soft agarose and tumor formation in nude mice by a xenotransplantation of cells, all of which suggested an epithelial origin. The tumors produced in nude mice by the inoculation of cell lines were demonstrated by light an electron microscopy to be derived from RCC. The doubling time of these cell lines were 180.0 h, in SMKT-R-1, 56.4 h in SMKT-R-2 and 55.7 h in SMKT-R-3. The cell lines were aneuploid in their chromosomal analysis, and SMKT-R-2 and SMKT-R-3 also had three and two marker chromosomes, respectively. The different biological characters of these cell lines from the others so far established would be of benefit in the future study of human RCC.

Enhanced Anti-Tumor Effects of Recombinant Human Tumor Necrosis Factor Plus VP-16 On Metastatic Renal Cell Carcinoma in a Xenograft Model

A nude mouse renal subcapsular and subcutaneous implantation xenograft model utilizing the SN12C human renal carcinoma cell line was investigated. In the absence of treatment, renal subcapsular implantation of SN12C resulted in metastatic spread (lung, liver and lymph nodes) and death of all animals. Radical nephrectomy of the tumor-bearing kidney after various periods of tumor implantation demonstrated that surgery alone after 18 days of tumor growth resulted in no statistically significant increase in survival with 100% of the nephrectomized animals succumbing to local recurrence and distant metastases.Recombinant human tumor necrosis factor (rTNF) and VP16 (etoposide), both well known cytotoxic and cytostatic anticancer agents, were tested singly and in combination against this metastatic model of human renal adenocarcinoma. Single agent rTNF or VP16 therapy after radical nephrectomy demonstrated only minimal efficacy with no significant decrease in local recurrence and distant metastases as compared to nephrectomy only control animals. In contrast, the combination of rTNF plus VP 16 when given after nephrectomy resulted in a significant decrease in local recurrence and no gross evidence of metastasis in any animal. Subcutaneously growing SN12C tumor nodules were also treated with the same rTNF, VP16 and combination regimens. Regression in tumor size was noted only in the combination treatment group. rTNF or VP 16, as single agents, demonstrated only slight growth inhibition that was not statistically significant.These results suggest that by combining TNF plus VP16, a synergistic enhancement of antineoplastic activity against local as well as metastatic human renal cell carcinoma can be produced.

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.

In vitro antiproliferative effect of cancer chemotherapeutic agents and their combination with interferon in renal cell carcinoma

We studied an antiproliferative effect of cancer chemotherapeutic agents, interferon (IFN) and, in particular, their combination effect on renal cell carcinoma. Either of colony formation assay (CFA) or [3H]-thymidine incorporation assay ([3H]-TdR assay) was employed as an in vitro chemosensitivity testing system. When compared, these two systems produced similar results with a good correlation (r = 0.97, p less than 0.01), in the antiproliferative effect of 30 drugs for 4 primary renal cell carcinomas and 5 xenotransplantable renal cell carcinomas. In vitro chemosensitivity test (CFA or [3H]-TdR assay) screened successfully only 5 "sensitive" drugs (7.9%) out of a total 63 cancer chemotherapeutic agents for 24 human renal cell carcinoma. This confirmed the findings reported by others. In the study of the antiproliferative effect of a cancer chemotherapeutic agent, human lymphoblastoid interferon (HLBI) and their combination on human renal cel carcinoma cell line (SMK-R2). Each of VBL, MTX or HLBI tended to suppress [3H]-TdR uptake in a dose-dependent manner. The combination of VBL (0.05 microgram/ml) and HLBI (10(2) or 10(3) IU/ml) produced a subadditive effect, and that of MTX (0.1 microgram/ml) and HLBI 10(2) IU/ml produced a synergistic effect on the human renal cell carcinoma cell line, the effect which is evaluated by Valeriote and Lin's criteria of combination. In particular, the synergistic effect by MTX and HLBI under the clinically achievable drug concentration seems important, when its clinical application is considered.