Human Renal Carcinoma Cell Line Research Articles

Apoptosis induction in renal cell carcinoma by TRAIL and gamma-radiation is impaired by deficient caspase-9 cleavage.

TNF-related apoptosis-inducing ligand (TRAIL APO-2L) is a member of the TNF family and induces apoptosis in cancer cells without affecting most non-neoplastic cells. The present investigation is focused on apoptosis induction by combined exposure to TRAIL and ionising radiation (IR) in human renal cell carcinoma (RCC) cell lines. Here, we demonstrate that all RCC cell lines coexpress TRAIL and the death-inducing receptors, TRAIL-R1 and TRAIL-R2. Exposure to TRAIL alone induced marked apoptosis in three out of eight RCC cell lines. Combined exposure to TRAIL and IR resulted in a sensitisation to TRAIL-induced apoptosis in one RCC cell line only. Enhanced apoptosis induction by TRAIL in combination with IR was paralleled by an increase in PARP cleavage and activation of executioner caspase-3, whereas caspases-6 and -7 were not involved. Moreover, exposure to TRAIL and/or IR resulted in a marked activation of initiator caspase-8, possibly augmented by the observed reduction of inhibitory c-FLIP expression. In contrast to other tumour types, activation of initiator caspase-9 was not detectable in our RCC model system after exposure to TRAIL and/or IR. This lack of caspase-9 activation might be related to an impaired ‘crosstalk’ with the caspase-8 pathway as suggested by the missing Bid cleavage and to the appearance of an XIAP cleavage product known to inhibit caspase-9 activation. Deficient activation of caspase-9, therefore, might contribute to the clinically known resistance of human RCC against IR and also argues against an effective combination therapy with TRAIL and IR in this tumour type.

Overexpression of hepatocyte growth factor receptor in renal carcinoma cells indirectly stimulates tumor growth in vivo

We examined the role of increased expression of HGFR kinase in in vivo growth of renal carcinoma. Human renal carcinoma cell line, ACHN cells, was transfected with plasmid encoding wild-type HGFR gene to generate cell lines with increased HGFR protein. ACHN cells with elevated HGFR expression, denoted clones 8 and 10, respectively, showed higher basal kinase activities of HGFR and PI3-kinase than those of empty-vector (mock)-transfected cells. Clone 8 and 10 cells grew similar to mock cells in culture. In mice, tumors of these clones grew more rapidly than those of mock cells. Microvessel density of clone 8 or 10 tumors was higher than that of mock tumors. Clone 8 and 10 cells secreted vascular endothelial growth factor-A (VEGF-A) more than mock cells and the secretion was PI3-kinase inhibitor, LY294002-sensitive. Anti-VEGF-A neutralizing antibody significantly inhibited tumor growth of clones 8 and 10 in mice. These results indicate for the first time that overexpression of HGFR tyrosine kinase in renal carcinoma cells participates in rapid tumor growth in vivo.

Expression of urotensin II and its receptor in adrenal tumors and stimulation of proliferation of cultured tumor cells by urotensin II

Urotensin II is a potent vasoactive peptide, which was originally isolated from fish urophysis. We studied expression of urotensin II and its receptor mRNAs in the tumor tissues of adrenocortical tumors, pheochromocytomas and neuroblastomas. Effects of exogenously added urotensin II on cell proliferation were studied in a human adrenocortical carcinoma cell line, SW-13 and a human renal cell carcinoma cell line, VMRC-RCW. The reverse transcriptase polymerase chain reaction (RT-PCR) showed expression of urotensin II and its receptor mRNAs in all the samples examined; seven pheochromocytomas, nine adrenocortical adenomas (four with primary aldosteronism, four with Cushing syndrome and one with non-functioning adenoma), four adrenocortical carcinomas, one ganglioneuroblastoma and five neuroblastomas, as well as four normal portions of adrenal glands (cortex and medulla). Urotensin II-like immunoreactivity was detected in one of eight adrenocortical adenomas, two of four adrenocortical carcinomas, one of six pheochromocytomas, and one of five neuroblastomas by radioimmunoassay, but not in normal portions of adrenal glands (detection limit; 0.2 pmol/g wet weight). Treatment with urotensin II for 24 h significantly increased number of SW-13 cells (at 10 −8 and 10 −7 mol/l) and VMRC-RCW cells (at 10 −8 mol/l). These findings raise the possibility that urotensin II may act as an autocrine/paracrine growth stimulating factor in adrenal tumors.

Activation of Ras/Erk Pathway by a Novel MET-interacting Protein RanBPM

MET is a receptor protein-tyrosine kinase (RPTK) for hepatocyte growth factor (HGF), which is a multifunctional cytokine controlling cell growth, morphogenesis, and motility. MET overexpression has been identified in a variety of human cancers. Oncogenic missense mutations of the tyrosine kinase domain of the MET gene have been identified in human papillary renal cell carcinomas. In this study, RanBPM, also known as RanBP9, is identified as a novel interacting protein of MET through yeast two-hybrid screen. RanBPM contains a conserved SPRY (repeats in splA and RyR) domain. We demonstrate that RanBPM can interact with MET in vitro and in vivo, and the interaction can be strengthened by HGF stimulation. RanBPM interacts with the tyrosine kinase domain of MET through its SPRY domain. We show that RanBPM can induce GTP-Ras association and Erk phosphorylation and elevate serum response element-luciferase (SRE-LUC) expression, indicating that RanBPM can activate the Ras-Erk-SRE pathway. We demonstrate that RanBPM, which itself is not a guanine exchange protein, stimulates Ras activation by recruiting Sos. On the cellular level, A704 cells, a human renal carcinoma cell line, transfected with RanBPM exhibit increased migration ability. Our data suggest that RanBPM, functioning as an adaptor protein for the MET tyrosine kinase domain, can augment the HGF-MET signaling pathway and that RanBPM overexpression may cause constitutive activation of the Ras signaling pathway.

BCG effects on telomerase activity in bladder cancer cell lines

Although intravesical bacillus Calmette-Guerin (BCG) therapy is very effective in the treatment of and prophylaxis against superficial bladder cancer, its exact mechanism of action is not clear. In this study, the effect of BCG on telomerase activity was examined in the T24 and J82 bladder cancer cell lines, the ACHN human renal cell carcinoma cell line, and the PC-3 human prostate cancer cell line. T24 and J82 cells were cocultured with BCG for 5 days. Telomerase activity was measured by telomeric repeat amplification protocol. Cell-cycle phase was determined by FACS analysis. Telomerase activity in all cell lines provided high absorbance. Telomerase activity in BCG-treated T24 and J82 cells was significantly decreased when compared with that in the nontreated cells. On the other hand, telomerase activity did not change in ACHN and PC-3 cells after they had been cocultured with BCG. In BCG-treated T24 cells and J82 cells, apoptotic cells were markedly increased compared with those in the nontreated cells. These results suggest that the reduction of telomerase activity is related to the mechanism of BCG effects. Possible mechanisms to be considered are either that BCG inhibits telomerase first, or that it induces apoptosis and decreases telomerase activity as a result of this induction.

Novel glutathione-dependent thiopurine prodrugs: evidence for enhanced cytotoxicity in tumor cells and for decreased bone marrow toxicity in mice.

Elevated glutathione (GSH) levels have been detected in many tumors compared with the healthy, surrounding tissue. Often, this GSH up-regulation is associated with drug resistance. The prodrugs 6-(2-acetylvinylthio)guanine (AVTG) and 6-(2-acetylvinylthio)purine (AVTP) contain a novel butenone moiety that allows the prodrugs to react selectively with sulfhydryl nucleophiles to release the chemotherapeutic drug 6-thioguanine (6-TG) or 6-mercaptopurine (6-MP), respectively. The cellular uptake and metabolism of trans-AVTG in two human renal carcinoma cell lines that were used as models were rapid and associated with depletion of intracellular GSH. Formation of 6-TG from trans-AVTG correlated positively with intracellular GSH concentrations, and was significantly reduced by diethyl maleate pretreatment. Intracellular concentrations of 6-TG after incubations with trans-AVTG were significantly higher than the 6-TG concentrations obtained after incubations with equimolar concentrations of 6-TG; thus, the prodrug delivered more 6-TG to the cell than did 6-TG itself. Cytotoxicity studies demonstrated that AVTG and AVTP had similar IC(50) values that were comparable with those of 6-TG, but were significantly lower than those of 6-MP. Furthermore, after in vivo treatment of mice with the prodrugs, no reduction was observed in circulating white blood cell counts, whereas white blood cell counts of mice treated with equimolar or 60% lower doses of 6-TG were reduced by 50 to 60%. Collectively, the results show that AVTG and AVTP are novel potential chemotherapeutic agents that may target tumors with up-regulated levels of GSH, and may exhibit less systemic toxicity than the parent thiopurines.

Transient activation of the c-Jun N-terminal kinase (JNK) activity by ligation of the tetraspan CD53 antigen in different cell types.

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. We have studied the implication of CD53 antigen in signal transduction by determining the effect of its ligation on the c-Jun N-terminal kinase (JNK) in different cell types. Ligation of the rat or human CD53 antigen induces a three- to fourfold transient activation of JNK activity that peaks at 3-5 min. The effect was detected by assaying the endogenous or exogenous (transfected) JNK activity. The JNK response was detected in IR938F cells, a rat B-cell lymphoma, and in Jurkat cells derived from a human T-cell lymphoma. This JNK activation was not mediated by the vav oncogene, and CD53 does not cooperate with CD3 for vav activation. A similar JNK activation was also detected in a human renal carcinoma cell line that was transiently transfected with the human CD53 cDNA to mimic the CD53 ectopic expression in carcinomas. In stable CD53-transfected cells it stimulated Jun-dependent transcriptional activity. We conclude that parts of the cell responses modulated by the CD53 are mediated by JNK activation, and this activation is independent of the different protein interactions that the CD53 protein has on specific cell types.

The serine proteinase thrombin promotes migration of human renal carcinoma cells by a PKA-dependent mechanism

In this study, we demonstrated that thrombin activates protein kinase C (PKC), mitogen activated protein kinases (MAP kinases), transcription factor nuclear factor-κB (NF-κB), and cAMP-dependent protein kinase (PKA) in the human renal carcinoma cell line A-498. In addition, it enhanced the migratory capacity, but had no effect on the proliferation of A-498 cells. The effect of thrombin on migration could be blocked by the PKA inhibitor H-89 but was not influenced by inhibition of PKC, MAP kinases or NF-κB. We concluded, that thrombin acts as a regulator on human A-498 renal carcinoma cell migration including PKA.

Expression of three vasoactive peptides, urotensin-II, adrenomedullin, and endothelin-1, in a human renal cell carcinoma cell line, VMRC-RCW

Background. The kidney produces and secretes various vasoactive peptides, such as endothelin-1 and adrenomedullin, and expresses receptors for these peptides. These vasoactive peptides are considered to have autocrine or paracrine growth-regulatory activities in the kidney, in addition to actions in the renal circulation, and water and electrolyte transport. It was reported that renal cell carcinoma cell lines, such as VMRC-RCW cells, secreted some autocrine growth factors. The production of these peptides by VMRC-RCW cells has not been studied, however. Methods. We studied the expression of three vasoactive peptides, urotensin-II, adrenomedullin, and endothelin-1, in VMRC-RCW human renal cell carcinoma cells by radioimmunoassay, Northern blot analysis, and reverse transcriptase polymerase chain reaction (RT-PCR). Results. Northern blot analysis showed the expression of adrenomedullin mRNA in VMRC-RCW cells. RT-PCR analysis showed the expression of urotensin-II and endothelin-1 mRNAs in VMRC-RCW cells. Immunoreactive adrenomedullin and immunoreactive endothelin were detected in the culture medium (17.5 ± 0.70 fmol/105 cells per 24 h, and 0.75 ± 0.03 fmol/105 cells per 24 h, respectively, mean ± SEM; n = 6). The adrenomedullin levels in the medium were higher in VMRC-RCW cells than in DLD-1 colonic carcinoma cells and HeLa cervical cancer cells, but the endothelin levels were lower in VMRC-RCW cells. Reverse-phase high performance liquid chromatogra-phy suggested that VMRC-RCW cells secreted both endothelin-1 and endothelin-2. On the other hand, no significant amount of urotensin-II was detected in the culture medium (<0.5 fmol/105 cells per 24 h). Conclusions. VMRC-RCW human renal cell carcinoma cells express three vasoactive peptides; urotensin-II, adrenomedullin, and endothelin-1. The secreted peptides may be related to tumor biology as well as renal pathophysiology.

Increase in hepatocyte growth factor receptor tyrosine kinase activity in renal carcinoma cells is associated with increased motility partly through phosphoinositide 3-kinase activation.

Dysregulated cell motility is one of the major characteristics of invasion and metastatic potentials of malignant tumor cells. Here, we examined the hepatocyte growth factor (HGF)-induced cell motility of two human renal carcinoma cell lines, ACHN and VMRC-RCW. Scattering and migration was induced in ACHN in an HGF-dependent manner, whereas they were maintained in VMRC-RCW even in the absence of HGF. In VMRC-RCW, HGF receptor (HGFR) tyrosine kinase was constitutively active, and sequence analysis showed N375S, A1209G and V1290L mutations. However, transfection experiments using porcine aortic endothelial (PAE) cells demonstrated that no single mutation or combination of two or three mutations caused HGF-independent constitutive activation. Conversely, the expressed amount of receptor protein had a pivotal role in the basal kinase activity. With respect to downstream signaling molecules of HGFR in ACHN or VMRC-RCW, the Ras-MAPK pathway was downregulated, whereas phosphoinositide 3-kinase (PI3-kinase) was not further activated by HGF-treatment in VMRC-RCW cells. The PI3-kinase inhibitors, wortmannin and LY294002 strongly inhibited spontaneous migration of VMRC-RCW. One transfected PAE cell line with massive overexpression of HGFR demonstrated scattered morphology and increased PI3-kinase activity in association with increased motility, which was partially inhibited by LY294002. Taken together, our results indicate that the overexpression of HGFR causes increase in cellular motility and PI3-kinase shows the important contribution on the increased motility of renal carcinoma cells.

Rac affects invasion of human renal cell carcinomas by up-regulating tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 expression.

Rho-like GTPases, including Cdc42, Rac1, and RhoA, regulate distinct actin cytoskeleton changes required for adhesion, migration, and invasion of cells. Tiam1 specifically activates Rac, and earlier studies have demonstrated that Tiam1-Rac signaling affects migration and invasion in a cell type- and cell substrate-specific manner. In the present study, we examined the role of Tiam1-Rac signaling in migration and invasion of human renal cell carcinomas. Stable overexpression of Tiam1 or constitutively active V12-Rac1 in a human renal cell carcinoma cell line (clearCa-28) strongly inhibited cell migration by promoting E-cadherin-mediated cell-cell adhesion. Blocking E-cadherin-mediated adhesion by E-cadherin-specific HAV peptides allowed cells to migrate, but was not sufficient to antagonize Tiam1- and V12-Rac1-induced inhibition of Matrigel invasion, suggesting that Rac may influence invasion also through other mechanisms. Indeed, Tiam1-mediated Rac activation induced transcriptional up-regulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) and post-transcriptional up-regulation of TIMP-2, whereas secretion and activity levels of their counterparts, matrix metalloproteinase-9 and matrix metalloproteinase-2, respectively, were not affected. Application of recombinant TIMP-1 and TIMP-2 proteins significantly inhibited invasion of mock-transfected clearCa-28 cells, supporting a role of TIMPs in Rac-mediated inhibition of invasion. To our knowledge, this is the first evidence that increased Rac signaling may inhibit invasion of epithelial tumor cells by up-regulation of TIMP-1 and TIMP-2.

Upregulation of the gene encoding a cytoplasmic dynein intermediate chain in senescent human cells

Normal human somatic cells, unlike cancer cells, stop dividing after a limited number of cell divisions through the process termed cellular senescence or replicative senescence, which functions as a tumor-suppressive mechanism and may be related to organismal aging. By means of the cDNA subtractive hybridization, we identified eight genes upregulated during normal chromosome 3-induced cellular senescence in a human renal cell carcinoma cell line. Among them is the DNCI1 gene encoding an intermediate chain 1 of the cytoplasmic dynein, a microtubule motor that plays a role in chromosome movement and organelle transport. The DNCI1 mRNA was also upregulated during in vitro aging of primary human fibroblasts. In contrast, other components of cytoplasmic dynein showed no significant change in mRNA expression during cellular aging. Cell growth arrest by serum starvation, contact inhibition, or gamma-irradiation did not induce the DNCI1 mRNA, suggesting its specific role in cellular senescence. The DNCI1 gene is on the long arm of chromosome 7 where tumor suppressor genes and a senescence-inducing gene for a group of immortal cell lines (complementation group D) are mapped. This is the first report that links a component of molecular motor complex to cellular senescence, providing a new insight into molecular mechanisms of cellular senescence.

Bcl-2 modulates Fas-mediated apoptosis in human renal cell carcinoma cell lines.

The objective of this study was to characterize the role of Bcl-2 expression in Fas-mediated apoptosis in human renal cell carcinoma (RCC) cell lines. RT-PCR analyses showed that 10 different RCC cell lines expressed Fas, but not Fas ligand. Seven of 10 cell lines expressed Fas strongly, while 3 cell lines weakly expressed Fas by flow cytometric analyses. Measurement of the LDH concentration in the culture supernatant revealed that 6 of the 7 cell lines which expressed Fas strongly were sensitive to treatment with an agonistic anti-Fas monoclonal antibody (CH11), whereas all cell lines which weakly expressed Fas did not show Fas-mediated cell death. Furthermore, Bcl-2 expression was found only in three cell lines which were all susceptible to CH11, and the downregulation of Bcl-2 protein by treatment with antisense oligodeoxynucleotide targeting Bcl-2 gene resulted in an enhancement of cell death induced by CH11. These findings suggest that strong Fas expression is necessary for Fas-mediated cell death in RCC cell lines, and Bcl-2 has a protective role against treatment with an anti-Fas monoclonal antibody, despite the fact that Bcl-2 expression was observed only in sensitive cell lines to Fas-mediated cell death.

Mapping and expression pattern analysis of key components of the major histocompatibility complex class I antigen processing and presentation pathway in a representative human renal cell carcinoma cell line.

Renal cell carcinoma (RCC) represent approximately 5% of all cancer deaths. At the time of presentation, over 50% of the patients have already developed locally advanced or metastatic disease with five-year survival rates of less than 20%. Although relative resistant to conventional regimens, RCC are partially susceptible to T cell-based immunotherapy. To further develop this treatment modality, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied for both the mapping of the key components of the major histocompatibility complex (MHC) class I antigen processing and presentation machinery (APM) and the characterization of the constitutive and cytokine-regulated protein expression profiles in a representative human RCC cell line. The latter aspect is based on the fact, that the expression level of some of the APM components can be altered in response to interferon (IFN)-gamma treatment. Total cell lysates from untreated and IFN-gamma-treated tumor cells were separated on 2-D PAGE gels using broad range immobilized pH gradient (IPG) strips. Serial Western blot analyses using sets of APM-specific antibodies were performed to target the relevant protein spots. Protein verification was mostly accomplished via peptide mass finger-printing using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). To date, the majority of the APM-related components have been identified and mapped. In addition, the different protein expression profiles of untreated and IFN-gamma-treated RCC cells are under investigation.

Synergistic antitumor effect of ionomycin and cisplatin against renal cell carcinoma in vitro and in vivo

Objectives. To characterize the synergistic antitumor effects of the calcium ionophore, ionomycin, and of cisplatin against human renal cell carcinoma cell line, ACHN, both in vitro and in vivo. Methods. The in vitro growth rate of ACHN after exposure to these compounds was measured, using the MTT assay. The apoptotic features in ACHN were evaluated by DNA ladder analysis and flow cytometric analysis. Bcl-2 and Bax expression levels in ACHN after treatment were examined by Western blot. The synergistic antitumor effects of ionomycin and cisplatin against the growth of established ACHN tumors in athymic nude mice were then tested. Results. The in vitro growth rate of ACHN was suppressed more by ionomycin and cisplatin in combination than by either alone. DNA ladder and fragmentation were more obvious when the cells were incubated with ionomycin and cisplatin together than with either reagent alone. Ionomycin treatment increased the expression level of Bax protein, whereas Bcl-2 expression was not influenced. Although an intraperitoneal injection of cisplatin or an intratumoral injection of ionomycin against subcutaneous ACHN tumors somewhat reduced tumorigenicity in nude mice, the effect was significantly enhanced by a combination of these drugs. Conclusions. The synergistic antitumor effects suggest that ionomycin-based therapy could be a novel therapeutic strategy with which to treat advanced renal cell carcinoma.

Enhancement of sensitivity to capecitabine in human renal carcinoma cells transfected with thymidine phosphorylase cDNA.

The purpose of the present study was to examine directly the role of thymidine phosphorylase (TP) in the sensitivity of renal cell carcinoma (RCC) to a novel fluoropyrimidine carbamate, capecitabine. TP cDNA-transfected RCC are used in these experiments to provide a basis for improved therapeutic benefit in chemoimmunotherapy. Human RCC line KU2 cells were transfected with pcDNA3.1/zeo(+) with or without human TP cDNA by the lipofectin method. We established a clone transfected with pcDNA3.1/zeo(+)/TP (KU2-TP15) and a clone transfected with pcDNA3.1/zeo(+) as a control (KU2-C1). TP expression levels (mean +/- SD) examined by enzyme-linked immunosorbent assay (ELISA) were 1.3 +/- 0.14 U/mg protein in KU2, 1.6 +/- 0.57 U/mg protein in KU2-C1 and 216 +/- 25.6 U/mg protein in KU2-TP15. Immunohistochemical staining of subcutaneous tumors established in Balb/c nu/nu mice showed that KU2-TP15 was strongly positive for TP expression, whereas KU2 and KU2-C1 were negative. Sensitivities in vitro to 5-fluorouracil (5FU), 5'-deoxy-5-fluorouridine (5'DFUR) and capecitabine in KU2-TP15 were significantly enhanced compared with those in KU2 or KU2-C1. A moderate but statistically significant bystander effect was observed in vitro. KU2-TP15 tumors showed significant increase in the in vivo sensitivities to 5'DFUR and capecitabine as compared with the vehicle alone while KU2-C1 tumors did not. The difference in tumor-free rate in mice bearing KU2-TP15 at 2 months after the cessation of treatment was statistically significant between the capecitabine treatment group and the controls, the 5FU treatment group and the 5'DFUR treatment group. The present study clearly provides direct evidence for the role of TP in mediating the sensitivity of RCC to capecitabine.

Activation of the MNCA9 gene is associated with hypomethylation in human renal cell carcinoma cell lines

The MN/CA9 (G250) gene expressed in the normal alimentary tract in a tissue-specific manner is often activated in renal cell carcinomas. To cast light on the activation mechanism, we examined the methylation status of this gene in seven human renal cell carcinoma cell lines (SKRC-01, -06, -10, -12, -14, -44, and -59) and three normal kidney tissue samples by using the bisulfite genomic sequencing protocol. CpG methylation was measured at seven locations in the MN/CA9 5′ region. MN/CA9 transcripts were detected by reverse transcription–polymerase chain reaction in five of the renal cell carcinoma cell lines (SKRC-01, -06, -10, -44, and -59). These MN/CA9 positive cell lines showed hypomethylation, whereas the remaining two cell lines (SKRC-12, and -14), and three normal kidney tissue samples without transcripts demonstrated hypermethylation. Treatment with the demethylating agent 5-aza-2′-deoxycytidine resulted in activation of the MN/CA9 gene in the negative cell lines (SKRC-12 and -14). These data suggest that hypomethylation in the 5′ region may have a major role in expression of the MN/CA9 gene in renal cell carcinoma cells. Mol. Carcinog. 27:184–189, 2000. © 2000 Wiley-Liss, Inc.

Activation of theMN/CA9 gene is associated with hypomethylation in human renal cell carcinoma cell lines

The MN/CA9 (G250) gene expressed in the normal alimentary tract in a tissue-specific manner is often activated in renal cell carcinomas. To cast light on the activation mechanism, we examined the methylation status of this gene in seven human renal cell carcinoma cell lines (SKRC-01, -06, -10, -12, -14, -44, and -59) and three normal kidney tissue samples by using the bisulfite genomic sequencing protocol. CpG methylation was measured at seven locations in the MN/CA9 5' region. MN/CA9 transcripts were detected by reverse transcription-polymerase chain reaction in five of the renal cell carcinoma cell lines (SKRC-01, -06, -10, -44, and -59). These MN/CA9 positive cell lines showed hypomethylation, whereas the remaining two cell lines (SKRC-12, and -14), and three normal kidney tissue samples without transcripts demonstrated hypermethylation. Treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in activation of the MN/CA9 gene in the negative cell lines (SKRC-12 and -14). These data suggest that hypomethylation in the 5' region may have a major role in expression of the MN/CA9 gene in renal cell carcinoma cells.

Protein kinase C in human renal cell carcinomas: role in invasion and differential isoenzyme expression.

The role of protein kinase C (PKC) in in vitro invasiveness of four different human renal cell carcinoma (RCC) cell lines of the clear cell type was investigated. Different PKC-inhibitors markedly inhibited invasiveness of the highly invasive cell lines, suggesting an invasion-promoting role of PKC in human RCC. Analysis of PKC-isoenzyme expression by protein fractionation and immunoblotting revealed that all cell lines expressed PKC-α, -ɛ, -ζ, -μ and -ι as known from normal kidney tissue. Interestingly, PKC-δ, known to be expressed by normal kidney epithelial cells of the rat, was absent on protein and RNA levels in all RCC cell lines investigated and in normal human kidney epithelial cells. PKC-ɛ expression levels correlated positively with a high proliferation activity, but no obvious correlation between expression levels of distinct PKC-isoenzymes and in vitro invasiveness was observed. However, by immunofluorescence microscopy, membrane localisation of PKC-α and PKC-ɛ reflecting activation of the enzymes, was associated with a highly invasive potential. In conclusion, our results suggest a role for PKC in invasion of human RCCs and might argue in favour of a particular role of PKC-α and PKC-ɛ. Our results further suggest that organ-specific expression patterns of PKC-isoenzymes are not necessarily conserved during evolution. © 2000 Cancer Research Campaign

Modulation of telomerase activity by zinc in human prostatic and renal cancer cells

Because the up-regulation of telomerase in most cancer tissues is considered to be responsible for the unlimited proliferation of cancer cells, suppression of telomerase activity is an attractive potential target for cancer therapy. The mechanism for the activation of telomerase in cancer cells, however, is still unclear. In the present study, we demonstrated that Zn induces an enhancement of telomerase activity in the human renal cell carcinoma (NRC-12) and prostatic cancer (DU145) cell lines. The maximum elevation of the activity was observed 6 hr after treatment with 100 μM Zn; it was diminished by the addition of either metal chelator or cycloheximide. Other metals such as Cd and Cu also enhanced telomerase activity but to a lesser extent, and no correlation between the activation of telomerase and the induction of metallothionein was observed. Our findings provide the first evidence that metals, especially Zn, can modulate telomerase activity in cancer cells.