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

Summary 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 xenotranaplantation of cells, all of which suggested an epithelial origin. The tumors produced in nude mice by the innoculation of cell lines were demonstrated by light an electron microscopy tobe 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.     

Establishment and characterization of seven human renal cell carcinoma cell lines

OBJECTIVE: To establish human renal cell carcinoma (RCC) cell lines, and to investigate the cell phenotypes and molecular characteristics of human RCC cell lines and their corresponding tumour tissues. MATERIALS AND METHODS: Seven human RCC cell lines from pathologically proven RCCs were established. The histopathology of the primary tumours, in vitro growth characteristics and status of tumour suppressor genes, mismatch repair genes and microsatellite instability (MSI) were examined in cell lines and their corresponding tumour tissues. Five of the cell lines were derived from clear cells (SNU-228, -267, -328, -349, and -1272), one from granular cells (SNU-482), and one from mixed clear and granular cell types (SNU-333). The mutational status was compared for von Hippel-Lindau (VHL), p53, TGF-beta type II receptor (TGF-betaRII), hMSH2, and hMLH1 genes in the cell lines and their corresponding tumour tissues. The MSI status of the cell lines was determined by screening for adenine repeat sequences, e.g. BAT-25, BAT-26, and BAT-40. RESULTS: All lines showed different doubling times and were confirmed by DNA fingerprinting analysis to be unique. Contamination by mycoplasma or bacteria was excluded. In two cell lines (SNU-349 and -1272) and their tumour tissues, mutations in the VHL gene were found. The SNU-267 line had a frameshift mutation in the p53 gene. A missense mutation of the TGF-betaRII gene was detected in the SNU-1272 line and the corresponding tissue. Analysis of the repeat sequences showed one cell line (SNU-349) to have MSI and the other six to have microsatellite stability. As MSI is a hallmark of the inactivation of mismatch repair genes, the presence of hMSH2 and hMLH1 mutations was investigated in all seven cell lines. An inactivating homozygous single base-pair deletion of the hMLH1 gene was found only in the SNU-349 cell line and corresponding tissue. Moreover, a frameshift mutation within an 8-bp polyadenine repeat present in the hMSH3 coding region was found only in the MSI cell line and tumour tissue. CONCLUSION: These newly established RCC cell lines should provide a useful in vitro model for studies related to human RCC. The SNU-349 cell line should be especially useful for studies of MSI and mismatch repair-defective RCCs.     

Inflammatory Protein Serum Amyloid A1 Marks a Subset of Conventional Renal Cell Carcinomas with Fatal Outcome

Background

Conventional renal cell carcinoma (RCC) is the most common renal cancer. As the metastatic conventional RCC is practically incurable, there is a need for markers to estimate the tumour aggressiveness.

Objective

To identify and characterise new marker(s) associated with the poor prognosis of conventional RCC.

Design, Setting, and Participants

RNA from 24 conventional RCCs was analysed for global gene expression by Affymetrix U133 Plus 2.0 arrays. Tissue microarrays containing 224 renal tumours including 87 conventional RCCs were used for immunohistochemistry. Cell lines HD2, HD48, HA344 and HA465 established in our laboratory were used for invasion assay and zymography.

Measurements

Serum amyloid A 1 (SAA1) was found to be upregulated in conventional RCCs and it has been analysed by quantitative RT-PCR and immunohistochemistry on TMAs to establish the correlation between SAA1 protein expression and patient survival by uni and multivariate analysis. The effect of SAA1 on tumour cell behaviour in vitro has also been examined by invasion assay and zymography.

Results and Limitations

SAA1 RNA is expressed in conventional RCC samples of patients with poor prognosis. Immunohistochemistry of 72 conventional RCCs with a 5 yr follow up showed a correlation between SAA1 expression and the clinical outcome of disease. Stimulation of conventional RCC cell lines with recombinant SAA1 increased the expression of metalloproteinase (MMP)-9 and the invasive potential of tumour cells. Limitation of the study is a relatively small number (72) of patients having follow up.

Conclusion

SAA1 seems to be a useful marker to estimate the prognosis of conventional RCCs.     

TGF-beta promotes the establishment of renal cell carcinoma bone metastasis.

Bone metastases develop in approximately 30% of patients with RCC, and the mechanisms responsible for this phenomenon are unknown. We found that TGF-beta1 stimulation of RCC bone metastasis cells promotes tumor growth and bone destruction possibly by stimulating paracrine interactions between tumor cells and the bone. INTRODUCTION: Bone metastasis is a frequent complication and causes marked morbidity in patients with renal cell carcinoma (RCC). Surprisingly, the specific mechanisms of RCC interaction with bone have been scarcely studied despite the inability to prevent or effectively treat bone metastasis. Bone is a reservoir for various growth factors including the pleiotropic cytokine TGF-beta1. TGF-beta1 has been shown to have tumor-supportive effects on advanced cancers and evidence suggests its involvement in promoting the development of breast cancer bone metastasis. Here, we studied the potential role of TGF-beta1 in the growth of RCC bone metastasis (RBM). MATERIALS AND METHODS: To inhibit TGF-beta1 signaling, RBM cells stably expressing a dominant-negative (DN) TGF-betaRII cDNA were generated. The in vivo effect of TGF-beta1 on RBM tumor growth and osteolysis was determined by histological and radiographic analysis, respectively, of athymic nude mice after intratibial injection of parental, empty vector, or DN RBM cells. The in vitro effect of TGF-beta1 on RBM cell growth was determined after TGF-beta1 treatment by MTT assay. RESULTS: TGF-beta1 and the TGF-beta receptors I and II (TGF-betaRI/II) were consistently expressed in both RBM tissues and cell lines. Inhibition of TGF-beta1 signaling in RBM cells significantly reduced tumor establishment and osteolysis observed in vivo after injection into the murine tibia, although no effect on tumor establishment was observed after injection of RBM cells subcutaneously or into the renal subcapsule. Treatment of five RBM cell lines with TGF-beta1 in vitro either had no effect (2/5) or resulted in a significant inhibition (3/5) of cell growth, suggesting that TGF-beta1 may promote RBM tumor growth indirectly in vivo. CONCLUSIONS: TGF-beta1 stimulation of RBM cells plays a role in promoting tumor growth and subsequent osteolysis in vivo, likely through the initiation of tumor-promoting paracrine interactions between tumor cells and the bone microenvironment. These data suggest that inhibition of TGF-beta1 signaling may be useful in the treatment of RBM.     

TGFβ_1及PCNA在肾细胞癌中的表达及其临床意义

目的 :探讨肾细胞癌中转化生长因子 β1 (TGFβ1 )及增殖细胞核抗原 (PCNA)的表达及其价值。方法 :采用免疫组织化学技术 (SP法 )对 46例肾细胞癌和 11例正常肾组织标本中 TGFβ1 、PCNA进行检测 ,并结合临床资料进行分析。结果 :肾癌组 TGFβ1 表达量较正常组高 (P <0 .0 5 ) ;PCNA在肾癌组高表达 (P <0 .0 1) ,且在高分期、高分级肾细胞癌中表达量较低分期、低分级肾细胞癌高 (P <0 .0 5 ) ;TGFβ1 表达量与 PCNA表达量呈负相关关系 (P <0 .0 1) ;TGFβ1 表达量低及 PCNA表达量高的肾癌患者预后差 (P <0 .0 5 )。结论 :TGFβ1 对处于增殖状态的肾细胞癌是一种重要负性调节因子 ,可抑制肾癌发展 ;TGFβ1 及 PCNA表达可作为判断肾细胞癌预后的指标     

In vitro effects of epidermal growth factor (EGF) on growth of urological malignant tumor cells

It is well known that Epidermal Growth Factor (EGF) is a cell-regulating factor for variety of tissues in vitro including normal and malignant cells. Furthermore, Takano et al reported that a decreased expression of EGF receptor in clones of human cancer KB cell line might be one of the pleiotropic properties of multidrug-resistant cells. However, both the influence of EGF on human urological cancer cell lines and the relation between EGF receptors and sensitivities of antitumor drugs on these cell lines have not been fully described. We have studied the effects of EGF on growth of 4 transitional carcinoma cell lines of bladder (TCCaB), 1 squamous cell carcinoma cell line of bladder (SCCaB), 5 renal cell carcinoma cell lines (RCC) and 3 prostatic carcinoma cell lines (CaP), as well as the relationship between the number of EGF receptors and drug sensitivities of these cell lines in vitro against methotrexate, vinblastine, adriamycin, cisplatin and etoposide (VP16). The present results determined by the in vitro colony forming efficiency method showed that exogenous addition of EGF to cell cultures at 0.1 ng/ml stimulated the growth of SCCaB by 169.0%, and at 1 ng/ml inhibited that of RCC by 2.9%-79.0%, relative to control. The more EGF receptors by 125I-EGF binding assay, the higher inhibition of VP16 on the growth of these cell lines. These results suggested that EGF stimulated the growth of SCCaB and inhibited the growth of RCC in vitro, and we found that these phenomena were correlated with neither the number of EGF receptors nor affinities of that receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

P-170 glycoprotein, glutathione and associated enzymes in relation to chemoresistance of primary human renal cell carcinomas

High intrinsic chemoresistance contributes to the dismal outcome of patients with disseminated renal cell carcinoma (RCC). In experimental cell lines, two defined defence mechanisms, P-170 glycoprotein and glutathione metabolism, have been established in multidrug resistance, a cross-resistance to cytotoxic compounds without functional or structural similarities. In 21 primary human RCCs, P-170 expression was examined, glutathione content and activities of related enzymes determined and the results were correlated to the degree of in vitro chemoresistance. P-170 was found in 10 of 17 resistant tumors but in none of the sensitive cases. The glutathione content was significantly higher and the related enzyme distinctively enhanced in resistant RCCs. Both mechanisms occurred independently and may well explain the multidrug resistance of RCC. Therefore, reversal of one or both systems may have a clinical impact on the chemotherapy of RCCs.     

Glioblastoma and cerebral microvascular endothelial cell migration in response to tumor-associated growth factors

OBJECTIVE: Glioma cell migration is determined by a complex interplay between soluble motogens and extracellular matrix components. Several growth factors are thought to be involved in glioma cell migration; however, little is known about their motogenic potency relative to one another. METHODS: Using modified Boyden chamber assays, we compared the chemotactic effects of scatter factor/hepatocyte growth factor (SF/HGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, epidermal growth factor (EGF), fibroblast growth factor (FGF)-1, FGF-2, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF), pleiotrophin (PTN), and midkine (MK) in concentrations ranging from 1 pmol/L to 50 nmol/L on three different human glioblastoma cell lines. Checkerboard analyses distinguished between chemotaxis and chemokinesis. We further investigated the motogenic effects on human cerebral microvascular endothelial cells and analyzed receptor expression profiles. RESULTS: SF/HGF was the most potent chemotactic factor for all three glioblastoma cell lines, inducing up to 33-fold stimulation of migration. TGF-alpha showed the second strongest effect (up to 17-fold stimulation), and FGF-1 was also chemotactic for all three glioblastoma cell lines analyzed (maximal 4-fold effect). EGF, FGF-2, IGF-1, IGF-2, TGF-beta1, and TGF-beta2 were chemotactic for one or two of the cell lines (2- to 4-fold effects), whereas PDGF-AA, PDGF-BB, VEGF, PTN, and MK had no effect. In contrast, the most potent stimulators of cerebral microvascular endothelial cell migration were PDGF-AA (4-fold) and PDGF-BB (6-fold). CONCLUSION: The expression levels of SF/HGF and TGF-alpha as well as their respective receptors, MET and EGFR, are known to correlate with glioma malignancy grade. The particularly strong motogenic effects of these two growth factors suggest that they could be promising targets for an antimigratory component of glioma therapy, at least in comparison with the 12 other factors that were analyzed.     

Expression of the SART1 tumor rejection antigen in renal cell carcinoma

We have previously described the SART1 gene, which encodes both the SART1₂₅₉ antigen expressed in the cytosol of the majority of squamous cell carcinomas and some adenocarcinomas and the SART1₈₀₀ antigen expressed in the nucleus of the majority of proliferating cells. The SART1₂₅₉ antigen is recognized by HLA-A24 and A26-restricted cytotoxic T lymphocytes (CTLs). The present study investigated the expression of these two antigens in renal cell carcinomas (RCCs) in order to identify an appropriate molecule for use in specific immunotherapy for RCC patients. These two antigens were detected in all RCC cell lines and cells of the primary cultures of the RCCs tested. Further, they were detectable in cells of the primary cultures of non-tumorous kidney tissues. In contrast to these cultured cells, SART1₂₅₉ was detectable in only a few uncultured RCC tissues (5/20, 25%) and was undetectable in non-tumorous kidney tissues. SART1₈₀₀ was also scarcely detectable in uncultured RCC tissues (3/20, 15%) and non-tumorous kidney tissues (4/20, 20%). HLA-A2402-restricted and tumor-specific CTL (KE4-CTL) used for the cloning of the SART1 gene showed significant levels of cytotoxicity to both the cells from the RCC cell line and the cells from the primary cultures of RCC tissues, but did not lyse any normal cells, including cells from the primary cultures of non-tumorous kidney tissues. The SART1-derived peptide at positions 690–698 induced HLA-A24 restricted CTLs cytotoxic to RCC cells from peripheral blood mononuclear cells (PBMCs) of RCC patients. Therefore, the SART1 peptide could be an appropriate molecule for use in peptide-based specific immunotherapy for RCC patients. Received: 22 October 1999 / Accepted: 11 January 2000     

舒尼替尼治疗转移性非透明细胞肾癌的疗效观察

目的 初步探讨舒尼替尼治疗转移性非透明细胞肾癌的疗效.方法 非透明细胞肾癌22例.男14例,女8例.年龄29～76岁,中位年龄46岁.根治性肾切除术后出现转移14例,初诊时诊断为肾癌伴转移行减瘤性肾切除术8例.病理证实乳头状癌12例,嫌色细胞癌1例,集合管癌3例,未分类癌6例.转移部位包括肺、淋巴结、肾上腺,骨和肝脏.舒尼替尼50mg/d,口服,每天1次,治疗4周休息2周.治疗时间4.5 ～24.0个月,中位时间11个月.随访4.5 ～25.0个月,中位时间14个月.结果 22例疾病控制率为73％ (16/22).部分缓解4例(18％),其中乳头状癌3例,嫌色细胞癌1例,转移灶位于肺或肺加腹膜后淋巴结,或腹膜后淋巴结.疾病稳定＞3个月12例(55％),用药3个疗程内疾病进展6例(27％).结论 舒尼替尼治疗转移性肾乳头状癌、嫌色细胞癌、集合管癌、未分类癌有效,对淋巴结转移及肺转移者的疗效相对较好.     

肿瘤转移相关基因1在人骨肉瘤细胞的表达与转移侵袭的关系

目的比较肿瘤转移相关基因(MTA1)在人骨肉瘤细胞高低转移株的表达水平,探讨MTA1表达水平与骨肉瘤细胞转移潜能的相关性。方法采用半定量逆转录-聚合酶链反应 (RT-PCR)检测MG-63骨肉瘤细胞高低转移株MTA1的表达情况,用Boyden小室体外侵袭实验检测两株MG-63细胞的体外侵袭力;用脂质体介导的MTA1基因转染MG-63低转移株细胞,通过 RT-PCR检测MTA1的表达;Boyden小室体外侵袭实验检测转染前后细胞侵袭力的变化。结果 RT-PCR结果显示MTA1在MG-63低转移细胞株中表达水平低(1．32),在高转移细胞株中表达水平高(6．27,P<0．05);Boyden小室体外侵袭实验显示MG-63高转移株细胞体外侵袭力强,其穿膜细胞相对百分率为(46．3±2．4)％,低转移株细胞体外侵袭力较弱,其穿膜细胞相对百分率(12．6± 1．1)％,两者差异有统计学意义(P<0．05);转染MTA1基因后,低转移细胞株转移潜能较未转染细胞明显增高。结论 MTA1与人骨肉瘤细胞转移潜能有密切关系。     

Epidermal growth factor stimulation can substitute for c-Src overexpression in promoting breast carcinoma invasion

BACKGROUND AND AIMS: Cancer progression is in large part dependent on the complex process of cell invasion, involving adhesion, motility, and enzymatic proteolysis. Overexpression of the Src proto-oncogene (c-Src), a nonreceptor tyrosine kinase, has been implicated in the progression of both colon and breast cancer. Our group has previously reported that overexpression of c-Src leads to a significant gain in invasive cell behavior in vitro. In this study, we sought to assess the relative importance of epidermal growth factor (EGF) stimulation and c-Src overexpression in conferring an invasive phenotype. METHODS: Breast carcinoma cells and colon epithelial cells which naturally express low levels of c-Src were used for these studies. The cells were transfected so that they overexpressed c-Src; the mock-transfected parent lines were used as controls. Transfectants were tested for changes in invasion patterns after Src inhibition and EGF stimulation. RESULTS: Invasion assays in both cell systems confirmed the importance of c-Src in determining invasive potential. A significant correlation was shown between c-Src kinase protein and cell invasion. Furthermore, Src inhibition significantly inhibited invasion in a dose-dependent manner. To clarify the relative contribution of EGF and c-Src to cell invasion, the ability of cells to invade through growth-factor-reduced matrigel, with or without EGF added, was compared to invasion through intact matrigel. The breast and colon cell lines behave quite differently in this regard. In the colon model, overexpressed c-Src is critical for cell invasion and stimulation with EGF is synergistic with c-Src overexpression. Conversely, the breast carcinoma cells transfected with c-Src were unable to invade without EGF stimulation and did not demonstrate the same synergistic relationship between c-Src and EGF. Instead, our results indicate that in BT474 breast carcinoma cells, EGF can substitute for c-Src in promoting breast cancer cell invasion. CONCLUSIONS: Because most breast carcinomas overexpress c-Src, it behooves one to question the extent to which reducing the amount of EGF and consequent EGFR activity will decrease invasion. In this study, the effects of EGF on cell invasion were determined in light of a single alteration in c-Src expression. Our results show that EGF enhances the impact of c-Src overexpression on invasion. In breast cancer cells, EGF is capable of inducing invasion to the same extent as c-Src overexpression. This suggests that anti-EGFR therapies will be efficacious in retarding breast carcinoma invasion and metastasis.     

Role of squamous cell carcinoma antigen 1 expression in the invasive potential of head and neck squamous cell carcinoma

BACKGROUND: Serine proteases have important roles in tumor invasion and metastasis, and their inhibitors, serine protease inhibitors (serpins), are attractive targets for therapeutic strategies. On chromosome 18q21, there is a cluster of serpins: maspin, headpin, and squamous cell carcinoma antigen 1 (SCCA1)/SCCA2. Others and we have reported that the expression of these serpins is down regulated in head and neck squamous cell carcinoma (HNSCC) cells compared with normal squamous epithelial cells. In this study, we hypothesized that expression of SCCA1 is biologically disadvantageous to HNSCC cells. METHODS: HNSCC cell lines were transfected with a mammalian expression vector with SCCA1 cDNA. In vitro proliferation, migration, or invasive potential (matrigel assay) of the transfectants were assayed. In addition, the in vivo growth and invasion was analyzed using the floor-of-mouth model of nude mice. RESULTS: SCCA1 expression did not alter the in vitro growth rate of established HNSCC cells. However, SCCA1 expression significantly inhibited the in vitro invasion in matrigel assays. Furthermore, the in vivo growth and invasion in nude mice was also inhibited by SCCA1 expression. CONCLUSIONS: Overexpression of SCCA1 in a HNSCC cell line inhibited its invasive potential. Loss of expression of the serpin SCCA1 may play a role in the malignant progression of HNSCC.     

应用抑制性消减杂交技术克隆肾癌转移相关基因

目的:应用抑制性消减杂交技术构建人高侵袭性肾癌细胞和低侵袭性肾癌细胞间差异表达的cD-NA消减文库,筛选并克隆肾细胞癌转移相关基因.方法:采用涂有Matrigel胶的Transwell小室分离回收高、低侵袭性肾癌细胞,分别从高侵袭性肾癌细胞与低侵袭性肾癌细胞中提取polyA RNA,合成双链cDNA,经RsaI酶切后将高侵袭性肾癌细胞cDNA分为两组并加上接头1和接头2,再与过量低侵袭性肾癌细胞cDNA进行两次消减杂交及两次抑制性PCR,PCR产物与pMD18-T载体连接并转化JM109大肠杆菌构建成cDNA消减文库,文库扩增后随机挑取克隆进行测序及同源性分析.结果:文库共包含185个阳性克隆,随机挑取50个阳性克隆分析,其中45个克隆有插入片段.将其中15个有插入片段的克隆进行测序,表明源于7个已知基因.结论:利用抑制性消减杂交技术,从一对同源的高低转移表型差异的细胞株中获得了7条可能与肾癌转移相关的cD-NA序列,他们可能在促进肾癌转移中起到重要作用.     

Role of hepatocyte growth factor in invasion of prostate cancer cell lines through tumor-stromal interaction

We examined how prostate stromal cell-derived hepatocyte growth factor (HGF) affects invasion of prostate cancer cells through tumor-stromal interaction. The effects of HGF, various growth factors [transforming growth factor (TGF)-alpha, TGF-beta 1, basic fibroblast growth factor, keratinocyte growth factor, and platelet-derived growth factor], and conditioned medium (CM) from prostate stromal cells (PrSC) on prostate cancer cells (LNCaP, PC-3 and DU145) were determined by collagen gel invesion assay. DU145 cells and PrSC were co-cultured for matrigel invasion chamber assay. LNCaP and PC-3 cells did not respond to any of the factors examined. Invasion of DU145 cells into the collagen gel matrix was induced by HGF and TGF-beta 1, but not by any of the other factors tested. When DU145 cells were cultured in CM from PrSC or co-cultured with PrSC, the cells acquired invasive potential, and this invasion was inhibited by an antibody against HGF, but not against TGF-beta 1. Induction activity of CM from cancer cells to stimulate HGF production by PrSC was studied by ELISA method and Western blotting. Native type HGF production in PrSC was enhanced by some unknown inducer(s) produced by cancer cells. In summary, PrSC-derived HGF enhanced invasive activity of the prostate cancer cell line DU145 through tumor-stromal interaction wherein DU145 cells secreted some HGF-inducer(s) for PrSC.     

siRNA沉默HYAL1基因表达对人乳腺癌细胞侵袭力的影响

目的研究RNA干扰对透明质酸酶基因HYAL1的表达以及对人乳腺癌细胞侵袭力的影响。方法体外化学合成HYAL1序列特异性双链RNA（dsRNA）,在脂质体（SiPORT Lipid）的介导下转染人人乳腺癌细胞株MDA-MB-231、MDA—MB-453S、ZR-75和ZR-75-30。荧光共聚焦显微镜下观察转染效率,RT—PCR分析HYAL1 mRNA的表达,体外实验测定肿瘤细胞穿透matrigel的能力。结果4种乳腺癌细胞株的HYAL1基因的表达均被HYAL1-siRNA有效封闭,HYAL1 mRNA相对水平明显降低（P〈0．05）。转染48h后4种人乳腺癌细胞的侵袭力均明显降低（P〈0．01）。结论HYAL1-siRNA能有效抑制人乳腺癌细胞株HYAL1基因的表达,降低人乳腺癌细胞的侵袭力。     

Histological subtyping and nuclear grading of renal cell carcinoma and their implications for survival: a retrospective nation-wide study of 629 patients

OBJECTS: The aim of this study was to evaluate the prognostic significance of the current WHO histological subtyping and Fuhrman nuclear grading on the survival of patients with renal cell carcinoma (RCC). MATERIALS AND METHODS: A retrospective population-based study was carried out on all patients with a histopathologically confirmed diagnosis of RCC in Iceland between 1971 and 2000. Fuhrman grade, TNM stage, and survival were evaluated and multivariate analysis applied in order to determine prognostic factors. RESULTS: Out of 629 patients (387 males, 242 females, mean age 64 years), 558 (88.7%) had clear cell, 53 (8.4%) papillary, and 13 (2.1%) chromophobe RCC. Patient demographics were comparable for the two major subtypes, but chromophobe RCCs were larger in size and were diagnosed at a younger age. Clear cell RCCs were more often of higher grades (G3+G4, 48.4%) and at advanced TNM stages (III+IV, 59.3%) than papillary RCCs (22.6% and 34% respectively, p<0.001). Linear regression analysis showed a strong correlation between grade, tumor size, and stage (p<0.001). Chromophobe RCCs had a better survival in univariate analysis than both papillary and clear cell RCCs (84.6% vs. 66.5% and 54.9% 5-year disease specific survival, p<0.001). However, in the multivariate analysis, only the patient's age, calendar year of diagnosis, TNM stage, and nuclear grade were independent prognostic factors of survival. CONCLUSION: In this complete nation-wide series nuclear grading is important in predicting survival of patients with RCC. It is strongly related to both tumor size and stage, with stage being by far the strongest prognostic factor. Different histological subtypes confer different survival. However, in spite of the distinctive cytogenetic and molecular characteristics of the subtypes, the survival difference is to a large extent due to differences in grade and particularly stage.     

Pyrrolidine dithiocarbamate exerts anti-proliferative and pro-apoptotic effects in renal cell carcinoma cell lines.

BACKGROUND: The activation of nuclear factor-kappaB (NF-kappaB) has been implicated in the development, progression and metastasis of renal cell carcinoma (RCC). This study investigates the effect of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, on two metastatic human RCC cell lines, ACHN and SN12K1. METHODS: RCC cell lines and normal cells were exposed to 25 or 50 microM of PDTC. Apoptosis was measured by flow cytometry and TdT-mediated nick end labelling methods. Cell viability and proliferation were measured by MTT and BrdU assays, respectively. Expression of NF-kappaB subunits, IkappaBs, IkappaB Kinase (IKK) complex and apoptotic regulatory proteins were analysed by western blotting and/or immunofluorescence. DNA-binding activity of NF-kappaB subunits were measured by ELISA. RESULTS: RCC cell lines had a higher basal level expression of all the five subunits of NF-kappaB than normal primary cultures of human proximal tubular epithelial cells or HK-2 cells. PDTC decreased the viability and proliferation of RCC, but not normal cells. Of the two RCC cell lines, ACHN had a higher basal level expression of all the five NF-kappaB subunits than SN12K1 and was more resistant to PDTC. While PDTC induced an overall decrease in expression of all the five NF-kappaB subunits in both RCC cell lines, unexpectedly, it increased the nuclear expression of NF-kappaB in ACHN, but not in SN12K1. PDTC reduced the DNA-binding activity of all the NF-kappaB subunits and the expression of the IKK complex (IKK-alpha, IKK-beta and IKK-gamma) and the inhibitory units IkappaB-alpha and IkappaB-beta. PDTC induced a significant increase in apoptosis in both RCC cell lines. This was associated with a decrease in expression of the anti-apoptotic proteins, Bcl-2 and Bcl-(XL), without marked changes in the pro-apoptotic protein Bax. CONCLUSION: These data suggest that PDTC has the potential to be an anticancer agent in some forms of RCC.     

Role of membrane type 1-matrix metalloproteinase and gelatinase A in head and neck squamous cell carcinoma invasion in vitro.

The proteolytic activity of gelatinase A, a member of the matrix metalloproteinase (MMP) family, is considered to be a critical factor in tumor cell penetration of the extracellular matrix. To express catalytic activity, however, gelatinase A requires activation by another MMP, membrane type 1-matrix metalloproteinase (MT1-MMP). The head and neck squamous cell carcinoma cell line, UM-SCC-1, forms a quiescent monolayer atop collagen unless stimulated with epidermal growth factor (EGF; 3.5 nmol/L), which induces single cell invasion within 48 hours. To determine the role of the MT1-MMP/gelatinase A protease system in an in vitro stromal invasion model, expression vectors for MT1-MMP and gelatinase A were transfected into UM-SCC-1 (SCC-1/MT and SCC-1/gelA, respectively). SCC-1/MT tumor cells were found to invade in the absence of growth factor stimulation. Additionally, these cells displayed shorter onset to invasion and penetrated deeper into the collagen gel with EGF stimulation than did control vector transfectants. SCC-1/gelA cells similarly demonstrated invasion in the absence of EGF and a heightened invasive potential under EGF-stimulated conditions. These results suggest that the MT1-MMP/gelatinase A protease system participates in squamous cell carcinoma invasion of collagenous matrices.