Human Kidney Tumor Research Articles

Molecular cloning and characterization of human GIPC2, a novel gene homologous to human GIPC1 and Xenopus Kermit

PDZ-domain protein GIPC1/GIPC interacts with GTPase-activating protein RGS-GAIP, TGFbeta type III receptor, and integrin alpha6A subunit. Kermit, a Xenopus orthologue of human GIPC1, interacts with some class of WNT receptor. In this study, we identified a novel GIPC1-related gene in human genome by using bioinformatics, and isolated GIPC2 cDNAs by using cDNA-PCR. GIPC2 encoded a 315-amino-acid protein with a central PDZ domain, which showed 62.0% total-amino-acid identity with GIPC1. Both GIPC2 gene on human chromosome 1 and GIPC1 gene on human chromosome 19p13.1 were found to consist of 6 exons. Exon-intron structures of GIPC2 gene and GIPC1 gene were well conserved. GIPC2 mRNA was relatively highly expressed in ascending colon, followed by kidney and pancreas. Among 3.7-, 2.6-, and 1.7-kb GIPC2 mRNAs generated due to alternative polyadenylation, 2.6-kb GIPC2 mRNA was the major GIPC2 transcript in adult kidney. Expression of GIPC2 mRNA was significantly down-regulated in 6 out of 14 cases of primary kidney tumors, in 5 out of 11 cases of primary colon tumors, and in 1 out of 7 cases of primary rectal tumors. Because GIPC2 might bind to TGFbeta type III receptor or some class of WNT receptor, just like GIPC1 or Kermit, down-regulation of GIPC2 mRNA in human primary tumors might lead to interference of TGFbeta signaling or some class of WNT signaling. This is the first report on molecular cloning of GIPC2, the second member of the GIPC gene family, which is down-regulated in human primary kidney and colorectal tumors.

Expression of the membrane-associated carbonic anhydrase isozyme XII in the human kidney and renal tumors.

Carbonic anhydrase isozyme XII (CA XII) is a novel membrane-associated protein with a potential role in von Hippel-Lindau carcinogenesis. Although Northern blotting has revealed positive signal for CA XII in normal human kidney, this is the first study to demonstrate its cellular and subcellular localization along the human nephron and collecting duct. Immunohistochemistry with a polyclonal antibody (PAb) raised against truncated CA XII revealed distinct staining in the basolateral plasma membrane of the epithelial cells in the thick ascending limb of Henle and distal convoluted tubules, and in the principal cells of the collecting ducts. A weak basolateral signal was also detected in the epithelium of the proximal convoluted tubules. In addition to the normal kidney specimens, this immunohistochemical study included 31 renal tumors. CA XII showed moderate or strong plasma membrane-associated expression in most oncocytomas and clear-cell carcinomas. The segmental, cellular, and subcellular distribution of CA XII along the human nephron and collecting duct suggests that it may be one of the key enzymes involved in normal renal physiology, particularly in the regulation of water homeostasis. High expression of CA XII in some renal carcinomas may contribute to its role in von Hippel-Lindau carcinogenesis.

Expression of MUC1, Thomsen-Friedenreich-related antigens, and cytokeratin 19 in human renal cell carcinomas and tubular clear cell lesions.

The expression of MUC1, MUC2, mucin-associated Thomsen-Friedenreich-related antigens (TF, sialosyl-TF, Tn, and sialosyl-Tn), and cytokeratin 19 (CK19) was systematically investigated in situ in 58 resected human kidney tumours, surrounding tissue of normal appearance, and two normal kidneys obtained at autopsy, using monoclonal antibodies. In kidney tissues of normal appearance, TF, s-TF, MUC1 and CK19 were positive in distal tubules and collecting ducts but negative in proximal tubules. In contrast, MUC2, Tn, and s-Tn were negative throughout the normal renal tubular system. Almost all renal cell carcinomas (RCCs) showed strong immunoreactivity for MUC1, but all were negative for MUC2. Some RCCs expressed TF, Tn, s-Tn, and CK19. In addition, the immunomorphological characteristics of the majority of clear-cell RCCs and clear/granular RCCs with anti-MUC1 and anti-CK 19 closely resembled those of the collecting duct and the distal tubule rather than the proximal tubule. In the renal tissue of otherwise normal appearance adjacent to clear-cell RCCs and clear/granular RCCs, clear cells with excessive storage of glycogen were often found in the collecting duct system, but only rarely in the proximal tubules. These results suggest that the majority of clear-cell RCCs and clear/granular RCCs may originate from the collecting duct system.

25] Evaluation of biological role of c-Jun N-terminal kinase using an antisense approach

Publisher Summary The mammalian mitogen-activated protein (MAP) kinase signaling pathway links extracellular stimuli to the regulation of cellular responses, including cellular growth, differentiation, and apoptosis. MAP kinase family members are a conserved group of serine/threonine protein kinases that include c-Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK), and p38 MAP kinases. An antisense approach is a useful strategy to potently and specifically inhibit gene expression to delineate the precise functional role of a target protein and identify potential pharmacological targets. Treatment of cultured cells with oligonucleotides targeted to either human JNK1 or human JNK2 results in a potent inhibition of JNK messenger ribonucleic acid (mRNA) and protein expression, as well as JNK kinase activity. Inhibition of JNK gene expression by antisense oligonucleotides reveals a significant suppression of hypoxia/reoxygenation-induced apoptosis in human kidney cells and tumor necrosis factor (TNF)- α -induced E-selectin expression in human endothelial cells. These results suggest that the activation of JNK is an important signal transduction protein for apoptosis induced by the cellular stress of hypoxia/reoxygenation. In addition, the results demonstrate that JNK2 is the JNK isoform responsible for TNF- α -mediated induction of E-selectin in endothelial cells.

HER-2/neu is expressed in human renal cell carcinoma at heterogeneous levels independently of tumor grading and staging and can be recognized by HLA-A2.1-restricted cytotoxic T lymphocytes

The HER-2/neu oncoprotein, a 185 kDa membrane-associated tyrosine kinase with extensive homology to the epidermal growth factor receptor (EGF-R), is overexpressed in breast and ovarian carcinomas. Its overexpression is closely associated with poor prognosis in the course of disease. Here we demonstrate HER-2/neu overexpression in both established cell lines and biopsy material obtained from renal epithelial tumors. Immunohistochemical analysis of human kidney tumor lesions using 2 HER-2/neu-specific antibodies revealed HER-2/neu expression in more than 40% of primary epithelial renal tumors and more than 30% of primary renal cell carcinoma (RCC) specimens. A distinctive HER-2/neu expression pattern was found in different subtypes of kidney tumors with the highest frequency in chromophilic and chromophobic RCC, but neither associated with disease stage nor tumor grade. Eight of 10 RCC cell lines expressed significant levels of HER-2/neu mRNA and protein, but at a lower level compared with HER-2/neu overexpressing ovarian carcinoma cells. To evaluate the immune response against HER-2/neu expressing HLA-A2-positive (HLA-A2(+)) RCC cells, allogeneic HLA-A2-restricted cytotoxic T-lymphocyte (CTL) lines generated by pulsing dendritic cells with 3 different HER-2/neu-derived peptides, (HER-2(9.369), HER-2(9.435) and HER-2(9.689), were utilized in chromium-release assays. Specific lysis of HER-2/neu expressing HLA-A2(+) RCC cell lines was mediated by CTL lines specific for each of these 3 HER-2/neu-derived epitopes. The fine specificity of 2 CTL clones was defined to the epitopes HER-2(9.435) and HER-2(9.689). Their specificity was then confirmed by cold target inhibition assays. In addition, CTL-mediated lysis was enhanced by pulsing tumor cells with exogenous HER-2/neu-specific peptides. Our data suggest that (i) HER-2/neu is heterogeneously expressed in different subtypes of RCC, (ii) HER-2/neu is naturally processed by RCC and (iii) HER-2/neu epitopes presented by RCC can be recognized by HLA-A2-restricted, HER-2/neu-specific CTL.

A new antigen recognized by cytolytic T lymphocytes on a human kidney tumor results from reverse strand transcription.

By stimulating blood lymphocytes from a renal cell carcinoma patient in vitro with the autologous tumor cells, we obtained cytolytic T lymphocyte (CTL) clones that killed several autologous and allogeneic histocompatibility leukocyte antigen (HLA)-B7 renal carcinoma cell lines. We identified the target antigen of these CTLs by screening COS cells transfected with the HLA-B7 cDNA and with a cDNA library prepared with RNA from the tumor cells. The antigenic peptide recognized by the CTLs has the sequence LPRWPPPQL and is encoded by a new gene, which we named RU2. This gene is transcribed in both directions. The antigenic peptide is not encoded by the sense transcript, RU2S, which is expressed ubiquitously. It is encoded by an antisense transcript, RU2AS, which starts from a cryptic promoter located on the reverse strand of the first intron and ends up on the reverse strand of the RU2S promoter, which contains a polyadenylation signal. This mechanism of antigen expression is unprecedented and further illustrates the notion that many peptides recognized by T cells cannot be predicted from the primary structure of the major product of the encoding gene. Antisense transcript RU2AS is expressed in a high proportion of tumors of various histological types. It is absent in most normal tissues, but is expressed in testis and kidney, and, at lower levels, in urinary bladder and liver. Short-term cultures of normal epithelial cells from the renal proximal tubule expressed significant levels of RU2AS message and were recognized by the CTLs. Therefore, this antigen is not tumor specific, but corresponds to a self-antigen with restricted tissue distribution.

Inhibitory effect of shark liver oil on cutaneous angiogenesis induced in Balb/c mice by syngeneic sarcoma L-1, human urinary bladder and human kidney tumour cells.

The effect of shark liver oil on cutaneous angiogenesis induced in mice by intradermal grafting of tumour cells was evaluated. It was shown that this substance (Ecomer) suppressed neovascular response in mice grafted with sarcoma L-1 syngeneic cells, human kidney cancer and human urinary bladder cancer cells. In addition, strong stimulatory effect of this drug on mice blood granulocyte number and their metabolic activity was observed.

Characterization of an HLA-A3 restricted human kidney specific T cell clone

Background. The tissue specificity of a cytolytic T lymphocyte is determined by the MHC class I bound peptide it recognizes. We have developed an allorestricted human CTL clone, DBS 1.5, that recognizes an epitope found on HLA-A3+ kidney epithelial cells but not on HLA identical B-lymphoblastoid cells. The peptide recognized by this clone has been isolated from HPLC separated, acid eluted peptides from purified HLA class I molecules from HLA-A3+ kidney tissue. This peptide shares no sequence homology with any known protein. Methods. To confirm the tissue specificity of the HLA-A3 restricted clone and the peptide it recognizes we have transfected the gene for HLA-A3 into a number of tumor cell lines both human and murine not expressing this antigen. The resulting transfected lines, confirmed by immunofluorescent staining, were used as targets to determine if expression of HLA-A3 alone was sufficient to allow recognition and lysis by the HLA-A3 restricted T cell clone. Results. The HLA-A3 restricted T cell clone recognized HLA-A3 when expressed on human kidney epithelial cells and to a lesser extent on human lung epithelium and human epidermal cells. Of the tumor lines transfected with HLA-A3 only the human kidney tumor cell line was lysed at a level equal to the original kidney epithelial cell used to develop the clone. Conclusion. These results confirm that this allorestricted human CTL clone is tissue specific recognizing a peptide found in human epithelial tissue that must be presented in the context of HLA-A3 for recognition.

Differential expression of CYP1A1, CYP1A2, CYP1B1 in human kidney tumours

The presence of mRNA of individual members of the CYP1 gene family in normal and neoplastic kidney has been investigated by RTPCR. CYP1B1 mRNA was consistently expressed in both normal and neoplastic kidney while CYP1A1 was present in the majority of normal and neoplastic whereas CYP1A2 was infrequently expressed. Expression of the Ah receptor and Arnt which are involved in the transcriptional activation of the CYP1 genes was also studied. The Ah receptor mRNA and Arnt mRNA were consistently expressed both in kidney tumours and normal kidney. These results indicate differential expression of individual members of the CYP1 gene family in normal and neoplastic kidney and suggest that several mechanisms including the Ah receptor complex could be involved in their regulation.

Analysis of native WT1 protein from frozen human kidney and Wilms' tumors.

The Wilms' tumor susceptibility gene, WT1, is altered in a subset of Wilms' tumors and encodes a transcription factor with four zinc fingers. Here we describe the isolation of native WT1 protein from frozen normal human kidney and Wilms' tumor samples. Through size exclusion chromatography and Western blot analysis we determined the elution pattern of WT1. The majority of WT1 from adult kidney and Wilms' tumor specimens was found to elute at a size of approximately 120 kDa, consistent with a WT1 homodimer and some WT1 protein was also found in a higher molecular weight complex. In 14 week fetal kidney the majority of the WT1 protein eluted at a size of 80 kDa, suggesting that at this developmental stage the WT1 protein is not present as a homodimer. The identity of complexing partners can now be studied using this approach.

TSC1 and TSC2 gene mutations in human kidney tumors.

TSC1 and TSC2 gene mutations in human kidney tumors.

Pathogenesis of renal cell adenomas and carcinomas in animal models.

Renal neoplasms rarely develop spontaneously in laboratory animals, but have been induced in different species, particularly in small rodents, by a variety of oncogenic agents including chemicals, hormones, radiation and viruses [1–5]. Although more than 100 chemicals, different types of highenergy radiation and viruses have been identified as renal carcinogens in rodents, only a few animal models of renal carcinogenesis have been analyzed in greater detail. The main reason for this selective approach seems to be that phenotypic similarities rather than differences are characteristic of neoplastic development induced by the various oncogenic agents [1]. A hereditary renal cell carcinoma was described in rats by Eker [6] and has been thoroughly studied as a model of dominantly inherited predisposition to a specific tumor type [7–9]. Even if such genetic events are involved in the evolution of renal cell carcinomas, phenotypic similarities with certain types of tumors induced by exogenous agents are obvious [9, 10]. The spectrum of renal neoplasms observed in animal models is nearly as broad as that known from human pathology [4]. Whereas experience in human pathology has guided the classification and histogenetic derivation of experimental renal tumors for a long time, several experimental findings had a strong impact on the classification and elucidation of the pathogenesis of human kidney tumors in the past two decades [1, 4, 5]. As to the variety of etiologic agents producing renal neoplasms the reader is referred to a number of reviews [1–5, 11, 12]. This article will be focused on some animal models which have significantly contributed to our under-

Mutations in OGG1, a gene involved in the repair of oxidative DNA damage, are found in human lung and kidney tumours.

The human OGG1 gene encodes a DNA glycosylase activity catalysing the excision of the mutagenic lesion 7,8-dihydro-8-oxoguanine from oxidatively damaged DNA. The OGG1 gene was localized to chromosome 3p25, a region showing frequent loss of heterozygosity (LOH) in lung and kidney tumours. In this study, we have analysed by RT-PCR the expression of OGG1 in 25 small cell lung cancers, in 15 kidney carcinomas and the 15 normal kidney counterparts. The results show that OGG1 messenger RNA can be detected in all tumours tested and that no significant difference was observed in the level of expression between normal and tumoral kidney tissues. Denaturing gradient gel electrophoresis (DGGE) was used to screen this series of human tumours for alterations in the OGG1 cDNA. The study revealed homozygous mutations in three tumours, two from lung and one from kidney. Sequencing analysis of the mutants identified a single base substitution in each of the three cases: two transversions (GC to TA and TA to AT) and one transition (GC to AT). All three substitutions cause an amino acid change in the hOgg1 protein. For the mutant kidney tumour, the normal tissue counterpart shows a wild-type profile. These results suggest a role for OGG1 mutations in the course of the multistage process of carcinogenesis in lung or kidney.

Theta-class glutathione S-transferases in human kidney and renal tumours.

Glutathione S-transferases are a family of dimeric multi-functional enzymes involved in the cellular detoxification and excretion of endogenous substances and xenobiotics. The theta-class of GSTs is composed of two homodimeric isoenzymes designated GSTTl-I[ I] and GSTT2-2 [2]. GSTs are thought to be involved in the sensitivity of tumours to the therapeutic effects of anticancer drugs. It is therefore important to characterize the level of expression of these enzymes in both normal and neoplastic tissues. This is especially important in renal tissue since renal tumonrs are particularly refractory to chemotherapy. Cytosolic proteins f?om normal cortex and matched tumour tissues from nephrectomy patients (n=31) were resolved using a 15% SDS-PAGE system and transferred to nitrocellulose membranes using standard procedures. Theta-class GST expression was determined by directing primary antibodies against GSTTl-1[3] (monoclonal) and GSTT2-2 [4] (polyclonal) respectively. Membranes were subsequently incubated with HRP-conjugated secondary antibodies, rabbit anti-mouse Ig (GSTTI-I) and goat anti-rabbit Ig (GSTT2-2) and visualised using DAB. lmmunohistochemical localisation of theta-class GSTs was determined using the SABC-AP system.

P XVI A.13 - P XVI A.13 Evidence that DNA adducts in some human kidney tumours in France are related to ochratoxin A

P XVI A.13 - P XVI A.13 Evidence that DNA adducts in some human kidney tumours in France are related to ochratoxin A

Substrate/Inhibitor Properties of Tumour Purine Nucleoside Phosphorylase

Abstract Substrate/inhibitor properties of purine nucleoside phosphorylase (PNP), isolated from human lung and kidney tumour tissues, have been characterised and compared with those of the enzyme from the corresponding normal organs.

Asymmetric Expression of Protein Kinase CK2 Subunits in Human Kidney Tumors

Renal clear cell carcinomas and the corresponding ipsilateral control tissues were investigated for protein kinase CK2 activity and subunit ratio. The average protein kinase CK2 activity from 21 different kidney samples was 318 U/mg and that from the corresponding tumors 610 U/mg. The subunit ratio of protein kinase CK2α in tumors/normal tissue (T/N) was 1.58 and that of the protein kinase CK2β (T/N) was 2.65. The data suggest that the generally described increase in protein kinase CK2 activity in tumor cells may to some extent result from a deregulation in subunit biosynthesis or degradation. This at least partly owing to the presence of excess enzymatically active protein kinase α-subunit but also to a significantly higher presence of the non-catalytic β-subunit.

Binding of urinary protein C inhibitor to cultured human epithelial kidney tumor cells (TCL-598). The role of glycosaminoglycans present on the luminal cell surface.

Binding of urinary protein C inhibitor (PCI) to cultured human epithelial kidney tumor cells (TCL-598) was studied. Binding was dose-dependent, time-dependent, and saturable. Heparin interfered in a dose-dependent way with PCI binding to TCL-598 as did heparan sulfate and to a lesser degree also dermatan sulfate. Pretreatment of TCL-598 with protamine sulfate inhibited subsequent binding of PCI in a dose-dependent manner and > 100 micrograms/ml protamine sulfate reduced binding of PCI to < 10% of the control. Binding of 125I-PCI was specific, and bound 125I-PCI was recovered from the cells by heparin treatment or detached together with intact cells by EDTA treatment, migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the same mobility (M(r) = 57,000) as unbound 125I-PCI. Furthermore, cell-bound PCI was functionally active as judged from its ability to inhibit the amidolytic activity of urokinase, and its inhibitory activity was stimulated approximately 3-4-fold as compared to fluid-phase PCI. Immunogold electron microscopy revealed that PCI-antigen presented to the cells from the luminal side bound exclusively to that surface in native as well as in prefixed cells. This binding of PCI was abolished in the presence of heparin (50 micrograms/ml) and after pretreatment of the cells either with protamine sulfate (400 micrograms/ml) or with heparinase III (0.5 unit/ml). A slight decrease in PCI binding was seen after pretreatment of the cells with chondroitinase ABC and chondroitinase AC. In contrast, binding of PCI to extracellular matrices of TCL-598 was decreased to approximately 70% after chondroitinase ABC treatment of the extracellular matrices, whereas both heparinase III or chondroitinase AC treatment only reduced matrix-bound PCI to approximately 95%. These data suggest that heparan sulfate-containing proteoglycans are predominantly involved in binding of PCI to the luminal side of TCL-598, while dermatan sulfate-containing proteoglycans, the overall predominant PCI-binding proteoglycans in TCL extracts, are responsible for PCI binding to the extracellular matrix. Heparan sulfate, however, exposed to an environment containing PCI under physiological conditions, might localize PCI and modulate its target enzyme specificity in vivo.

Activity and immunohistochemistry of DT-diaphorase in hamster and human kidney tumours.

We have studied the biochemical and immunohistochemical changes of DT-diaphorase in diethylstilbestrol (DES)-induced hamster kidney tumours and human biopsies from normal kidneys and renal clear cell carcinoma. The activities of primary and secondary antioxidants in these hamster and human tissues are also reported. DT-diaphorase is decreased in the different subcellular fractions of hamster and human tissues. In hamster kidney the activities of the one-electron quinone reductases show a nearly two-fold increase. Immunohistochemical findings confirm the decrease in DT-diaphorase in hamster and human tissues. This image is of special interest in the case of nephroblastoma (Wilms' tumour), since it has been proposed that the DES-induced tumour is a 'nephroblastoma-like' one. Primary anti oxidant enzymatic activities, i.e. superoxide dismutase and glutathione peroxidase, are increased in hamster kidney bearing DES-induced tumours and decreased in human renal clear cell carcinoma. Glutathione disulphide reductase is decreased in hamster and human tumours. The role of these enzymatic activities in the carcinogenic process is also discussed.

Structure and functional expression of a complementary DNA for porcine growth hormone-releasing hormone receptor

Growth hormone-releasing hormone (GHRH) belongs to the family of gutneuropeptide hormones which also includes glucagon, secretin and vasoactive intestinal peptide (VIP). All receptors for this peptide hormone family seem to involve similar signal transduction pathways. Upon hormone binding, these receptors interact with guanine nucleotide binding protein ‘Gs’ and cause the stimulation of adenylate cyclase. The secretin and VIP receptor cDNAs have recently been cloned and found to be homologous to those of calcitonin and parathyroid hormone receptors. Based on cDNA sequences of these receptors, we designed several oligonucleotide primers which were used to amplify two novel porcine pituitary cDNA fragments by the polymerase chain reaction. One novel receptor cDNA fragment was used to screen a porcine pituitary cDNA library and a full-length cDNA encoding a putative porcine GHRH receptor of 451 amino acids was isolated. This putative receptor mRNA is present specifically in porcine anterior pituitary cells and not in eight other porcine tissues as shown by Northern hybridization analysis. The receptor cDNA was subsequently cloned into a mammalian cell expression vector containing the cytomegalo-virus promoter. A human kidney tumor cell line (293) stably transfected with this vector was found to express the receptor efficiently and to bind [ 125I]-GHRH specifically. Furthermore, challenge of the 293 cells expressing the receptor by GHRH leads to efficient stimulation of cytoplasmic cAMP production.