Human Kidney Tumor Cell Research Articles

Comparison of the Expression Levels of Senescence Genes between Human Normal Kidney Cells and Kidney Tumor Cells According to Age

Comparison of the Expression Levels of Senescence Genes between Human Normal Kidney Cells and Kidney Tumor Cells According to Age

Optimization of the Extraction Process of Bioactive Compounds from Zingiber officinale Roscoe, Evaluation of Acetylcholinesterase Enzyme Inhibition and Cytotoxic Activity of the Free and Encapsulated Extract

The present study optimized the extraction process of bioactive compounds present in ginger (Zingiber officinale) dried at 80 °C, using ethanol:water 70:30 (v/v) as solvent. The extracts were evaluated for antioxidant activity by the 2,2’-azino-bis(3-ethylbenzothiazoline6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical reduction methods and by the chelating activity of FeII ions. It was found that the extraction condition with a temperature of 60 °C and an extraction time of 12 h showed better responses to the tests. Then, the characterization of the compounds was carried out by mass spectrometry and thermal analysis (thermogravimetric (TG), differential thermal analysis (DTA), and differential scanning calorimetry (DSC)), identifying that the main compounds of ginger were gingerols and shogaols, being confirmed by the intensities and characteristics of the thermal graphs. The inhibition of the enzyme acetylcholinesterase (AChE) was evaluated using the Ellman test, which did not show an inhibitory action. Regarding cytotoxic activity, the free extract and encapsulated in liposomes were tested, showing antiproliferative effect at different concentrations for human kidney tumor cells (786-0), liver cells (HUH7.5), and Macaca mullata normal kidney cells (LLC-MK2). Given the results obtained, ginger presents itself as a renewable source of bioactive compounds and can be indicated for applications in the pharmaceutical industry

Toxicogenic effects of the mushroom Ganoderma lucidum on human liver and kidney tumor cells and peripheral blood lymphocytes.

Ganoderma lucidum (Curtis) P. Karst., a bioactive mushroom with medicinal properties, is known to exert immunomodulatory, anti-inflammatory, hypocholesterolemic, hypoglycemic, and hepatoprotective effects. In this study, the effects of the G. lucidum fruiting body dry extract (GLE) on human liver (HepG2/C3A) and kidney (786-O) tumor cells and peripheral blood lymphocytes were evaluated. MTT-based cytotoxicity, trypan blue-based cell viability, comet, and cytokinesis-block micronucleus cytome assays were performed, and the production of reactive oxygen species was evaluated in vitro. GLE was toxic to the tumor cells, decreasing their viability by increasing their production of reactive oxygen species and inducing damage to their DNA. By contrast, only high concentrations of GLE were toxic to lymphocytes and decreased their viability, whereas low concentrations increased lymphocyte viability. Moreover, primary DNA damage was induced by GLE only at the highest concentration tested. G. lucidum shows potential antitumor effects against cancerous kidney and liver cells, exhibiting cytotoxic and genotoxic activity at low concentrations, whereas the same effects in lymphocytes are mediated only at high concentrations. This mushroom has the potential to be biotechnologically developed into a therapeutic agent for diseases, such as cancer.

Imaging of Clear Cell Renal Carcinoma with Immune Checkpoint Targeting Aptamer-Based Probe.

Immune checkpoint targeting immunotherapy has revolutionized the treatment of certain cancers in the recent years. Determination of the status of immune checkpoint expression in particular cancers may assist decision making. Here, we describe the development of a single-stranded aptamer-based molecular probe specifically recognizing human PD-L1. Target engaging aptamers are selected by iterative enrichment from a random ssDNA pool and the binding is characterized biochemically. Specificity and dose dependence is demonstrated in vitro in the cell culture using human kidney tumor cells (786-0), human melanoma cells (WM115 and WM266.4) and human glioblastoma LN18 cancer cells. The utility of the probe in vivo is demonstrated using two mouse tumor models, where we show that the probe exhibits excellent potential in imaging. We postulate that further development of the probe may allow universal imaging of different types of tumors depending on their PD-L1 status, which may find utility in cancer diagnosis.

Antitumoral activity, antioxidant capacity and bioactive compounds of ginger (Zingiber officinale)

Zingiber officinale, popularly known as ginger, it is in National Relations of Medicinal Plants of Interest to SUS (RENISUS) aiming at the appropriate and safe use of medicinal plants. Then, it is necessary to characterize and determine their bioactive compounds, their cytotoxic, mutagenic and/or antitumor properties, in order to guarantee food safety and its beneficial coadjuvant effects. The ginger rhizomes were dried at temperatures 40, 60, and 80°C and the bioactive compounds were extracted with methanol and ethanol (70:30 and 95:5). The rhizomes showed to be intermediate source of vitamin C. The highest levels of anthocyanins and yellow flavonoids were dried at 40°C. The highest content of phenolic compounds was for the extracts MeOH 70:30 and EtOH 70:30 at 80°C and for flavonoids MeOH 95:5 at 40°C. Data showed a positive correlation with the content of bioactive compounds and the antioxidant activity. The EtOH 70:30 extract at 80°C, did not present a cytotoxic effect, by calculating the mitotic index, or mutagen, by the evaluation of the chromosomal alterations, for the Allium cepa L. This extract showed a cytotoxic effect for human kidney tumor cells, by MTT test, for all concentrations tested and in the two evaluation times. The data serve as basis for the consumption of ginger, with a high content of bioactive compounds, with no harmful effect for the normal cells studied and with antitumor potential.

Isolevuglandins as a gauge of lipid peroxidation in human tumors

The cellular production of free radicals or reactive oxygen species (ROS) can lead to protein, lipid or DNA modifications and tumor formation. The cellular lipids undergo structural changes through the actions of enzymes (e.g. cyclooxygenases) or free radicals to form a class of compounds called Isolevuglandins (IsoLGs). The recruitment and continued exposure of tissue to ROS and IsoLGs causes increased cell proliferation, mutagenesis, loss of normal cell function and angiogenesis. The elevated concentration of ROS in cancerous tissues suggests that these mediators play an important role in cancer development. We hypothesized that tumors with elevated ROS levels would similarly possess an increased concentration of IsoLGs when compared with normal tissue. Using D11, an ScFv recombinant antibody specific for IsoLGs, we utilized immunohistochemistry to visualize the presence of IsoLG in human tumors compared to normal adjacent tissue (NAT) to the same tumor. We found that IsoLG concentrations were elevated in human breast, colon, kidney, liver, lung, pancreatic and tongue tumor cells when compared to NAT and believe that IsoLGs can be used as a gauge indicative of lipid peroxidation in tumors.

Abstract 1375: Contrasting effects of VEGF pathway inhibitors in primary and metastatic disease using perioperative neoadjuvant mouse models

Abstract Recent clinical trial failures with antiangiogenic therapy in patients with early-stage disease have raised the possibility that drug efficacy in preclinical studies, typically involving models of localized primary grown tumors, may not always predict for efficacy in treating metastatic disease, which is the most common cause of mortality in patients. Moreover, depending on disease stage and treatment circumstances, the efficacy of VEGF-pathway inhibitors administered as monotherapies may be offset in certain settings by increased aggressive invasiveness and augmented metastatic potential. Thus, with hundreds of trials underway in neoadjuvant and adjuvant settings to evaluate the efficacy of antiangiogenic therapy in blocking or slowing micrometastatic disease and eventual tumor recurrence, there is an urgent need clarify the utility of such drugs in all stages of tumor progression. Herein we describe a novel preclinical neoadjuvant therapeutic methodology used to compare the effects of short-term VEGF pathway inhibition in highly metastatic human breast, melanoma, and kidney tumor cells grown orthotopically in SCID mice before and after surgical resection. While cytotoxic chemotherapy administered in the maximum tolerated dose could slow tumor growth and lead to a prolongation of survival after resection, similar significant benefits by various VEGF inhibitors in primary tumor inhibition did not consistently translate into significant prevention of recurrent local and distant metastasis after treatment cessation. Furthermore, reductions in primary tumor growth by neutralizing antibodies to VEGF or VEGFR-2 were more predictive of modest survival benefits after tumor resection, compared to the VEGFR tyrosine kinase inhibitor (TKI) sunitinib, which had either no effect or decreased survival compared to control - despite significant reductions in primary tumor growth after neoadjuvant treatment. Potential tumor-independent ‘host’ drug responses could account for this difference as short-term treatment of immunocompromised nu/nu mice prior to intravenous tumor inoculation lead to modest or negligible benefits with VEGF or VEGFR2 antibodies compared to worse outcomes in VEGFR TKI pre-treated animals. Taken together, preclinical neoadjuvant therapy allows for the distinction of ‘anti-primary’ and ‘anti-metastatic’ effects and may serve as a predictive tool to model ongoing clinical trials as well as indicate potential drug combinations which may be used to overcome limitations when such drugs are used as a monotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1375. doi:1538-7445.AM2012-1375

Abstract 3640: Potential cooperation of monoclonal antibody based targeting of gamma-glutamyl transferase (GGT1) with chemotherapy in kidney tumor xenografts

Abstract Gamma-glutamyl transferase (GGT1) is a cell membrane bound enzyme that functions in gamma-glutamyl cycling, to provide cells with intracellular glutathione (GSH). GGT1 has been recognized to participate in chemotherapeutic resistance of tumors to carboplatin and various alkylating agents by increasing GSH levels intracellularly, thus allowing the tumor to escape electrophilic damage. Cysteinylglycine generated by membrane bound GGT1 can bind to extracellular cisplatin and prevent it from entering the cell. Mechanisms that target GGT1 and alter its function have been postulated to increase tumor sensitivity to conventional chemotherapies. We have generated a novel monoclonal antibody (mAb), 8G9 [IgG2a, k1] that recognizes GGT1. An immunohistochemical (IHC) survey of benign adult human tissues revealed an antigen distribution in formalin-fixed, paraffin embedded tissues consistent with previous reports of GGT1 expression. A subsequent IHC screen of nearly 100 kidney tumors revealed that scoring based on 8G9 immunoreactivity showed a preference for clear cell (94.45%), multilocular cystic (100%) and papillary cell (95.83%) kidney tumors. A statistical association of 8G9 immunostaining with Fuhrman nuclear grade or pathology staging was not evident. FACS analysis confirmed that 8G9 recognizes a cell surface epitope. Cross-immunoprecipitations performed with 8G9 and a commercially available GGT1 antibody confirmed that 8G9 recognizes GGT1. 8G9 exhibited a unique preference for native GGT1 likely through recognition of a combined epitope formed by the association of the GGT1 heavy and light chains. Because 8G9 appears to recognize a native, surface accessible GGT1 epitope, we performed xenograft studies to test the efficacy of 8G9 in blocking kidney tumor growth either as a single agent or in combination with cisplatin or carboplatin. Athymic nude mice were engrafted with the human kidney tumor cell line Caki-1. Upon successful tumor engraftment, mice were treated with 8G9, cisplatin or carboplatin as single agents or with 8G9+ ciplatin or 8G9 + carboplatin. As a single agent, 8G9 treatment did not result in a reduction of relative tumor volume. Likewise, there was little difference in relative tumor volume in mice treated a combination of 8G9 and carboplatin. However, 8G9 + cisplatin treatment exhibited a modest reduction in tumor volume. These preliminary data suggest that targeting GGT1 may have a cooperative effect in reducing tumor growth in the presence of cisplatin, and that the efficacy of chemotherapy combined with targeting GGT1 may be particularly sensitive to the mechanistic action of the chemotherapeutic agent. Further studies are needed to fully evaluate GGT1 as a potential drug target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3640. doi:10.1158/1538-7445.AM2011-3640

In Vitro and In Vivo Studies on the Effects of Bone Morphogenetic Protein-7 on Human Kidney and Lung Tumor Cells

Breast, kidney, lung, and prostate cancers are among the human cancers that show high propensity to form bone metastasis. Bone morphogenetic protein (BMP) -2 and -7 are two members of the BMP superfamily which show the most potent biological activity in stimulating bone differentiation and repair. These proteins have been used in clinical treatment of orthopedic diseases and have also been studied in different types of cancer. We report here detection of mRNA coding for three type I and one type II BMP receptors in G-402 kidney tumor cells and A-549 lung tumor cells, suggesting that these cells are responsive to BMPs. We then observed that BMP-7 inhibited cell proliferation of both cell lines in a protein concentration dependent manner in vitro. Additionally, when BMP-7-treated cells were implanted into the flank region of male nude mice, smaller tumors, compared to those formed with the untreated cells, were observed. Histological analysis showed that the masses formed at the site of implantation displayed significantly less number of tumors than the control and exhibited significant ectopic bone formation. These findings raise the possibility of BMP-7 as a therapeutic agent for kidney and lung cancers.

Immunotherapy with Cytokine-Induced Killer Cells in Metastatic Renal Cell Carcinoma

Cytokine-induced killer (CIK) cells have shown antitumor activity against several tumor cells both in vitro and in vivo. This study reports on the large-scale expansion of CIK cells and also present preliminary results from a pilot clinical trial. Sixteen (16) patients with renal cell carcinoma (RCC), all of whom had metastases after radical nephrectomy and adjuvant therapy using interferon-alpha (IFN-alpha) and/or interleukin-2 (IL-2), were treated with CIK cells. CIK cells were generated from peripheral blood mononuclear cells (PBMCs) and incubated in the presence of IFN-gamma followed by OKT3 and IL-2. Treatment schedule consisted of two to three cycles of CIK cell infusions at an interval of 3 weeks. A total of 46 infusions were administered to 16 metastatic RCC (mRCC) patients. The median number of transferred cells per treatment was 6.7 x 10(9) (range, 2.5-12.3). At a 60:1 effector-target cell ratio, CIK cells killed 51.4% and 32.1% of two human kidney tumor cell lines (293 and SK-RC-42), respectively. After CIK cell infusion, the percentage of CD3(+), CD8(+), CD3(+)CD56(+), and NKG2D(+) cells and the intracellular products of two type 1 cytokines (IFN-gamma and tumor necrosis factor alpha) significantly increased in the patients' PBMCs. Toxicity was minimal, and there were no immediate adverse reactions to the infusions. Three (3) patients had complete response, 1 patient had partial response, and 6 patients had stable disease. These results showed that adoptive CIK cell immunotherapy is a safe and effective treatment, which may have essential benefits for the improvement of the immunologic function in mRCC patients and play an important role in the treatment of mRCC.

Chmp1A functions as a novel tumor suppressor gene in human embryonic kidney and ductal pancreatic tumor cells

Chmp1A (Chromatin modifying protein 1A/Charged multivesicular protein 1A) is a member of the ESCRT-III (Endosomal Sorting Complex Required for Transport) family that was shown to function in endosome-mediated trafficking via multivesicular body (MVB) formation and sorting. Recent reports suggest that ESCRT complexes are also involved in cell cycle progression and tumor development. Using in vitro and in vivo model systems, we provide evidence that Chmp1A is a novel tumor suppressor, especially in the pancreas. We demonstrated that short hairpin RNA (shRNA) mediated stable silencing of Chmp1A in HEK 293T cells resulted in an increase of anchorage-independent growth in soft agar assay and tumor formation in xenograft assay. To investigate the involvement of Chmp1A in human tumor development we screened human cancer arrays and pancreatic tissue arrays. We discovered that Chmp1A mRNA and protein was reduced and/or altered (protein) in various human pancreatic tumors. To investigate the biological implication of these data, we either over-expressed or silenced Chmp1A in human pancreatic ductal tumor cells (PanC-1) and studied the effect of these manipulations on cell and tumor growth respectively. Stable over-expression of Chmp1A in PanC-1 cells resulted in cell growth inhibition and tumor xenograft inhibition respectively. In contrast, silencing of Chmp1 in PanC-1 cells resulted in the elevation of cell growth in vitro. Mechanistically, over-expression of Chmp1A strongly increased the protein level of P53 and phospho-P53. Taken together, our data indicates that Chmp1A is a novel tumor suppressor, especially in pancreas and that Chmp1A regulates tumor growth potentially through P53 signaling pathway.

Transcriptional activation of caspase-6 and -7 genes by cisplatin-induced p53 and its functional significance in cisplatin nephrotoxicity

This study examined the role of cisplatin-induced p53 activation in regulation of caspases and cellular injury during cisplatin nephrotoxicity. The executioner caspase-6 and -7 but not caspase-3 were identified as transcriptional targets of p53 in cisplatin injury as revealed by chromatin immunoprecipitation, a reporter gene and electrophoretic mobility shift assays, and real-time PCR following overexpression and inhibition of p53. DNA binding by p53 involved the first introns of the human and mouse caspase-7 gene and the mouse caspase-6 gene. Studies in human kidney, breast, ovary, colon, and prostate tumor cell lines also validated these findings. Treatment of p53 (-/-) cells with cisplatin did not induce caspase-6 and -7 expression and subsequent activation. In caspase-3 (-/-) cells, inhibition of caspase-6 and -7 activations markedly prevented cisplatin-induced cell death. In an in vivo model of cisplatin nephrotoxicity inhibition of p53 activation by a p53 inhibitor suppressed transactivation of the caspase-6 and -7 genes and prevented renal failure. p53 (-/-) mice were resistant to cisplatin nephrotoxicity as assessed by renal function and histology. These studies provide first evidence for p53-dependent transcriptional control of the caspase-6 and -7 genes and its functional significance in cisplatin injury to renal cells and functional implication of cisplatin-induced p53 induction in vitro and in vivo in cisplatin nephrotoxicity.

Secretion of the Human T Cell Leukemia Virus Type 1 Transactivator Protein Tax

The HTLV-1 Tax protein is well known as a transcriptional transactivator and inducer of cellular transformation. However, it is also known that extracellular Tax induces the production and release of cytokines, such as TNF-a and IL-6, which have adverse effects on cells of the central nervous system. The cellular process by which Tax exits the cell into the extracellular environmment is currently unknown. This study characterizes the process of Tax secretion from the cell. Specifically, cytoplasmic Tax was demonstrated to localize to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex. Additionally, it was demonstrated that full-length Tax was secreted from both baby hamster kidney cells and a human kidney tumor cell line. Tax secretion was partially inhibited by brefeldin A, suggesting that Tax migrated from ER to Golgi complex. The combined treatment of Tax-transfected cells with PMA and ionomycin resulted in a small increase in the amount of Tax secreted suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies provide a link between Tax accumulation in the cytoplasm, the detection of Tax in the extracellular environment. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Secretion of the Human T Cell Leukemia Virus Type I Transactivator Protein Tax

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I protein Tax is well known as a transcriptional transactivator and inducer of cellular transformation. However, it is also known that extracellular Tax induces the production and release of cytokines, such as tumor necrosis factor-alpha and interleukin-6, which have adverse effects on cells of the central nervous system. The cellular process by which Tax exits the cell into the extracellular environment is currently unknown. In most cell types, Tax has been shown to localize primarily to the nucleus. However, Tax has also been found to accumulate in the cytoplasm. The results contained herein begin to characterize the process of Tax secretion from the cell. Specifically, cytoplasmic Tax was demonstrated to localize to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex. Additionally, it was demonstrated that full-length Tax was secreted from both baby hamster kidney cells and a human kidney tumor cell line, suggesting that Tax enters the secretory pathway in a leaderless manner. Tax secretion was partially inhibited by brefeldin A, suggesting that Tax migrated from the endoplasmic reticulum to the Golgi complex. In addition, combined treatment of Tax-transfected BHK-21 cells with phorbol myristate acetate and ionomycin resulted in a small increase in the amount of Tax secreted, suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies begin to provide a link between Tax localization to the cytoplasm, the detection of Tax in the extracellular environment, its possible role as an extracellular effector molecule, and a potential role in neurodegenerative disease associated with HTLV-I infection.

Identification of the RNA polymerase II subunit hsRPB7 as a novel target of the von Hippel-Lindau protein.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the hereditary VHL disease and sporadic clear cell renal cell carcinomas (CCRCC). VHL-associated tumors are highly vascularized, a characteristic associated with overproduction of vascular endothelial growth factor (VEGF). The VHL protein (pVHL) is a component of the ubiquitin ligase E3 complex, targeting substrate proteins for ubiquitylation and subsequent proteasomic degradation. Here, we report that the pVHL can directly bind to the human RNA polymerase II seventh subunit (hsRPB7) through its beta-domain, and naturally occurring beta-domain mutations can decrease the binding of pVHL to hsRPB7. Introducing wild-type pVHL into human kidney tumor cell lines carrying endogenous mutant non-functional pVHL facilitates ubiquitylation and proteasomal degradation of hsRPB7, and decreases its nuclear accumulation. pVHL can also suppress hsRPB7-induced VEGF promoter transactivation, mRNA expression and VEGF protein secretion. Together, our results suggest that hsRPB7 is a downstream target of the VHL ubiquitylating complex and pVHL may regulate angiogenesis by targeting hsRPB7 for degradation via the ubiquitylation pathway and preventing VEGF expression.

Protein-tyrosine Phosphatase α, RPTPα, Is a Helicobacter pylori VacA Receptor

Helicobacter pylori vacuolating cytotoxin, VacA, induces vacuolation, mitochondrial damage, cytochrome c release, and apoptosis of gastric epithelial cells. To detect gastric proteins that serve as VacA receptors, we used VacA co-immunoprecipitation techniques following biotinylation of the cell surface and identified p250, a receptor-like protein-tyrosine phosphatase beta (RPTP beta) as a VacA-binding protein (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). VacA causes vacuolation of G401 cells, a human kidney tumor cell line, although they do not express RPTP beta. By co-immunoprecipitation with VacA, we identified p140 as a potential receptor in those cells. p140 purified by chromatography on a peanut agglutinin affinity matrix contained internal amino acid sequences of RGEENTDYVNASFIDGYRQK and AEGILDVFQTVK, which are identical to those in RPTP alpha. The peptide mass fingerprinting of p140 by time of flight-MS analysis also supported this identification. Treatment of G401 cells with RPTP alpha-morpholino antisense oligonucleotide before exposure to toxin inhibited vacuolation. These data suggest that RPTP alpha acts as a receptor for VacA in G401 cells. Thus, two receptor tyrosine phosphatases, RPTP alpha and RPTP beta, serve as VacA receptors.

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.

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.

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.

Application of quantitative image analysis to a mammalian cell line grown on microcarriers

Quantitative image analysis has been applied to the monitoring of cultures of a mammalian cell line on microcarriers. Procedures have been developed to investigate microcarrier colonization and cluster formation and to determine the eventual modification of cell size during cultivation using scanning electron microscopy (SEM) microphotography. The human kidney tumor cells (TCL 598) on which the procedures were tested underwent a slight size decrease during the development of the first cell layer on the microcarriers. The cluster size and the cell size remained constant during the culture stationary phase.