ABSTRACT
This study describes the establishment of a rat kidney cortical collecting duct (CCD) clonal cell line (RCCD1 cells) that maintains high transepithelial resistance and specific hormonal sensitivities. Immortalized cells were obtained by infection of primary cultured CCD cells with the wild-type simian virus 40. Grown on Petri dishes, RCCD1 cells are organized as monolayers of cuboid cells separated by tight junctions and form domes. Grown on permeable filters, confluent RCCD1 cells exhibit high transepithelial resistance (Rt: 2390 +/- 140 omega. cm2), transepithelial potential difference (PD) of -10.5 +/- 1.2 mV lumen negative, an associated short-circuit current (Isc) of 4.3 +/- 0.5 microA/cm2, and generated significant Na+, K+, H+ and HCO3- gradients, reflecting Na+ and H+ reabsorption and K+ and HCO3- secretion. RCCD1 cells exhibit features of both principal (PC) and intercalated (IC) cells. Consistent with PC phenotype, about 50% of the cells were positively stained by a PC-specific agglutinin. In situ hybridization studies revealed the presence of alpha, beta and gamma subunit mRNAs of the amiloride-sensitive epithelial Na+ channel and alpha 1 and beta 1 subunits of Na(+)-K(+)-ATPase. Moreover, Na(+)-K(+)-ATPase was immunolocalized at the basolateral side of the cells. Arginine vasopressin (AVP) induced a significant increase in both cellular cAMP content and Isc. Amiloride decreased in a dose-dependent manner Isc from untreated and AVP-treated RCCD1 cells. In addition, a barium-sensitive K+ conductance was evidenced in the apical side of the cells. Consistent with IC phenotype, isoproterenol (ISO) provoked a large increase in cellular cAMP and stimulated Isc. The effect of ISO on Isc was blocked by 5 x 10(-3) M DPC, a chloride channel blocker. Finally, AVP plus ISO had additive effect on Isc. Taken together, these results provide evidence that the RCCD1 cell line has maintained many of the original properties of rat CCD from which they were derived.
Subject(s)
Cell Line , Kidney Cortex/cytology , Kidney Cortex/metabolism , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/metabolism , Animals , Arginine Vasopressin/pharmacology , Cell Polarity , Cyclic AMP/metabolism , Electric Impedance , Epithelial Cells , Epithelium/metabolism , In Situ Hybridization , Ion Transport , Isoproterenol/pharmacology , Kidney Cortex/drug effects , Kidney Tubules, Collecting/drug effects , Membrane Potentials , Microscopy, Electron , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sodium-Potassium-Exchanging ATPase/geneticsABSTRACT
P44 Ro (Mel) is a human malignant melanoma cell line derived from a testicular metastasis in a DNA repair deficient, xeroderma pigmentosum patient. This line harbors a N-ras gene mutated in codon 61. To investigate other cellular genes possibly contributing to the expression of its transformed phenotype, four XP44 revertant cell lines were isolated by different selection procedures and the association of the level of expression of various oncogenes (including N-ras) and tumor suppressor genes with the selection for the revertant phenotype was determined. The revertants exhibited a significant but variable degree of phenotypic reversion, according to the selective pressure to which they were submitted, and a phenotypic stability dependent on their constant maintenance in selective medium. Back-revertant lines were isolated by culturing revertant lines in control medium for several weeks. The comparison between parental, revertant and back-revertant cells has revealed that, beyond the mutation in codon 61 of N-ras, two groups of genes appear to be also implicated in the transformation process of XP44 RO (Mel) cells: one group, comprising pim A, trk, Rb and p53, whose expression is independent of the cell selection conditions; the other group, comprising Ha-ras, N-ras, neu 1, fos and met H, whose expression is more or less dependent upon such conditions. The myc gene is apparently not involved in this phenomenon. These results, besides strengthening the concept that carcinogenesis is a multigenic process, suggest that diverse mechanisms can lead to the transformed phenotype, but that these mechanisms might have some pathway(s) in common.
Subject(s)
Cell Transformation, Neoplastic , Genes, Tumor Suppressor , Melanoma/genetics , Oncogenes , 3T3 Cells , Adult , Animals , DNA Repair , Genes, Retinoblastoma , Genes, p53 , Genes, ras , Humans , Male , Melanoma/pathology , Mice , Tumor Cells, CulturedABSTRACT
The hormonal responsiveness profile of the cortical collecting duct varies from one species to another. To identify the hormones and agonists that modulate the functions of this tubule segment in the human species, we generated a cell line (HCD) immortalized by SV40 virus. The tubular origin of this cell line was assessed by the expression of collecting duct-specific antigens and the ability of vasopressin to increase by nine-fold cAMP synthesis. Glucagon and adenosine stimulated cAMP synthesis, and atrial natriuretic peptide stimulated cGMP synthesis in a concentration-dependent manner. Bradykinin, adenosine and angiotensin increased intracellular calcium concentration ([Ca2+]i). Because adenosine can regulate tubular functions, we examined its role on glucagon-induced cAMP synthesis. Using adenosine analogs, we demonstrated that HCT cells both expressed adenosine type-2 (A2) receptors which stimulated cAMP production, and adenosine type-1 (A1) receptors linked to [Ca2+]i increase which inhibited glucagon-stimulated cAMP synthesis. The inhibitory effect was abolished by pertussis toxin, and was neither due to [Ca2+]i increase nor to protein kinase C activation, which indicated that some A1 adenosine receptors were directly negatively coupled to adenylyl cyclase. These results suggest that adenosine can modify human cortical collecting duct functions in opposite ways according to the adenosine receptor activated.
Subject(s)
Adenosine/physiology , Cyclic AMP/biosynthesis , Glucagon/pharmacology , Kidney Tubules, Collecting/metabolism , Analysis of Variance , Animals , Atrial Natriuretic Factor/pharmacology , Calcium/metabolism , Cell Line , Cyclic GMP/biosynthesis , Humans , Intracellular Fluid/drug effects , Intracellular Fluid/metabolism , Kidney Tubules, Collecting/drug effects , Kidney Tubules, Collecting/immunology , Rabbits , Rats , Receptors, Purinergic P1/metabolism , Vasopressins/pharmacologyABSTRACT
The middle ear epithelium and respiratory epithelia share basic properties such as homeostasis of air-filled cavities and mucociliary clearance toward the pharynx. With the middle ear SV40-transformed (MESV) cell line, we used the short-circuit current (Isc) technique to investigate changes in ion transport induced by oxidants. Xanthine and xanthine oxidase on the basal side of the monolayers dramatically increased Isc up to 50%. This effect was not affected by superoxide dismutase or mannitol, but could be blunted by catalase or 1,3-dimethyl-2-thiourea. Increasing concentrations of H2O2 from 10(-5) to 5 x 10(-4) M produced a dose-dependent increase in Isc from 0.26 +/- 0.16 up to 4.21 +/- 0.43 microA/cm2 (P < 0.05, n = 5). Concentration of half-maximal stimulation (EC50) was 4.68 x 10(-5) M. This effect was inhibited by indomethacin and was related to a sodium transport, since the H2O2-induced increase in Isc could be prevented or abolished by 1) apical addition of benzamil (10(-6)M) and 2) substitution of sodium with N-methyl-glucamine. H2O2 exposure also induced indomethacin-sensitive increase in released prostaglandin (PG) E2 (EC50 = 5.62 x 10(-5) M) and in cAMP content (EC50 = 3.95 x 10(-5) M) with similar kinetics. These results suggest that exposure of MESV cells to oxidants stimulates the production of PGE2, which in turn increases the transepithelial sodium transport rate.
Subject(s)
Dinoprostone/physiology , Ear, Middle/metabolism , Reactive Oxygen Species/metabolism , Sodium/metabolism , Animals , Biological Transport/drug effects , Cell Line, Transformed , Cell Membrane Permeability/drug effects , Cyclic AMP/metabolism , Ear, Middle/ultrastructure , Electric Conductivity , Epithelium/metabolism , Epithelium/ultrastructure , Gerbillinae , Glucose/metabolism , Glucose Oxidase/metabolism , Hydrogen Peroxide/pharmacology , Mannitol/metabolism , Microscopy, Electron , Superoxides/pharmacology , Xanthine , Xanthine Oxidase/metabolism , Xanthines/metabolismABSTRACT
We have analyzed the effects of SV40 large-T oncogene on proteoglycan (PG) synthesis in a temperature-sensitive SV40-transformed renal cell line. Cells shifted from permissive (33 degrees C) to restrictive (39.5 degrees C) temperature, acquired within 48 h the phenotype of principal cells of the renal collecting tubule. They then synthesized hyaluronan, a large-sized PG, small amounts of free GAG chains, and a major approximately 130-kDa heparan sulfate-PG. Sulfated PGs were localized in a basement membrane-like layer and possessed the same core protein (61-70 kDa) derived from perlecan. Expression of large-T oncogene at the permissive temperature induced dramatic alterations of the extracellular matrix, and a 4- and 12-fold reduction in cell-associated and medium-released sulfated PGs, due to a approximately 50% decrease in perlecan mRNA content and gene transcription. This contrasted with a 2-fold increase in actin gene transcription and a 10- and 2-fold rise in the hyaluronan content in cells and medium, respectively. These alterations did not occur in a control cell line (RC.SV3) derived from the same tubule segment but infected with wild-type SV40 strain. They are thus most likely related to the functional state of large-T oncogene and may take part in the early steps of transformation.
Subject(s)
Antigens, Polyomavirus Transforming/genetics , Heparan Sulfate Proteoglycans , Heparitin Sulfate/genetics , Hyaluronic Acid/biosynthesis , Kidney Tubules, Collecting/metabolism , Oncogenes , Proteoglycans/genetics , Transcription, Genetic , Animals , Cell Line, Transformed , Chromatography, Ion Exchange , Glycosaminoglycans/metabolism , Heparitin Sulfate/metabolism , Hot Temperature , Hyaluronic Acid/isolation & purification , Immunohistochemistry , Kidney Tubules, Collecting/cytology , Proteoglycans/metabolism , Rabbits , Sulfuric Acids/metabolismABSTRACT
INTRODUCTION: Otitis media with effusion is a disease of the middle ear epithelium resulting from a decreased sol layer as well as increased mucus secretion and plasma-derived protein transudation, which causes mucus plugging. Because the epithelium keeps the middle ear cavities fluid-free and air-filled, we investigated its fluid transport capacities, which may be involved in both efficacy of the mucociliary clearance and drying-out of the posterior ear cavities (Yen PT et al: Acta Otolaryngol (Stockh) 113, 1993). We have established the absorptive capacity of middle ear epithelial cells in primary culture (Herman P, et al: Am J Physiol 262, 1992). However, the paucity of cells obtained by enzymatic digestion led us to develop a new model for further investigation of middle ear epithelial cell. METHODS: We established a middle ear cell line (MESV) using simian virus 40 (SV40) infection of middle ear epithelial cells from the Mongolian gerbil. RESULTS: Investigation of the transport processes using the short-circuit current technique showed that MESV cells retain most characteristics of the original middle ear epithelial cells. Transepithelial sodium transport from the apical to the basal side was responsible for the transepithelial lumen-negative potential difference. CONCLUSION: The presence of high concentrations of prostaglandin E2 in the middle ear effusions has been documented. This work investigates the effect of prostaglandin E2 on the rate of transepithelial ion transport of MESV cells. Prostaglandin E2 increased the rate of electrogenic sodium transport by means of increase in the intracellular cyclic adenosine monophosphate (cAMP) content. Such a modulation of sodium transport in the course of otitis media could be responsible for the reduced periciliary sol layer that impairs the mucociliary clearance.
Subject(s)
Dinoprostone/physiology , Ear, Middle/physiopathology , Otitis Media with Effusion/physiopathology , Animals , Cell Line , Cell Transformation, Viral , Cells, Cultured , Chronic Disease , Cyclic AMP/metabolism , Ear, Middle/ultrastructure , Electric Stimulation , Electrophysiology , Epithelium/physiopathology , Epithelium/ultrastructure , Gerbillinae , Ion Transport , Models, Biological , Mucociliary Clearance , Otitis Media with Effusion/pathology , Sodium/metabolismABSTRACT
We used a dual immunomorphological and physiological approach to demonstrate that the RC.SVtsA58 rabbit cortical cell line exhibits features of highly differentiated cortical collecting tubule (CCT) principal cells (PC). First, we raised monoclonal antibodies against RC.SVtsA58 cells and screened their reactivity with the rabbit kidney: three were specific for the basolateral domain of CCT PC and bound to 100% of RC.SVtsA58 cells. Second, we showed that bradykinin, atrial natriuretic peptide, and prostaglandin E2 increased intracellular Ca2+, guanosine 3',5'-cyclic monophosphate, and adenosine 3',5'-cyclic monophosphate (cAMP), respectively. In addition, 10 nM bradykinin inhibited desmopressin-elicited cAMP production by > or = 40%; this effect was suppressed by 10 microM of indomethacin and was reproduced with 1 nM of prostaglandin E2, indicating the conservation of arginine vasopressin-related regulatory loops described in microdissected CCT and freshly isolated cells. However, RC.SVtsA58 cells also express intercalated cell markers even after repeated cloning, which suggests that tsA58, a temperature-sensitive strain of simian virus-40, has transformed a multipotent type of PC in keeping with the cell interconversion hypothesis.
Subject(s)
Antigens/immunology , Hormones/physiology , Kidney Cortex/immunology , Kidney Cortex/physiology , Simian virus 40/physiology , Temperature , Animals , Antibodies, Monoclonal/immunology , Biological Transport , Cell Line, Transformed , Electrophysiology , Kidney Cortex/cytology , Rabbits , Sodium/metabolismABSTRACT
We have analyzed the functional and morphological effects of corticosteroid hormones in a SV40-transformed rabbit cortical-ascending-limb (CAL) cell line (RC.SV2, Vandewalle et al., 1989) having mineralocorticoid (MR) and glucocorticoid (GR) receptors (Rafestin-Oblin et al., 1993). Both aldosterone and dexamethasone (5 x 10(-8) M) induced a marked increase in (3H)ouabain binding (used to quantify membrane Na(+)-K+ ATPase) detectable as early as 6 hours and maximal at 24 hours (+56-57%) (due to a 1.6-1.8-fold increase in cell membrane binding sites without Kd alteration), and significantly augmented the ouabain-sensitive component of Rb+ influx. Triiodothyronine (T3, 10(-9) M) also stimulated ouabain binding by 21% but was not permissive for steroid action, whereas 5 micrograms/ml insulin had no effect. Both steroid hormones, T3 and insulin induced the formation of domes that was tightly correlated with ouabain binding (r = 0.949) except for insulin. The effects of aldosterone and dexamethasone on cell monolayers and cell ultrastructure were, however, strikingly different as aldosterone induced a marked amplification of basolateral areas with appearance of large intercellular spaces, reminiscent of the changes observed in deoxycorticosterone-treated rats, whereas dexamethasone predominantly influenced cell height. This discrepancy might be due to specific occupancy of MR and GR by aldosterone and dexamethasone, respectively, and/or to nongenomic effects of dexamethasone. We have thus characterized a cell culture model making it possible to analyze the actions of mineralocorticoid and glucocorticoid hormones in the mammalian kidney.
Subject(s)
Aldosterone/pharmacology , Dexamethasone/pharmacology , Kidney Tubules, Distal/chemistry , Kidney Tubules, Distal/cytology , Receptors, Steroid/analysis , Simian virus 40/physiology , Adrenal Cortex Hormones/analysis , Adrenal Cortex Hormones/metabolism , Animals , Cell Line, Transformed , Insulin/pharmacology , Kidney Tubules, Distal/ultrastructure , Microscopy, Electron , Ouabain/metabolism , Phenotype , Rabbits , Receptors, Mineralocorticoid , Receptors, Steroid/metabolism , Rubidium , Sodium-Potassium-Exchanging ATPase/analysis , Sodium-Potassium-Exchanging ATPase/drug effects , Sodium-Potassium-Exchanging ATPase/physiology , Triiodothyronine/pharmacologyABSTRACT
The activity of catalase, a key enzyme in cell detoxication of oxygen derivatives, was studied in SV40 transformed human fibroblasts. A cytogenetic study was performed in parallel to establish a quantification of 11p arm on which the corresponding gene is mapped. mRNA amounts were determined by Northern blotting. At early passages, catalase activity strongly decreased whereas the corresponding mRNA was present. No deletions of 11p arms were detected. At later passages, catalase activity remained low. 11p arm deletions were frequent, and the amount of mRNA was decreased. In these late passages, the good correlation between the number of 11p arms and catalase activity suggested a gene dosage effect. It is assumed that the decrease of catalase activity provides a selective advantage for the transformed cells. This decrease is related to a post-transcriptional change of regulation at early passages and to the loss of the corresponding gene at later passages.
Subject(s)
Catalase/genetics , Catalase/metabolism , Cell Transformation, Viral/genetics , Chromosome Deletion , Chromosomes, Human, Pair 11/physiology , Fibroblasts/physiology , Simian virus 40/physiology , Cell Line, Transformed , Cell Transformation, Viral/physiology , Fibroblasts/enzymology , Humans , Karyotyping , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic/geneticsABSTRACT
From an hepatocarcinoma cell line (LFCL.2A), unable to grow in a culture medium in which methionine was replaced by L-homocysteine, we had previously isolated revertant clones presenting a low growth rate, a loss of tumorigenicity and an inhibition of transcription of three oncogenes: c-Ki-ras, c-Ha-ras and c-myc. Here we showed that long-term deprivation of methionine led to a depletion of spermine, while putrescine and spermidine contents remained unchanged. When the revertant cells were shifted in a medium containing methionine, the oncogene transcription (except the p53 gene) started very rapidly in parallel with an increase in the putrescine content. By contrast, spermidine and spermine contents decreased during the first hours but were not significantly different from control values after numerous subcultures in methionine-containing medium.
Subject(s)
Biogenic Polyamines/biosynthesis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Gene Expression/genetics , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/metabolism , Methionine/deficiency , Oncogenes/genetics , Animals , Biogenic Polyamines/metabolism , Culture Media , Genes, myc/genetics , Genes, p53/genetics , Genes, ras/genetics , Methionine/pharmacology , Rats , Rats, Wistar , Time Factors , Transcription, Genetic/genetics , Tumor Cells, CulturedABSTRACT
The relative contributions of Na(+)-K(+)-ATPase pumps and Na(+)-K(+)-Cl- cotransport to total rubidium (Rb+) influx into primary cultures of renal tubule cells (PC.RC) and cells transformed either with the wild-type or a temperature-sensitive mutant of the simian virus 40 (SV40), were measured under various growth conditions. The Na(+)-K(+)-ATPase-mediated component represented 74% and 44-48% of total Rb+ influx into PC.RC and SV40-transformed cells, respectively. Proliferating transformed cells showed substantial ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx (41-45% of total) which indicated the presence of a Na(+)-K(+)-Cl- cotransport. The Or-Bs component of Rb+ influx was greatly reduced when temperature-sensitive transformed renal cells (RC.SVtsA58) grown in Petri dishes or on permeable filters were shifted from the permissive (33 degrees C) to the restrictive temperature (39.5 degrees C) to arrest cell growth. The ouabain-sensitive Rb+ influx mediated by the Na(+)-K(+)-ATPase, the total and amiloride-sensitive Na+ uptakes were not modified following inhibition of cell proliferation. A similar fall in the Or-Bs influx was obtained when renal tubule cells transformed by the wild-type SV40 (RC.SV) were incubated with the K+ channel blocker, tetraethylammonium (TEA) ion, which we had previously shown to arrest cell growth without affecting cell viability (Teulon et al.: J. Cell. Physiol., 151:113-125, 1992). Reinitiation of cell growth by removal of TEA or return to 33 degrees C of the temperature-sensitive cells restored the Or-Bs component of Rb influx. Taken together, these results indicate that the Na(+)-K(+)-Cl- cotransport activity is critically dependent on cell growth conditions.
Subject(s)
Carrier Proteins/metabolism , Kidney Tubules/metabolism , Potassium/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Bumetanide/pharmacology , Cell Division , Cell Line, Transformed , Cell Size , Cells, Cultured , Chlorides/metabolism , DNA/biosynthesis , Kidney Tubules/cytology , Mutation , Rabbits , Rubidium/metabolism , Simian virus 40/genetics , Simian virus 40/physiology , Sodium/metabolism , Sodium-Potassium-Chloride Symporters , Temperature , Tetraethylammonium , Tetraethylammonium Compounds/pharmacologyABSTRACT
The presence of mineralocorticoid (MR) and glucocorticoid (GR) receptors was investigated in two renal tubular cell lines, derived from primary cultures of isolated rabbit kidney cortical cells infected with the wild-type SV40 virus, which exhibit thick ascending limb (RC.SV2) and collecting tubule (RC.SV3) phenotypes (Vandewalle et al. J. Cell. Physiol. 141, 1989, 203-221). MR and GR were quantified, in cell monolayers and cell cytosolic fractions, with [3H]aldosterone, [3H]dexamethasone and [3H]RU486, an antiglucocorticoid with no affinity for MR. Cytosolic receptors from RC.SV2 and RC.SV3 cells labeled with [3H]aldosterone, [3H]dexamethasone or [3H]RU486 sedimented at approximately 8 S in a 15-40% glycerol gradient. All steroids displaced bound [3H]dexamethasone to the same extent, suggesting that dexamethasone bound to both MR and GR: under the conditions of assay, [3H]aldosterone binds exclusively to MR, and [3H]RU486 to GR. In both RC.SV2 and RC.SV3 cells, [3H]aldosterone bound to one class of high affinity sites (Kd 0.14-0.8 nM; Nmax 8 to 22 fmol/mg protein). In both cell lines, the number of high affinity binding sites for [3H]dexamethasone ranged from 9 to 18 fmol/mg protein with an affinity of 0.5-1.3 nM. Compared to renal cortex, the most striking observation was a marked decrease in [3H]dexamethasone binding in primary cultures and SV40-transformed cells. These results indicate that MR and GR are expressed in two established mammalian kidney tubular cell lines providing new models of cultured renal cells for studies on the physiological effects of corticosteroid hormones.
Subject(s)
Kidney Tubules/metabolism , Mineralocorticoids/metabolism , Receptors, Steroid/metabolism , Aldosterone/metabolism , Animals , Cell Line, Transformed , Cells, Cultured , Centrifugation, Density Gradient , Dexamethasone/metabolism , Kidney Tubules/cytology , Rabbits , Receptors, Glucocorticoid/metabolism , Receptors, MineralocorticoidABSTRACT
The activities of several enzymes involved in the antioxidant system of the cell were studied in parallel to cytogenetic alterations at various times after SV40 infection and transformation of human fibroblasts. At early passages after SV40 infection, glutathione reductase (GSR), glutathione peroxidase (GPX), glutathione transferase (GST) and glucose-6-phosphate dehydrogenase (G6PD) activities were decreased. This, associated with the low superoxide dismutase (SOD) and catalase activities previously noticed in these cells, suggested that they are in a highly pro-oxidant status. Although chromosomes carrying the genes encoding these enzymes are frequently underrepresented, there is no direct relationship between the number of chromosomes and enzyme activities. Except for GPX, all the activities tend to increase in established cell lines reaching levels comparable to those of non-transformed fibroblasts. The late increase of G6PD activity may correlate with the frequent duplication of the early replicating X. GSR seems to correlate with G6PD activity and GPX to SOD total activity. The most striking alterations affect mitochondrial and peroxisomal enzymes activities: SOD, GPX and catalase.
Subject(s)
Cell Transformation, Neoplastic , Cell Transformation, Viral , Glutathione/metabolism , Simian virus 40 , Cell Line , Chromosome Aberrations , Fibroblasts/enzymology , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase/metabolism , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Glutathione Reductase/genetics , Glutathione Reductase/metabolism , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , HumansABSTRACT
To characterize the sulfated proteoglycans (PGs) alterations associated with malignant transformation of epithelial cells in vitro, the localization, charge, size, and composition of cell-associated and secreted sulfated PGs have been compared in rabbit renal proximal-tubule cells in primary culture (Ronco et al., 1990) and in a derived SV-40 transformed cell line (RC.SV1) exhibiting a proximal phenotype and high tumor-inducing ability (Vandewalle et al., 1989). Both normal and transformed cells incorporated PGs into a thick basement membrane layer as shown by ruthenium red staining and immunodetection with a monoclonal antibody raised against the core protein of the bovine basement membrane heparan sulfate-PG (HS-PG). In primary cultures of normal cells, cell-associated PGs were almost identical to those extracted from renal tubule fractions in vivo by their size (Kav = 0.27 vs. 0.26 on Sepharose CL-6B) and composition characterized by the exclusive presence of heparan sulfate glycosaminoglycan (HS-GAG) chains. In addition, the cells secreted a HS-PG with similar biochemical characteristics (Kav = 0.29; 100% HS-GAG chains). The SV-40-transformed RC.SV1 cells also synthesized and secreted a unique PG with the same charge and Kav values and apparently the same core protein (35 kDa) as in nontransformed cells, but three major differences were observed: (i) an increased proportion of PG-associated [35S]sulfate radioactivity released into the culture medium (36 vs. 21%), (ii) the emergence of free GAG chains unincorporated into PGs and detected only in the cell-associated fraction, and (iii) a dramatic change in the composition of GAG chains in which chondroitin sulfate replaced heparan-sulfate. The latter finding is in keeping with the known chondroitin sulfate increase and heparan-sulfate decrease in epithelial tumors. The alterations of PGs observed in this study may play a role in the acquisition and/or maintenance of the malignant phenotype.
Subject(s)
Cell Transformation, Neoplastic , Kidney Tubules, Proximal/metabolism , Proteoglycans/biosynthesis , Simian virus 40/genetics , Antibodies, Monoclonal , Cell Line, Transformed , Cells, Cultured , Chromatography, Gel , Fluorescent Antibody Technique , Immunoenzyme Techniques , Kidney Tubules, Proximal/chemistry , Kidney Tubules, Proximal/cytology , Microscopy, Immunoelectron , Proteoglycans/isolation & purification , Sulfates/metabolism , Sulfur RadioisotopesABSTRACT
The expression of superoxide dismutases (SOD) 1 and 2 was studied in 4 clones of human fibroblasts after their infection by simian virus 40 (SV40), in parallel with the alterations of chromosomes 21 and chromosome 6q arms, carrying the genes that encode for SOD1 and SOD2 respectively. For all clones, a similar scheme with 2 main phases was observed for both chromosome and SOD variations. The first phase, defined as the pre-crisis phase, was characterized by chromosomal instability, but maintenance of normal numbers of chromosome 6q arms and chromosomes 21. The level of SOD2 mRNA was high, while SOD2 activity and immunoreactive protein were low. SOD1 protein and activity were decreased. In the second phase, defined as the post-crisis phase, the accumulation of clonal chromosomal rearrangements led to the loss of 6q arms, while the number of chromosomes 21 remained normal. SOD2 mRNA level was decreased and SOD2 immunoreactive protein and activity remained low. SOD1 protein and activity increased with passages, reaching values similar to those of control cells at late passages. As in established SV40-transformed human fibroblast cell lines, good correlation was found between SOD2 activity and the relative number of 6q arms. These results allow us to reconstruct the sequence of events leading to the decrease of SOD2, a possible tumor-suppressor gene, during the process of SV40-transformation of human fibroblasts.
Subject(s)
Cell Transformation, Viral , Superoxide Dismutase/metabolism , Cells, Cultured , Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 6 , Gene Expression , Humans , RNA, Messenger/genetics , Simian virus 40 , Superoxide Dismutase/geneticsABSTRACT
The hormonal regulation of a Na(+)-K(+)-Cl- cotransport was investigated in a renal tubule cell line (RC.SV2 cells) transformed by the simian virus 40. This cell line has the main characteristics of cells from the thick ascending limb of Henle, including the presence of Tamm-Horsfall protein and stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production by calcitonin (CT). Kinetic studies with 22Na+, 36Cl-, and 86Rb+ indicated the existence of a Na(+)-K(+)-Cl- cotransport with a stoichiometry of 1Na+:1K+: 2Cl-. All compounds stimulating cAMP production enhanced the ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx mediated by Na(+)-K(+)-Cl- cotransport. CT (100 ng/ml) increased the Or-Bs influx twofold by enhancing maximum velocity without changing the apparent Michaelis constant. The K(+)-channel blocker barium blunted the CT-stimulated Or-Bs influx by 64-74%, whereas the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate reduced the CT-stimulated influx by 28-40%. These results suggest that CT stimulates the Na(+)-K(+)-Cl- cotransport by a cAMP-dependent mechanism and that K+ recycling through K+ membrane channels is an important modulator of cotransporter-mediated ion fluxes.
Subject(s)
Calcitonin/pharmacology , Carrier Proteins/metabolism , Loop of Henle/metabolism , Animals , Cell Line, Transformed , Cyclic AMP/metabolism , Cyclic AMP/physiology , Cyclic GMP/metabolism , Cyclic GMP/physiology , Hormones/pharmacology , Kinetics , Loop of Henle/cytology , Mucoproteins/metabolism , Peptides/pharmacology , Rabbits , Sodium-Potassium-Chloride Symporters , UromodulinABSTRACT
Biology of glomerular visceral epithelial cells ("podocytes") and their role in inflammatory process remain obscure, partly because of the lack of well-differentiated podocyte cultures. We have established a human cell line by transfecting with a replication-defective SV40 plasmid (pSVHB1), a primary culture of podocytes derived from an enriched preparation of unencapsulated glomeruli free of tubule and Bowman's capsule contaminants. Podocyte specificity of the primary culture was assessed by a dual immunomorphological and functional approach. The resulting cell line (HGVEC.SV1) was cloned and the clonal cells were adapted to hormonally defined medium supplemented with only 2% newborn bovine serum. Clone A4 has been exhibiting over 35 passages, a combination of markers unique to podocytes, including expression of vimentin, podocalyxin, ectoenzymes (CALLA antigen and mRNA), heparan-sulfate proteoglycans (molecular mass of core protein = 75 kDa), and production of type IV collagen (alpha 1 and alpha 5 chains) established by immunoprecipitation and Northern blot analysis. Cytokeratin was detected in rare cellular foci and the search of Von Willebrand factor was negative. This clonal cell line has been used to demonstrate: (1) that human podocytes are highly sensitive to atrial natriuretic peptide (ANP) which induced a dose-dependent increase in cGMP production (x20 at 0.5 microM ANP), and (2) that secretion of ANP-stimulated cGMP is dramatically polarized as 93% of extracellular cGMP were released in the apical medium when filter-grown HGVEC. SV1A4 cells were stimulated at their basal pole.
Subject(s)
Atrial Natriuretic Factor/pharmacology , Cell Line, Transformed , Collagen/biosynthesis , Cyclic GMP/metabolism , Kidney Glomerulus/cytology , Antigens, Differentiation/analysis , Antigens, Neoplasm/analysis , Cell Polarity , Clone Cells , Culture Media , Cyclic AMP/metabolism , Dipeptidyl Peptidase 4 , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/analysis , Heparan Sulfate Proteoglycans , Heparitin Sulfate/analysis , Humans , Kidney Glomerulus/chemistry , Kidney Glomerulus/metabolism , Neprilysin , Proteoglycans/analysis , Simian virus 40 , Vimentin/analysisABSTRACT
A comparative study of chromosomal rearrangements occurring in 4 independent clones obtained from SV40-transformed cornea and skin human fibroblasts was performed. Rearrangements principally affect some constitutive heterochromatin and, to a lesser degree, telomeric regions. This results in multiple exchanges between a limited number of chromosome structures, i.e., in jumping translocations. Such rearrangements occur even at early passages and some of them give rise to clonal rearrangements that accumulate at late passages. This process is responsible for progressive modification of the karyotypes, principally characterized by deletion of a number of chromosome segments. Thus, clonal rearrangements are selected among many others not occurring at random. The selective pressure retaining clonal rearrangement seems to be similar for the 4 independent clones, since selection of the derivative chromosomes leads to the same imbalances, whatever the origin of the clone. This sequence of events recalls that of human solid tumors, since jumping rearrangements are generally observed in pre-malignant conditions or in low-grade malignancies, whereas clonal rearrangements leading to typical imbalances are detected in more advanced malignant tumors.
Subject(s)
Cell Transformation, Neoplastic , Cell Transformation, Viral , Gene Rearrangement , Simian virus 40/genetics , Translocation, Genetic , Cells, Cultured , Fibroblasts , HumansABSTRACT
We compared the pattern of K+ channels and the mitogenic sensitivity to K+ channel blocking agents in primary cultures of rabbit proximal tubule cells (PC.RC) (Ronco et al., 1990) and two derived SV-40-transformed cell lines exhibiting specific functions of proximal (RC.SV1) and more distal (RC.SV2) tubule cells (Vandewalle et al., 1989). First, K+ channel equipment surveyed by the patch-clamp technique was modified after SV-40 transformation in both cell lines; although a high conductance Ca(2+)-activated K+ channel [K+200 (Ca2+)] remained the most frequently recorded K+ channel, the transformed state was characterized by emergence of three Ca(2+)-insensitive K+ channels (150, 50, and 30 pS), virtually absent from primary culture, contrasting with reduced frequency of two Ca(2+)-sensitive K+ channels (80 and 40 pS). Second, quinine (Q), tetraethylammonium ion (TEA) and charybdotoxin (CTX), at concentrations not affecting cell viability, all decreased 3H-TdR incorporation and cell growth in PC.RC cultures, but only TEA had similar effects in transformed cells. The latter were further characterized by paradoxical effects of Q that induced a marked increase in thymidine incorporation. Q also exerted contrasting effects on channel activity: it inhibited the [K+200 (Ca2+)] when the channel was highly active, with a Ki (0.2 mM) similar to that measured for 3H-TdR incorporation in PC.RC cells (0.3 mM), but increased the mean current through poorly active channels. TEA blocked all K+ channels with conductance greater than or equal to 50 pS, including the [K+200 (Ca2+)], in a range of concentrations that substantially affected cell proliferation. The unique effect of TEA on SV-40-transformed cells might be related to broad inhibition of K+ channels.
Subject(s)
Calcium/pharmacology , Kidney Tubules, Proximal/cytology , Mitogens/pharmacology , Potassium Channels/physiology , Simian virus 40/physiology , Animals , Cell Division/drug effects , Cell Line, Transformed , Cell Transformation, Viral/drug effects , Cells, Cultured , Charybdotoxin , DNA/metabolism , Dose-Response Relationship, Drug , Drug Hypersensitivity , Epithelial Cells , Epithelium/physiology , Epithelium/ultrastructure , Kidney Tubules, Proximal/physiology , Kidney Tubules, Proximal/ultrastructure , Potassium Channels/drug effects , Quinine/pharmacology , Rabbits , Scorpion Venoms/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Tetraethylammonium Compounds/pharmacology , Thymidine/metabolism , TritiumABSTRACT
Eight LF x ICIG cell hybrid clones, isolated upon fusion of normal ICIG-7 human fibroblasts with tumorigenic, non-metastatic LF Cl.2A cells derived from a DAB-induced rat hepatocarcinoma, were studied. They were all highly tumorigenic and were capable of developing spontaneous lung metastases in syngeneic animals. All the hybrids were characterized by a rapid loss of human chromosomes. However, in long-term culture, they all revealed a persistence of human genetic information as assessed by Southern blotting. In hybrid lines in which human chromosomes were still visible, the most recurrent were numbers 7 and 9. Neither chromosome 7, previously reported to bear some of the genes controlling metastasis in human X mouse T-cell hybrids, nor chromosome 9 appeared to be correlated with the metastatic potential of LF X ICIG hybrids. The same conclusion applied (1) to a human 3.3-kb EcoRI DNA fragment which was amplified (approx. 10-fold) only in metastases induced by one out of 3 metastatic hybrids tested; (2) to the transcription level of c-Ha-ras and c-Ki-ras genes which was enhanced (approx. 4-fold) in metastatic and non-metastatic lines as well. Co-transfection of LF Cl.2A cells with pHSG 272 selectable marker DNA and genomic DNA from normal ICIG-7 human cells or from a hybrid-induced metastasis, reproducibly gave rise to geneticin-resistant transfectants capable of producing spontaneous lung metastases. Neither transfectants nor transfectant-induced metastases harbored detectable human DNA sequences but all harbored pHSG 272 DNA. These results again call for caution in gene transfer studies of the metastatic process.