Biomedical subjects

R Cassingena

Publications and source records attributed to R Cassingena.

At least 19 recordsLinked to original sources

PubMed1995

Oxygen metabolites modulate sodium transport in gerbil middle ear epithelium: involvement of PGE2.

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.

Animals↗

PubMed1994

Principal cell-specific antigen and hormonal regulatory network in RC.SVtsA58 cell line.

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.

Animals↗

PubMed1993

Mineralocorticoid receptors in SV40-transformed tubule cell lines derived from rabbit kidney.

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.

Aldosterone↗

PubMed1993

Alterations of the glutathione cycle enzymes during and after SV40-transformation of human fibroblasts.

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.

Cell Line↗

PubMed1993

Decrease in catalase activity and loss of the 11p chromosome arm in the course of SV40 transformation of human fibroblasts.

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.

Catalase↗

PubMed1992

Early superoxide dismutase alterations during SV40-transformation of human fibroblasts.

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.

Cell Transformation, Viral↗

PubMed1992

Jumping translocations originate clonal rearrangements in SV40-transformed human fibroblasts.

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.

Cell Transformation, Neoplastic↗

PubMed1992

Spontaneous metastatic potential of rat hepatocarcinoma cells after cell fusion or DNA transfection.

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.

Animals↗

PubMed1992

Transformation of renal tubule epithelial cells by simian virus-40 is associated with emergence of Ca(2+)-insensitive K+ channels and altered mitogenic sensitivity to K+ channel blockers.

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.

Animals↗

PubMed1992

Characterization of a simian virus 40-transformed human podocyte cell line producing type IV collagen and exhibiting polarized response to atrial natriuretic peptide.

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.

Antigens, Differentiation↗

PubMed1992

Regulation by calcitonin of Na(+)-K(+)-Cl- cotransport in a rabbit thick ascending limb cell line.

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.

Animals↗

PubMed1991

Establishment of permanent renal tubule cell lines by infection with the wild-type and a thermosensitive mutant of the simian virus 40.

This study summarizes the properties of rabbit renal tubule cell lines transformed by the simian virus 40 (SV40). By infection with the wild type SV40, it was possible to establish three different cell lines exhibiting the main functions of proximal (RC.SV1), thick ascending limb-distal (RC.SV2), and collecting (RC.SV3) tubules. Another cell line infected with a thermosensitive mutant of SV40 (RC.SVtsA58) was highly sensitive to arginine vasopressin when cells were cultured at the restrictive temperature of 39.5 degrees C, suggesting they derived from principal cells of the collecting tubule.

Animals↗

PubMed1991

Concomitant alterations of microfilaments and microtubules in human epithelial cells (HBL-100) in relation to their malignant conversion.

The organization of microfilaments and microtubules was studied in human epithelial HBL-100 cells at different steps in their malignant conversion. This conversion was obtained either spontaneously upon successive in vitro subcultures (approximately p. 70), or after superinfection of early passage non-tumorigenic cells (p. 28) with Kirsten murine sarcoma virus. Our results point out a clear relationship between the progression of cells toward malignancy, whatever the modality of malignant transformation, and severe cytoskeleton modifications. The fragmentation of F-actin fibers was the earliest event occurring at p. 46, before the appearance of tumorigenicity. In tumorigenic cells, intracytoplasmic F-actin was nearly completely fragmented, the bundle of peripheral actin was loosened and poorly stained whereas the tubulin network was more important and more largely extended throughout the cytoplasm than in non tumorigenic cells.

Actin Cytoskeleton↗

PubMed1990

Proto-oncogene amplification and homogeneously staining regions in human breast carcinomas.

Cytogenetic studies on fresh human breast cancers revealed that homogeneously staining regions (HSRs), which are assumed to represent DNA amplification, are observed in almost half of the cases. To search for a possible relationship between HSRs and proto-oncogene amplification, 16 proto-oncogenes, including ERBB2, were studied by Southern blot analysis in four tumors with two or three HSRs, and in three tumors without HSRs. Only four proto-oncogenes were found to be amplified in at least one tumor each: HST and INT2 (x3), MYC (x2-3), and FES (x greater than 10). The large sizes of the HSRs, which each corresponded to several percent of the haploid genome, were hardly compatible with the low rate of amplification, except for FES and then only if a large adjacent segment was co-amplified. This incomplete correlation was demonstrated by in situ hybridization, using biotinylated probes, which showed fluorescent spots on only one HSR for FES in one tumor and for INT2 in another one. Our results indicate that most of the large amplifications corresponding to HSRs do not involve the proto-oncogenes usually studied in breast cancer. The large amplification of FES, detected in one tumor, may be coincidental.

Blotting, Southern↗

PubMed1990

Methionine dependence of rat hepatocarcinoma cells in culture: relationship with tumourigenicity and oncogene expression.

The culture in methionine-deprived medium of a rat hepatoma cell line (LF) significantly decreases: (1) the growth in vitro of these cells; (2) their tumourigenicity; and (3) the expression of a panel of oncogenes. When the LF cells are replaced in a medium containing methionine, they maintain a diminished in vitro growth and tumourigenic capacity, despite an overexpression of the same oncogenes. These data point out the determinant role of a nutritional factor, methionine, in tumour growth.

Animals↗

PubMed1990

Methionine-independence, tumorigenicity and oncogene expression of rat hepatocarcinoma cells.

Methionine-dependent (Met-Hcy+) revertant sublines were isolated from a methionine-independent (Met+Hcy-) rat hepatocarcinoma cell line (LF). Characterization of these sublines has shown that long-term culture in methionine-deprived medium significantly decreases their ability to grow in vitro, their tumorigenicity in new-born syngeneic animals and their level of transcription of 3 oncogenes (c-Ki-ras, c-Ha-ras, c-myc) involved in hepatic growth. These data strongly support a relationship between methionine-dependence and ability to grow in vivo as tumors. A shift in methionine-supplemented medium for several cell generations of the various sublines grown in methionine deprived medium, has revealed that under these conditions, the cells maintain a diminished in vitro growth and tumorigenic capacity, despite an increased transcription of c-Ki-ras, c-Ha-ras and c-myc. This points out the complexity of the relationship between the level of activity of some oncogenes and the expression of the malignant phenotypes.

Animals↗

PubMed1989

Molecular cloning and characterization of endogenous SV40 DNA from human HBL-100 cells.

The human HBL-100 cell line harbours SV40 DNA integrated in tandem at a unique site. The SV40 T-antigen expressed in these cells is defective in a function essential to the replication of the viral genome. The integrated SV40 sequences were molecularly cloned in a bacteriophage, and a subclone (plasmid pSVHBI) containing a complete SV40 DNA was isolated. As compared to SV40 wild-type strain 776, sequence analysis of pSVHBI early region revealed the presence of several DNA alterations. Among these, a point mutation at position 3199, predicting a change at amino-acid 540 of arginine to isoleucine, was shown by marker rescue to be responsible for the deficiency of T-antigen. This novel mutation further delimits one of the T-antigen domains involved in SV40 DNA replication. Transfection experiments demonstrated that the transforming activity of the SV40 genome from HBL-100 cells is still preserved. Moreover, several transformed human cell clones thus obtained could be permanently established in culture.

Base Sequence↗

PubMed1989

Acquisition of tumorigenic potential in the human myoepithelial HBL100 cell line is associated with decreased expression of HLA class I, class II and integrin beta 3 and increased expression of c-myc.

The human breast cell line HBL100 acquires the capacity to invade normal tissues and to replace them by proliferation in vitro only at high passage levels (HPL). These cells therefore are a useful model for studying tumor progression in vitro. We have analyzed the expression of cell-surface markers supposed to be involved in the control of the neoplastic process. Quantitative flow cytometry has revealed that: (1) spontaneous expression of HLA class-I antigens strongly decreases in HPL HBL100 cells vs. LPL cells, which parallels amplification and over-expression of c-myc oncogene; (2) HLA DR antigens can be induced by IFN-gamma in LPL but not in HPL HBL100 cells; (3) HBL100 cells secrete a soluble protein factor which specifically inhibits HLA DR induction by IFN-gamma even in heterologous cell systems; (4) 50% of LPL HBL100 cells express integrin beta 3, whereas HPL HBL100 cells lose this antigen; (5) this cell line is myoepithelial in origin, since 100% of HBL100 cells exhibit the CD10 antigen. Our data stress a role of HLA antigens, of some integrins and of c-myc in the acquisition of malignant potential by myoepithelial mammary cells of the HBL100 line.

Biomarkers, Tumor↗