PubMed Health⌕ Search

Biomedical subjects

S Estrade

Publications and source records attributed to S Estrade.

At least 19 recordsLinked to original sources
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

Activation of the simian virus 40 (SV40) genome abrogates sensitivity to AVP in a rabbit collecting tubule cell line by repressing membrane expression of AVP receptors.

To analyze the role of SV40 genome in the phenotypic alterations previously observed in SV40-transformed cell lines, we infected rabbit renal cortical cells with a temperature-sensitive SV40 mutant strain (tsA58) and compared the cell phenotypes at temperatures permissive (33 degrees C) and restrictive (39.5 degrees C) for SV40 genome expression. At both temperatures, the resulting cell line (RC.SVtsA58) expresses cytokeratin and uvomorulin, but epithelial differentiation is more elaborate at 39.5 degrees C as shown by the formation of a well-organized cuboidal monolayer with numerous tight junctions and desmosomes. Functional characteristics are also markedly influenced by the culture temperature: cells grown at 33 degrees C respond only to isoproterenol (ISO, 10(-6) M) by a sevenfold increase in cAMP cell content above basal values; in contrast, when transferred to 39.5 degrees C, they exhibit increased sensitivity to ISO (ISO/basal: 19.1) and a dramatic response to 10(-7) M dDarginine vasopressin (dDAVP/basal: 18.2, apparent Ka: 5 X 10(-9) M) which peaks 48 h after the temperature shift. The latter is associated with membrane expression of V2-type AVP receptors (approximately 50 fmol/10(6) cells) which are undetectable when SV40 genome is activated (33 degrees C). Clonal analysis, additivity studies, and desensitization experiments argue for the presence of a single cell type responsive to both AVP and ISO. The characteristics of the RC. SVtsA58 cell line at 39.5 degrees C (effector-stimulated cAMP profile, lack of expression of brush-border hydrolases and Tamm-Horsfall protein) suggest that it originates from the cortical collecting tubule, and probably from principal cells.

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

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

Maintenance of proximal and distal cell functions in SV40-transformed tubular cell lines derived from rabbit kidney cortex.

This paper reports the preparation and describes the properties of three renal tubular cell lines derived using SV40 infection of primary cultures of rabbit kidney cortical cells, enriched in proximal cells. RC.SV1 was initially derived from cultures grown in the presence of fetal calf serum exhibiting a low degree of proximal differentiation. The cells were subsequently adapted to grow in serum-free hormonally defined medium and display basic properties of proximal tubule cells including well-developed apical microvilli, strong expression of brush-border hydrolases, Na+-coupled glucose uptake, and increased cyclic AMP production when exposed to PTH. The other two cell lines were derived from cultures in serum-free hormonally defined medium and propagated in the same medium. They are characterized by some common properties including rare and short microvilli, low expression of apical hydrolases, and low or undetectable Na+-dependent glucose uptake, but differ by their abilities to respond by an increase in cAMP to various hormonal stimuli. RC.SV2 cells are sensitive to calcitonin and to a lesser extent to isoproterenol and PTH, suggesting that they may originate from the thick ascending limb of Henle's loop and the bright portion of the distal tubule. RC.SV3 responds essentially to isoproterenol and arginine vasopressin, suggesting a more distal origin (late distal and initial collecting tubule). Emergence of distal cell lines from cultures exhibiting proximal characteristics may be related to distal cell overgrowth as suggested by analysis of growth kinetics and increased Na+/H+ exchanger activity in RC.SV2 compared with RC.SV1.

Animals↗
PubMed1988

Accelerated malignant conversion of human HBL-100 cells by the v-Ki-ras oncogene.

The human epithelial HBL-100 cell line harbors SV40 genetic information and has an unlimited growth potential. Despite displaying properties characteristic of transformation since its early in vitro passages, it is capable of producing progressively growing tumors in nude mice only after long-term culture. This is a reproducible phenomenon and apparently not the consequence of a selection of preexisting malignant cells. Superinfection of early passage nontumorigenic HBL-100 cells with Kirsten murine sarcoma virus, which contains a Ki-ras oncogene having undergone multiple activating events, induces morphologic alterations and rapidly converts the cells to neoplastic cells, further supporting the hypothesis of multistep carcinogenesis. The HBL-100 cell line might be useful in defining the oncogenes representative of different families, which are able to complement SV40 in this system.

Cell Line↗
PubMed1985

Epithelial HBL-100 cell line derived from milk of an apparently healthy woman harbours SV40 genetic information.

The epithelial HBL-100 cell line was established in vitro from milk of an apparently healthy woman. It exhibits characteristics of transformation from the very beginning and evolves during in vitro maintenance, until becoming tumorigenic in nude mice. This immortal cell line represents a useful model for studying the progression of human epithelial cells toward malignancy. In the course of our investigations we detected a 94K protein in HBL-100 cells obtained from four different sources. This protein is shown to be indistinguishable from the SV40 large T-antigen on the basis of: Recognition by polyclonal and different monoclonal antibodies. Partial peptide map analysis. Specific binding capacity to the SV40 DNA origin of replication. The presence of a tandemly integrated SV40 genome is demonstrated by Southern blotting. Successful rescue of SV40 DNA by fusion with permissive COS-7, but not CV-1 cells, indicates that the SV40 T-antigen from HBL-100 cells is defective in a function(s) essential to the replication of the viral DNA. The possible origin of the SV40 genetic information that we have detected in HBL-100 cells and the implications of this finding on studies involving this cell line are discussed.

Adenocarcinoma↗
PubMed1984

Expression of gamma-glutamyl transpeptidase in adult rat liver cells after transformation with SV40 virus.

By the use of histochemical techniques we have shown that the transformation of rat hepatocytes with simian virus 40 resulted in a large majority of cells producing gamma-glutamyl transpeptidase (gamma GT). Using a temperature-sensitive mutant of this virus we have shown that the percentage of cells exhibiting this enzyme in a temperature-sensitive SV40 mutant is much higher in cells grown at the permissive temperature of 33 degrees C than at the nonpermissive temperature of 40.5 degrees C. We thus conclude that, the correlation between gamma GT expression and chemical or spontaneous transformation of hepatocytes, previously described by other authors, can now be extended to viral transformation and that the enzyme activity is dependent on the expression of a transformed cell phenotype.

Animals↗
PubMed1977

Simian virus 40-Chinese hamster kidney cell interaction. III. Characteristics of chemical induction in a clone of virogenic transformed cells.

A number of chemical and physical agents were screened to determine their effectiveness in inducing simian virus (SV40) production in a virogenic clone of SV40-transformed Chinese hamster cells. Mitomycin C (MC) was the most effective inducing agent, and MC induction was further characterized. It was found that levels of infectious SV40 DNA were increased above control levels as early as 6 h after addition of MC to the culture medium and reached maximum levels by 48 h. Virus capsid (V) antigen and virions followed with a lag of about 24 h. V antigen production was sensitive to hydroxyurea, suggesting a dependence on virus DNA synthesis. The proportion of virus-producing cells (infectious centres) and the virus burst per cell were both stimulated by MC. Studies of 3H-thmidine incorporation demonstrated that the rate of SV40 DNA synthesis was maximal at 48 h post-induction, at which time cellular DNA synthesis was almost abolished. Caffeine, at doses not toxic to non-induced cells, strongly inhibited SV40production in both non-induced and induced cells, suggesting some role for DNA repair mechanisms.

Animals↗