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P Galand

Publications and source records attributed to P Galand.

At least 19 recordsLinked to original sources
PubMed1992

Expression of 27-kD heat-shock protein isoforms in human neoplastic and nonneoplastic liver tissues.

Previous study of rat liver during chemically induced hepatocarcinogenesis has shown that expression of isoforms of the 27-kD heat-shock protein was greater in neoplastic nodules and in hepatocellular carcinoma than in control livers. In this study, various human neoplastic and nonneoplastic liver tissues were investigated with electrophoresis after amino acid labeling to evaluate the expression of 27-kD heat-shock protein isoforms. This revealed that human liver contains 27-kD proteins that are recognized by a polyclonal antibody raised against human 27-kD heat-shock protein. Basal levels of fluorographical and immunostaining intensity of the 27-kD heat-shock protein spots (respectively, after [3H]leucine or 32P incorporation or as checked with a specific human 27-kD heat-shock protein antibody) were higher in hepatomas than in non-tumorous liver. Phosphorylation patterns of the 27-kD heat-shock protein isoforms were, however, similar in hepatocellular carcinoma and in uninvolved surrounding liver. Heat inducibility of the 27-kD heat-shock protein, tested in one case of liver cell adenoma and in the surrounding liver, was also preserved in both tissues. The role of the overexpression of 27-kD heat-shock protein in neoplastic liver tissues remains unknown. We propose, as a working hypothesis, that it is related to the resistant phenotype acquired by some tumors during malignant progression.

Adolescent↗
PubMed1992

Measurement of S phase duration in human epidermis using cyclin immunostaining and 3H-thymidine pulse labelling.

Cyclin/PCNA (proliferating cell nuclear antigen) is a nuclear protein strongly associated with DNA replication sites. Under methanol fixation, cyclin immunostaining might serve as a specific marker for S-phase cells. Associated with single labelling with 3H-thymidine, this immunodetection represents an easy method for measuring S-phase duration. Its application to six samples of normal human epidermis allowed us to measure an S phase of 9.7 +/- 0.3 h, a result in fair agreement with a previous estimate of 10.2 h obtained from a classical double-labelling technique with two different concentrations of 3H-thymidine.

Culture Techniques↗
PubMed1992

Apoptosis, cell proliferation and c-ras expression during and after cyproterone acetate (CPA) induced liver hyperplasia.

Cell proliferation and cell death appear in several systems as mutually exclusive, which raises the assumption that a same factor or secondary signal(s) might exert opposite control on the two processes. To test this assumption we investigated the time-course evolution of the S phase and apoptotic indices in rat liver during cyproterone acetate (CPA) induced hyperplasia and during the recovery of normal liver mass provoked, respectively, by cyproterone acetate (CPA) treatment and withdrawal. The levels of c-myc and c-ras transcripts were also followed in view of the indications of a positive role of these oncogenes in proliferation. The data showed that proliferation and cell death are not always mutually exclusive and that a high rate of cell death was indifferently associated with high or low c-ras expression. Our data are consistent with a role of this gene in proliferation but exclude that it plays an opposite role in controlling cell death.

Animals↗
PubMed1992

Kinetics of the calcium induced stratification of human keratinocytes in vitro.

In a low concentration of calcium (0.1 mM), keratinocytes form a monolayer with about 30% of cells synthesizing involucrin. After addition of calcium to the culture medium to a concentration of 1.2 mM, the monolayer stratifies within 24 h, with a preferential migration of involucrin positive keratinocytes. In the present study, we tried to determine if keratinocytes control the decision to migrate at a distinct cell cycle point. A percentage labelled mitosis (PLM) curve was constructed for keratinocytes grown in low calcium medium and values for the length of the cell cycle (47 h), S phase duration (11 h) and G2+M period (6 h), were obtained. Monolayer cultures at 80% confluence were switched to high calcium concentration at various times (from 0 to 48 h), after pulse labelling with [3H]-thymidine. Based on the PLM data, the behaviour of cells known to be in S, G1 and G2 at the time of the migration stimulus were followed. No significant difference in the percentage of labelled suprabasal cells was found for any point of the cell cycle. For cells submitting to stratification, in S phase involucrin staining showed that about 60% of the [3H]-thymidine labelled cells were also involucrin negative. These results indicate that upward migration of keratinocytes in cultured epithelium can be triggered at all points in the cell cycle with equal probability and is not restricted to those cells that already contained involucrin.

Calcium↗
PubMed1992

Renewal and differentiation of keratinocytes cultured on dead de-epidermalized dermis.

Human keratinocytes grown at an air-liquid interface on dead de-epidermalized dermis exhibit a pattern of organization similar to that seen in vivo. Cell renewal is limited to the basal layer. The cell cycle time determined after 7 days of culture, using a percentage labelled mitoses (PLM) technique, was about 15 h. This result is comparable with published data for cultivated keratinocytes but is shorter than the parameter proposed for epidermis in vivo. Appearance of labelled cells in the granular layer was observed 4 days after pulse labelling. Despite this high cell renewal, a normal cell differentiation with expression of various keratinization markers was maintained.

Adult↗
PubMed1992

Identification of the cellular protein encoded by the human Wilms' tumor (WT1) gene.

A putative tumor-suppressor gene (wt1) located at chromosome 11p13 and involved in Wilms' tumor development has recently been identified as a zinc finger polypeptide-encoding gene. The purpose of this study was to characterize the protein encoded by the human wt1 gene. The region spanning the entire zinc finger domain was amplified by polymerase chain reaction (PCR) and subcloned in the pATH 3 expression vector. Polyclonal antibodies against the fused TrpE-WT protein were raised. These antibodies immunoprecipitated a 49- to 51-kDa protein from hematopoietic tumor cells labeled in vivo with [35S]methionine. Subcellular fractionation and immunohistochemistry followed by confocal microscopy indicated that the Wilms' tumor gene product (WT1) is mainly localized within the nucleus.

Base Sequence↗
PubMed1991

Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro.

Tumor necrosis factor (TNF) is cytotoxic for many tumoral cell lines, whereas normal cells generally are considered resistant to this action. This study shows that this cytokine causes massive death of bovine endothelial cells in primary culture in a concentration- and time-dependent manner. Dying cells exhibit all the ultrastructural changes and the inter-nucleosome cleavage of DNA associated with apoptosis or 'programmed cell death.' This is the first report clearly showing a direct toxicity of TNF on endothelial cells and demonstrating that this results from the induction of the program of apoptotic death. Our observation raises the possibility that hemorrhagic necrosis in vivo, after treatment with TNF, might involve a direct cytocidal action on endothelial cells of the tumor neovasculature.

Animals↗
PubMed1990

Modulation by estrogen of the incidence of diethylnitrosamine-induced gamma-glutamyltranspeptidase-positive foci in rat liver.

We measured the number and size of foci of altered hepatocytes induced after 8 weeks by diethylnitrosamine (DENA) in the liver of rats pretreated with 17 beta-estradiol (E2), 1 or 24 h prior to the administration of the carcinogen. The average size of the lesions was the same in the E2 pretreated and unpretreated animals. The number of gamma-glutamyltranspeptidase (GGT)-positive foci per cm3 of liver increased from 364 +/- 57 in unpretreated animals to 1149 +/- 186 in animals receiving E2 24 h before DENA; it raised to 3779 +/- 280 when the hormone was injected 1 h before the carcinogen, i.e. about 25% of the number of foci scored in rats receiving the carcinogen 24 h after partial hepatectomy. The hypothesis is proposed that 1-h pretreatment with E2 increases hepatocyte susceptibility towards DENA action by enhancing the accessibility of the genome to the carcinogen.

Animals↗
PubMed1990

Enhanced expression of a 27 kD protein during diethylnitrosamine-induced hepatocarcinogenesis in rats.

Patterns of neosynthesized cellular proteins from normal rat liver and from diethylnitrosamine-induced neoplastic nodules and hepatocellular carcinomas were analyzed by radiolabeling and fluorography of two-dimensional gel electrophoregrams. Three proteins exhibited a significant and reproducible increase in labeling intensity in the nodules (n = 5) and in the tumors (n = 10) as compared to the normal liver (n = 10). Two of those proteins (MW 31 and 33 kD, pI of 5.25, 5.15 respectively) are secreted proteins and as yet, we have no clue as to their nature. The third one is an intracellular protein of 27 kD pI 5.5. Several similarities in physico-chemical properties (MW, pI, phosphorylated state, low methionine content) indicate that this 27 kD protein might be the 27 kD heat shock protein (27 HSP). This is further supported by our observation that the cadmium-induced 27 HSP comigrates with our 27 kD protein.

Animals↗
PubMed1990

Injury induced by fatty acids or bile acid in isolated human colonocytes prevented by calcium.

Measurement of the modulation of the growth fraction of isolated normal colonocytes from adult subjects in primary monolayer culture was used as a sensitive quantitative assay to evaluate toxic effects of several endogenous compounds found within the colon. This assay was used to study the role of CaCl2 in blocking cell injury. When added simultaneously with the injurious agent, 5-10 mM CaCl2 blocked the toxicity of physiological concentrations of deoxycholic acid, oleic acid, palmitic acid and linoleic acid.

Calcium Chloride↗
PubMed1990

S phase duration measurement by combined PCNA/cyclin immunostaining and radioautography after a single pulse-labelling with 3H-thymidine.

A method for measuring S phase duration is described and evaluated that combines single pulse labelling with 3H-thymidine (TdR), detected by radioautography, and proliferating cell nuclear antigen (PCNA)/cyclin immunostaining to replace the second pulse labelling of the classical double-labelling method. Conditions were set up in which nuclei showing one or both types of label were readily distinguished, hence allowing to verify that cell fluxes in and out of S phase were equal. S phase durations thus measured in different tissues of the mouse were concordant with those obtained by the double 3H-TdR labelling or from labelled mitoses curves. Our method might be used with archived samples of methanol-fixed cells or tissues, singly labelled with 3H-TdR or with bromodeoxyuridine.

Animals↗
PubMed1990

Effects of 17 beta-estradiol on c-myc and c-Ha-ras expression in the liver of ovariectomized female rats.

Twenty four hours after i.p. injection of 17 beta-estradiol (E2) (500 micrograms/100 g b.w.) to ovariectomized rats, the hepatocytes [3H]-thymidine pulse-labeling index (L.I.) was significantly increased, reaching a value of 4.3 +/- 1.6 percent (i.e. much lower than 32.0 +/- 2.0 percent 24 h. after partial hepatectomy -PH-). E2-treatment was followed by an increase in liver content in c-myc transcripts, with a peak at 650 percent basal value at 8 h, very similar to that observed after PH. In contrast, E2 induced an increase in liver c-Ha-ras expression with a similar time-course evolution but markedly lower amplitude than that seen after PH. These data are consistent with a role of c-myc in proliferative competence i.e. the ability to perform the Go-G1 transition, and with a role of c-ras in further progression of the cells in the cycle.

Animals↗
PubMed1990

Immunohistochemical detection of c-myc and c-erbA products in diethylnitrosamine-induced preneoplastic and neoplastic liver lesions in rats.

An immunohistochemical study of c-myc and c-erbA products (p-myc and p-erbA) in preneoplastic and neoplastic rat liver lesions showed that the longer the time of hepatocarcinogenic treatment, the higher the proportion of lesions, whatever their type, showing p-erbA positive cells (p-erbA+). The proportion of p-myc positive foci (the majority of which are also p-erbA+), which was low at the focus stage, increased at the nodule and tumor stages. The incidence of p-myc positivity (alone or combined with erbA positivity) in the nodules decreased from the nodule to the tumor stage. For tumors, three types of altered phenotypes were found, namely: p-myc+, p-erbA+ or p-myc+/p-erbA+. Nevertheless, there were regions in these tumors that were negative. At a given stage, all types of lesions exhibited about the same incidence of immunopositivity for the two oncogene products (expressed either alone or together), the presence of which did not correlate with proliferative activity. Since the fraction of lesions that undergo full malignant progression is much smaller than the proportion of lesions that express c-myc and/or c-erbA proteins, our data exclude the possibility that increased incidence of p-myc/ and/or p-erbA+ cells might be sufficient for inducing full malignancy. A significant proportion of foci and nodules, and of regions of these lesions and of tumors, were unlabeled, whatever the stage at which they are found. This indicates that, if implicated, the positive phenotype(s) would not be required for the maintenance of those liver alterations.

Animals↗
PubMed1990

Modulation by indomethacin of diethylnitrosamine-induced early changes in c-myc and c-ras expression and late incidence of preneoplastic lesions in rat liver.

We measured the levels of c-myc and c-ras expression before and after diethylnitrosamine (DENA) treatment in the liver of rats previously submitted to partial hepatectomy (PH), in the presence or absence of indomethacin (IMC), given at a dose that reduced by 75% the incidence of preneoplastic foci of altered hepatocytes scored 8 weeks after application of the carcinogen. The time-course evolution of c-myc response to PH was similar in IMC-treated and untreated rats (with a peak at 3-8 h at least as high in IMC-treated animals as in the hepatectomized reference group), whereas the overall c-ras response was significantly reduced by the IMC treatment, resulting in much lower c-ras expression at 18-24 h posthepatectomy. Treatment with DENA 24 h after PH did not significantly modify c-ras expression compared to partially hepatectomized controls. In contrast, DENA treatment resulted in a marked transient increase in c-myc expression that was at least as pronounced, if not the same, in the IMC-treated animals. These results leave open the possibility that increased c-myc expression under DENA influence might play a role in foci induction but exclude that this might be sufficient. They are consistent with a role for c-ras expression in determining the susceptibility of hepatocytes towards the carcinogenic action of DENA.

Animals↗
PubMed1990

Modulation by indomethacin or prostaglandin E2 of the incidence of diethylnitrosamine-induced gamma-glutamyltranspeptidase-positive foci in rat liver.

We investigated the effect of a pretreatment with indomethacin (IMC, ip 3.6 mg/kg body weight (bw)) or dimethylprostaglandin E2 (PGE2, ip 10 micrograms/kg bw) on the incidence and development of gamma-glutamyltranspeptidase (GGT)-positive foci of altered hepatocytes, scored 8 or 14 weeks after ip injection of diethylnitrosamine (DENA, 50 mg/kg bw) to rats submitted to two-thirds hepatectomy (PH) or sham operation (Sh). IMC reduced by about 4 times the incidence of DENA-induced GGT-positive foci per cm3 of liver tissue in sham-operated as well as in two-thirds hepatectomized rats, compared to the respective unpretreated controls. In contrast, PGE2 pretreatment increased the incidence of DENA-induced foci in both groups, this effect, in terms of absolute numbers of foci, being additive to that of PH alone. IMC pretreatment resulted in foci with lower average size in the Sh but not in the PH animals, whereas with PGE2 pretreatment the mean volume of the foci was increased in the two groups of rats. At the dose used, IMC did not modify the proliferative response of hepatocytes to PH, and PGE2 did not stimulate proliferation in the sham-operated animals. Altogether, these results indicate that: 1, the incidence of DENA-induced foci can be negatively modulated by interfering with the prostaglandins pathway through a mechanism that does not involve an action either on proliferative activity or on any other process that would be specific to the post-hepatectomy regenerative state; 2, positive modulation of the incidence of DENA-induced foci does not necessarily require stimulation of proliferation.

Animals↗
PubMed1989

Immunocytochemical detection of the onco-developmental protein oncomodulin in pre-neoplastic and neoplastic hepatocellular lesions during hepatocarcinogenesis in rats.

Oncomodulin is a calcium-binding protein, detectable in extra-embryonic human and rat placental cells and in a wide variety of tumors, but not in any normal embryonic or adult rodent or human tissues. It is also absent from proliferatively active fetal or regenerating adult rat liver. The presence of this oncodevelopmental marker was investigated in pre-neoplastic and neoplastic liver lesions during hepatocarcinogenesis induced in rats by DENA treatment, using an antibody raised against purified oncomodulin. Positive immunostaining was observed in foci of altered hepatocytes, in neoplastic nodules and in HC, but not in the histologically normal surrounding liver parenchyma. The proportion of oncomodulin-positive foci gradually rose from 20-25% at 2-3 months after DENA treatment, to about 88% at 6 months and later. The proportion of positive neoplastic nodules increased from 50% at 5 months to about 73% (range 36-100) at 9 months and later; 88% of the HC found 10 to 20 months after DENA treatment were also positive. That early neoplastic nodules are oncomodulin-positive in a proportion (50%) similar to that of foci after the same duration of treatment is consistent with a lineage relationship between them but makes it unlikely that oncomodulin expression conditions the focus-nodule transition. The role, if any, of oncomodulin in malignant progression remains to be elucidated. It seems out of the question that it is a simple correlate of proliferative activity.

Animals↗
PubMed1989

Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues.

Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidin-biotin-peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin-Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol-fixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanol-fixed tissues. The distribution of the positive nuclei in the two latter cases coincided with the proliferative compartment. On the other hand, double labelling with [3H]-thymidine and with the cyclin/PCNA antibody revealed that in methanol-fixed tissues the cyclin/PCNA labelling index did not differ by more than 6% from the [3H]-thymidine index. Besides the two labels overlapped in a proportion of labelled cells that was in reasonable agreement with expectation considering cells flow in and out of S phase since the time of [3H]-thymidine injection. This indicates that both labels recognize the same cells in this material. In contrast, in formaldehyde-fixed tissues, the cyclin/PCNA labelling index markedly exceeded the [3H]-thymidine labelling index. From this it is concluded that cyclin/PCNA immunostaining can be used: (1) In formaldehyde-fixed tissues (including existing material stored as paraffin blocks): for defining and mapping the proliferative (or germinative) compartment. (2) In methanol-fixed tissues as a substitute to the [3H]-thymidine autoradiographic labelling index. From this, a method is proposed (derived from classical 'double-labelling' technique) for measuring S phase duration in tissues fixed at a known interval time after a single labelling with [3H]-thymidine (or BrdU) and submitted to cyclin/PCNA immunocytochemical detection and to autoradiography (or to BrdU immunostaining).

Animals↗