Biomedical subjects

P Mehlen

Publications and source records attributed to P Mehlen.

34 records · Page 2Linked to original sources

PubMed1998

The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis.

The development of colonic carcinoma is associated with the mutation of a specific set of genes. One of these, DCC (deleted in colorectal cancer), is a candidate tumour-suppressor gene, and encodes a receptor for netrin-1, a molecule involved in axon guidance. Loss of DCC expression in tumours is not restricted to colon carcinoma, and, although there is no increase in the frequency of tumour formation in DCC hemizygous mice, reestablishment of DCC expression suppresses tumorigenicity. However, the mechanism of action of DCC is unknown. Here we show that DCC induces apoptosis in the absence of ligand binding, but blocks apoptosis when engaged by netrin-1. Furthermore, DCC is a caspase substrate, and mutation of the site at which caspase-3 cleaves DCC suppresses the pro-apoptotic effect of DCC completely. These results indicate that DCC may function as a tumour-suppressor protein by inducing apoptosis in settings in which ligand is unavailable (for example, during metastasis or tumour growth beyond local blood supply) through functional caspase cascades by a mechanism that requires cleavage of DCC at Asp 1,290.

Animals↗

PubMed1997

hsp27 as a switch between differentiation and apoptosis in murine embryonic stem cells.

Small stress proteins are developmentally regulated and linked to cell growth and differentiation. The early phase of murine embryonic stem (ES) cell differentiation, characterized by a gradual growth arrest, is accompanied with hsp27 transient accumulation. This differentiation process also correlated with changes in hsp27 phosphorylation and oligomerization. The role of hsp27 was investigated in ES clones stably transfected with murine or human hsp27 genes, placed in sense or antisense orientation. Several clones were obtained that either underexpressed endogenous murine hsp27 or overexpressed murine or human hsp27. Maintained undifferentiated, these clones showed similar growth rates. We report here that hsp27 constitutive overexpression enhanced the differentiation-mediated decreased rate of ES cell proliferation but did not alter morphological changes. In contrast, hsp27 underexpression, which attenuated cell growth arrest, induced differentiation abortion because of an overall cell death by apoptosis. Recently, we showed that hsp27 interfered with cell death probably because of its ability to modulate intracellular glutathione. hsp27 accumulation during ES cell differentiation was also correlated with an increase in glutathione, which was attenuated by hsp27 down-expression. Hence, hsp27 transient expression seems essential for preventing differentiating ES cells from undergoing apoptosis, a switch that may be redox regulated.

Animals↗

PubMed1997

Large unphosphorylated aggregates as the active form of hsp27 which controls intracellular reactive oxygen species and glutathione levels and generates a protection against TNFalpha in NIH-3T3-ras cells.

The mammalian small stress protein hsp27 is an oligomeric phosphoprotein which interferes with the cell death induced by several stimuli. In that sense, we and others have recently shown that human hsp27 expression induced cellular protection against tumor necrosis factor (TNFalpha), a protection which depends on the ability of hsp27 to decrease the level of reactive oxygen species and increase that of glutathione. Here, we have analyzed unphosphorylatable mutants of human hsp27 in which serines 15, 78, and 82 were replaced by alanines, glycines, or aspartic acids. Depending on the amino acid which was used to substitute the serine sites, a different pattern of hsp27 structural organization was observed. Alanine substitution generated large hsp27 aggregates while glycine and aspartic acid did the reverse. Hence, these phosphorylatable serine residues can be considered as key elements affecting hsp27 structural organization. Only the large aggregates of hsp27 were able to modulate reactive oxygen species and glutathione and generated cellular protection against TNFalpha. Moreover, using drugs that modulate the intracellular level of glutathione, we show that an increase in glutathione by itself was sufficient to generate large hsp27 structures while the reverse was observed in the case of glutathione deprivation.

3T3 Cells↗

PubMed1997

HSP27 as a mediator of confluence-dependent resistance to cell death induced by anticancer drugs.

Resistance of colorectal cancer cells to chemotherapeutic drugs increases as cells reach confluence. Here we show that the small stress protein HSP27, which has been described to block necrotic and apoptotic cell death, accumulates in confluent human colorectal cancer cell lines HT-29 and Caco2. Cell confluence also induces HSP27 phosphorylation and changes in its intracellular distribution. We also show that overexpression of human HSP27 by transfection of HT-29 cells increased the resistance of cells to doxorubicin or cisplatin and prevented drug-induced apoptosis. Interestingly, nonconfluent HSP27-transfected cells and confluent control cells in which HSP27 is expressed at the same level displayed a similar drug resistance. HSP27-transfected cells did not exhibit an enhanced resistance when they reached confluence, nor was there an increased accumulation of HSP27. We have previously shown that HSP27 expression blocks tumor necrosis factor-induced cell death as a result of decreasing intracellular reactive oxygen species (ROS). Here we show that HSP27 overexpression in HT-29 cells, obtained either by transfection or by growing the cells at high density, correlated with a significant ROS decrease. We conclude that cell confluent-dependent HSP27 accumulation, probably due to its ability to decrease ROS levels, is essential for the establishment of the resistance of colorectal cancer cells when reaching confluence.

Apoptosis↗

PubMed1996

Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1- and staurosporine-induced cell death.

Small stress protein expression enhances the survival of mammalian cells exposed to numerous injuries that induce necrotic cell death. The cell surface receptor Fas/APO-1 and its ligand have been recently identified as important mediators of apoptosis. Here, we show that constitutive expression of human heat shock protein (hsp)27 in murine L929 cells blocks Fas/APO-1-mediated cell death. Expression of human hsp27 prevented anti-APO-1-induced DNA fragmentation and morphological changes. These results strongly suggest that human hsp27 acts as a cellular inhibitor of Fas/APO-1-induced apoptosis. We also report that the expression of small stress proteins from different species, such as human hsp27, Drosophila Dhsp27, or human alphaB-crystallin, confers resistance to apoptotic cell death induced by staurosporine, a protein kinase C inhibitor. Hence, small stress proteins are novel regulators that are able to block apoptosis induced by different pathways.

Alkaloids↗

PubMed1996

Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death.

Expression of small stress proteins (shsp) enhances the survival of mammalian cells exposed to heat or oxidative injuries. Recently, we have shown that the expression of shsp from different species, such as human hsp27, Drosophila hsp27 or human alphaB-crystallin protected murine L929 cells against cell death induced by tumor necrosis factor (TNFalpha), hydrogen peroxide or menadione. Here, we report that, in growing L929 cell lines, the presence of these shsp decreased the intracellular level of reactive oxygen species (ROS). shsp expression also abolished the burst of intracellular ROS induced by TNFalpha. Several downstream effects resulting from the TNFalpha-mediated ROS increment, such as NF-kappaB activation, lipid peroxidation and protein oxidation, were inhibited by shsp expression. We also report that the expression of these different shsp raised the total glutathione level in both L929 cell lines and transiently transfected NIH 3T3-ras cells. This phenomenon was essential for the shsp-mediated decrease in ROS and resistance against TNFalpha. Our results therefore suggest that the protective activity shared by human hsp27, Drosophila hsp27 and human alphaB-crystallin against TNFalpha-mediated cell death and probably other types of oxidative stress results from their conserved ability to raise the intracellular concentration of glutathione.

Animals↗

PubMed1996

Inconstant association between 27-kDa heat-shock protein (Hsp27) content and doxorubicin resistance in human colon cancer cells. The doxorubicin-protecting effect of Hsp27.

To investigate the role of the small 27-kDa heat-shock protein (Hsp27) in the intrinsic resistance of colon cancer cells to doxorubicin, we modified Hsp27 expression either genetically by transfection or pharmacologically by cisplatin treatment. HT-29 cells were transfected with a full-length Hsp27 construct in the sense or antisense orientation. We found a good correlation between cell survival after doxorubicin treatment and Hsp27 content. A similar correlation was found for the thermoresistance of the Hsp27-transfected cells. In contrast, the sensitivity of the different transfected cells to 5-fluorouracil was not modified. cis-Platinum(II)diammine dichloride (cisplatin) treatment of HT-29 or Caco2 cells dramatically increased their Hsp27 mRNA and protein content. Accordingly, the cells became thermoresistant. Contrary to what has been previously assumed, however, cell resistance to doxorubicin was reduced. Our data suggest that the decreased resistance of the cells to doxorubicin may be due to a concomitant increase of topoisomerase II expression, the main target of anthracyclines. In conclusion, although Hsp27 seems to participate in the natural resistance of colon cancer cells to anthracyclines, its increase after cisplatin treatment is not associated with a decreased cytotoxicity to doxorubicin.

Cell Survival↗

PubMed1996

Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression.

We report here that both kappa B-dependent transactivation of a reporter gene and NF-kappa B activation in response to tumor necrosis factor (TNF alpha) or H2O2 treatments are deficient in human T47D cell transfectants that overexpress seleno-glutathione peroxidase (GSHPx). These cells feature low reactive oxygen species (ROS) levels and decreased intracellular ROS burst in response to TNF alpha treatment. Decreased ROS levels and NF-kappa B activation were likely to result from GSHPx increment since these phenomena were no longer observed when GSHPx activity was reduced by selenium depletion. The cellular contents of the two NF-kappa B subunits (p65 and p50) and of the inhibitory subunit I kappa B-alpha were unaffected by GSHPx overexpression, suggesting that increased GSHPx activity interfered with the activation, but not the synthesis or stability, of Nf-kappa B. Nuclear translocation of NF-kappa B as well as I kappa B-alpha degradation were inhabited in GSHPx-overexpressing cells exposed to oxidative stress. Moreover, in control T47D cells exposed to TNF alpha, a time correlation was observed between elevated ROS levels and I kappa B-alpha degradation. We also show that, in growing T47D cells, GSHPx overexpression altered the isoform composition of I kappa B-alpha, leading to the accumulation of the more basic isoform of this protein. GSHPx overexpression also abolished the TNF alpha-mediated transient accumulation of the acidic and highly phosphorylated I kappa B-alpha isoform. These results suggest that intracellular ROS are key elements that regulate the phosphorylation of I kappa B-alpha, a phenomenon that precedes and controls the degradation of this protein, and then NF-kappa B activation.

Cell Line↗

PubMed1996

Transient accumulation, phosphorylation and changes in the oligomerization of Hsp27 during retinoic acid-induced differentiation of HL-60 cells: possible role in the control of cellular growth and differentiation.

Expression of the mammalian small stress protein Hsp27 has been increasingly linked to cell growth regulation and differentiation. Hsp27 is a phosphoprotein which forms oligomers with native sizes ranging between 100 and 800 kDa. We have examined the fate of Hsp27 transiently expressed during the retinoic acid (tRA)-induced granulocytic differentiation of human leukemic HL-60 cells. We show that tRA, in addition to its effects on Hsp27 accumulation and phosphorylation, transiently increased the oligomerization state of this protein. While Hsp27 phosphorylation by tRA is an early phenomenon that takes place before cellular growth is altered, the redistribution of Hsp27 oligomers occurred later and concomitantly with the maximal accumulation of this protein. Hence, complex regulations of Hsp27 are induced by tRA which suggest that this protein plays a role in the pathway through which retinoids exert their effects. To approach Hsp27 function during HL-60 cell differentiation, experiments aimed at reducing the cellular content of this protein were performed by transiently inhibiting Hsp27 mRNA translation by a specific anti-sense oligonucleotide. This process, which decreased the basal level of Hsp27 by about 40%, did not interfere with the growth of undifferentiated HL-60 cells. In contrast, a decreased level of Hsp27 diminished the differentiation-mediated down-regulation of cell growth and altered some morphological changes induced by this retinoid. These results suggest that Hsp27 is a mediator of granulocytic differentiation.

Cell Differentiation↗

PubMed1995

Intracellular reactive oxygen species as apparent modulators of heat-shock protein 27 (hsp27) structural organization and phosphorylation in basal and tumour necrosis factor alpha-treated T47D human carcinoma cells.

The small stress protein heat-shock protein 27 (hsp27) is an oligomeric phosphoprotein, constitutively expressed in most human cells, which enhances cellular resistance to tumour necrosis factor alpha (TNF alpha). This phenomenon correlates with dramatic changes in hsp27 cellular location, structural organization and phosphorylation. To gain a better understanding of the molecular mechanisms regulating these properties of hsp27, we investigated whether they were a consequence of the intracellular production of reactive oxygen species (ROS) generated by TNF alpha. Here, we report that, in T47D carcinoma cell lines, the rapid burst of intracellular ROS production and changes in hsp27 locale, structural organization and phosphoisoform composition induced by TNF alpha were abolished by the overexpression of the antioxidant enzyme seleno-glutathione peroxidase (GSHPx). These effects were greatly diminished when GSHPx-expressing cells were grown in the absence of selenium, a cofactor that is essential for seleno-GSHPx activity, indicating that they are directly linked to the increased GSHPx activity. Moreover, in growing T47D cells, GSHPx expression induced intracellular redistribution of hsp27 and decreased the phosphorylation of this protein without altering its pattern of oligomerization. In contrast, the heat-mediated phosphorylation of hsp27 was not altered by decreased intracellular ROS levels. Hence, in growing and TNF-treated cells, several hsp27 properties appear to be modulated by fluctuations in intracellular ROS levels.

Animals↗

PubMed1995

Constitutive expression of human hsp27, Drosophila hsp27, or human alpha B-crystallin confers resistance to TNF- and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts.

Hyperthermia and other forms of stress that induce and/or stimulate heat shock or stress protein (hsp) expression enhance the cellular resistance to TNF-alpha. One of the stress proteins, hsp70, has been shown to participate in the molecular mechanisms that regulate this phenomenon. Here we have tested the capability of small hsps from different species to protect against this cytokine in the TNF-sensitive L929 fibrosarcoma cells. The genes that encode human hsp27, Drosophila hsp27, and human alpha B-crystallin were placed under the control of the constitutive SV40 early promoter and were stably introduced into L929 cells. We observed that all clones that constitutively expressed the exogenous small hsps exhibited a strong protection against TNF-mediated killing, which was proportional to the level of the expressed proteins. This phenomenon did not correlate with altered binding of TNF-alpha to its receptors, and no protection was observed as a consequence of the transfection or selection procedures. In addition, the overexpression of the exogenous small hsps did not modify the level of the endogenous stress proteins in the transfected clones. Remarkably, the small hsps also induced a protection against oxidative stresses generated by either hydrogen peroxide or menadione. In L929 cells, the killing induced by TNF-alpha and oxidative stress is thought to occur through the accumulation of intracellular reactive oxygen intermediates. Hence, our data suggest that the small hsps from different species share the property to protect L929 cells against the deleterious effects of reactive oxygen intermediates generated by either TNF-alpha or oxidative stress.

Animals↗

PubMed1995

Tumor necrosis factor-alpha induces changes in the phosphorylation, cellular localization, and oligomerization of human hsp27, a stress protein that confers cellular resistance to this cytokine.

The stress protein hsp27 is constitutively expressed in several human cells and shows a rapid phosphorylation following treatment with tumor necrosis factor-alpha (TNF-alpha). hsp27 usually displays native molecular mass ranging from 100 to 700 kDa. Here, we have analyzed the TNF-alpha-mediated changes in the phosphorylation, cellular localization, and structural organization of hsp27 in HeLa cells. We report that the TNF-alpha-mediated hsp27 phosphorylation is a long-lasting phenomenon that correlates with the cytostatic effect of this cytokine. Following TNF-alpha treatment, the rapid phosphorylation of hsp27 occurred concomitantly with complex changes in the intracellular distribution and structural organization of this protein. This resulted in the quantitative redistribution of hsp27 toward the soluble phase of the cytoplasm. In addition, during the first 2 h of TNF-alpha treatment, a transient increase in the native molecular mass of most hsp27 molecules (< or = 700 kDa) occurred. Then, by 4 h of TNF-alpha treatment, the native size of this stress protein drastically regressed (< 200 kDa). During this phenomenon, the phosphorylated isoforms of hsp27 remained concentrated in the small or medium-sized oligomers (< 300 kDa) of this protein. We also analyzed the properties of human hsp27 in transfected murine L929 cell lines that constitutively express this protein. In these cells, TNF-alpha induced modifications in the phosphorylation, intracellular distribution, and oligomerization of human hsp27 similar to those observed in HeLa cells. Moreover, the expression of hsp27 in L929 cells was found to correlate with a reduced cytotoxicity of this cytokine. Hence, the complex changes in the phosphorylation, intracellular locale and structural organization of human hsp27 may be related to the protective activity of this protein against the deleterious effects induced by TNF-alpha.

Animals↗

PubMed1994

The serum-induced phosphorylation of mammalian hsp27 correlates with changes in its intracellular localization and levels of oligomerization.

The oligomeric small heat-shock protein hsp27, also denoted hsp28, is constitutively expressed in several mammalian cells and displays a phosphorylation status that is related to cellular growth and differentiation. This protein is related to alpha-crystallin and has strong sequence similarity with an in vitro inhibitor of actin polymerization. Here, we have analyzed hsp27 phosphorylation, cellular localization and structural organization following serum stimulation of serum-starved HeLa cells. hsp27 is dephosphorylated in starved cells and quantitatively recovered in the form of small structures (< 200 kDa) present in the soluble phase of the cytoplasm. Immediately after the addition of serum to starved cells, a rapid phosphorylation and complex changes in the intracellular distribution and structural organization of hsp27 are observed. Phosphorylation essentially occurs at the level of small hsp27 structures (< 200 kDa) and is concomitant with the increased molecular mass (up to 700 kDa) of a fraction of this protein. Serum treatment also induced the detergent-sensitive association of another fraction of hsp27, still in the form of small and dephosphorylated structures, with cellular particulate fractions. Contrasting with these observations, hsp70 had the tendency to concentrate into nucleoli during serum starvation.

Blood↗

PubMed1994

Regulation of the 28 kDa heat shock protein by retinoic acid during differentiation of human leukemic HL-60 cells.

Dysregulation of hematopoietic cellular differentiation contributes to leukemogenesis. Unfortunately, relatively little is known about how cell differentiation is regulated. Considering that heat shock proteins (hsp) and specifically the small hsps have been increasingly linked to growth regulation, we sought to determine whether the mammalian small hsp (hsp28) is a growth-regulatory candidate during hematopoietic cell differentiation. Because of its effects on cell growth and differentiation and its increasing clinical use as a differentiating agent, we examined the effect of retinoic acid (RA) on hsp28 during differentiation of the human leukemic HL-60 cell line. Although hsp28 was constitutively expressed at low levels in untreated HL-60 cells, steady state hsp28 protein increased transiently, concomitant with the onset of G1 cell cycle arrest. Furthermore, hsp28 phosphorylation transiently increased within one hour following treatment with RA. Interestingly, in contrast to other differentiating agents the induction of hsp28 by RA was post-transcriptionally mediated with hsp28 protein and mRNA being discordantly regulated. These observations underscore the complex regulation of hsp28 by RA during granulocytic differentiation of human leukemic cells and indicate hsp28 as an intermediary in the pathway through which retinoids exert their growth and differentiative effects.

Cell Cycle↗

PubMed1993

Hela cells proteasome interacts with leucine-rich polypeptides and contains a phosphorylated subunit.

The proteasome is a multicatalytic proteinase complex composed of several non-identical protein subunits with molecular weights ranging from 20 to 35 kDa. To approach the mechanisms modulating the activity of this protease, we have investigated the possible interaction of this particle with specific polypeptides as well as the phosphorylation status of its subunits. A specific antiserum was used to immunoprecipitate this particle under native conditions. Three major polypeptides, characterized by molecular masses of 53, 59 and 77 kDa co-immunoprecipitated specifically with the proteasome. Labelling experiments indicated that these proteins are leucine-rich and contain very few methionine residues. None of them were phosphorylated in vivo in normal cell growth conditions, in contrast to one of the proteasome subunit (30 kDa). These results indicate that, in vivo, the proteasome is probably associated with leucine-rich polypeptides and that this protease is a kinase substrate.

Cysteine Endopeptidases↗

PubMed1993

Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of the Drosophila 27-kDa heat-shock protein.

The Drosophila melanogaster small heat-shock protein, hsp27 (Dhsp27) belongs to a family of polypeptides which shares a sequence related to alpha-crystallin and which protect cell against heat shock. Dhsp27 accumulates following heat shock and, in absence of stress, in the central nervous system, imaginal discs and the gonads of the developing fly. Two internal and adjacent deletion mutants in the conserved alpha-crystallin domain of Dhsp27 were constructed. Expression vectors containing either the coding sequence of Dhsp27 or that of the two deletion mutants linked to the Simian-Virus-40 late promoter were used to transfect monkey COS cells. The transient expression of Dhsp27 was found to decrease the sensitivity of COS cells to heat and hydrogen-peroxide stresses as judged by Trypan-blue staining and indirect immunofluorescence analysis. Using this rapid test, we observed that a deletion of 62 amino acids, which lies at the 5' end of the conserved alpha-crystallin domain and covers the first 41 amino acids of this region had only a weak effect on the protective activity of Dhsp27. This suggests that the N-terminal half of the conserved alpha-crystallin domain may not be essential for the protective activity of the small hsp. In contrast, Dhsp27 was no more active when the last 42 amino acids of the alpha-crystallin domain were deleted. Biochemical fractionation and indirect immunofluorescence analysis indicated that the protective function of Dhsp27 was localized at the level of the nucleus.

Animals↗