Biomedical subjects

Bernard Salles

Publications and source records attributed to Bernard Salles.

At least 19 recordsLinked to original sources

PubMed2006

Interplay between Ku, Artemis, and the DNA-dependent protein kinase catalytic subunit at DNA ends.

Repair of DNA double strand breaks (DSB) by the nonhomologous end-joining pathway in mammals requires at least seven proteins involved in a simplified two-step process: (i) recognition and synapsis of the DNA ends dependent on the DNA-dependent protein kinase (DNA-PK) formed by the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs in association with Artemis; (ii) ligation dependent on the DNA ligase IV.XRCC4.Cernunnos-XLF complex. The Artemis protein exhibits exonuclease and endonuclease activities that are believed to be involved in the processing of a subclass of DSB. Here, we have analyzed the interactions of Artemis and nonhomologous end-joining pathway proteins both in a context of human nuclear cell extracts and in cells. DSB-inducing agents specifically elicit the mobilization of Artemis to damaged chromatin together with DNA-PK and XRCC4/ligase IV proteins. DNA-PKcs is necessary for the loading of Artemis on damaged DNA and is the main kinase that phosphorylates Artemis in cells damaged with highly efficient DSB producers. Under kinase-preventive conditions, both in vitro and in cells, Ku-mediated assembly of DNA-PK on DNA ends is responsible for a dissociation of the DNA-PKcs. Artemis complex. Conversely, DNA-PKcs kinase activity prevents Artemis dissociation from the DNA-PK.DNA complex. Altogether, our data allow us to propose a model in which a DNA-PKcs-mediated phosphorylation is necessary both to activate Artemis endonuclease activity and to maintain its association with the DNA end site. This tight functional coupling between the activation of both DNA-PKcs and Artemis may avoid improper processing of DNA.

Amino Acid Sequence↗

PubMed2006

The loss of gammaH2AX signal is a marker of DNA double strand breaks repair only at low levels of DNA damage.

The induction of DNA double-strand breaks (DSBs) by genotoxic treatment leads to high toxicity and genetic instability. Various approaches have been undertaken to quantify the number of breaks and to follow the kinetic of DSB repair. Recently, the phosphorylation of the variant histone H2AX (named gammaH2AX), quantified by specific immunodetection approaches, has provided a valuable and highly sensitive method to monitor DSBs formation. Although it is admitted that the number of gammaH2AX foci reflected that of DSBs, contradictory reports leave open the question of a link between the disappearance of gammaH2AX signal and DSBs repair. We determined gammaH2AX expression (i) in cells either proficient or not in DSBs repair capacity, (ii) after exposure to ionizing radiation (IR) or calicheamicin gamma1, a radiomimetic compound, (iii) and by three different immunodetection methods, foci numbering, flow cytometry or Western blotting. We showed here that gammaH2AX loss correlates with DSB repair activity only at low cytotoxic doses, when less than 100-150 DSBs breaks per genome are produced, independently of the method used. In addition, in DNA repair proficient cells, the early decrease in the number and intensity of gammaH2AX foci observed after a 2 Gy exposure was not associated with a significant change in the global gammaH2AX level as determined by Western blotting or flow cytometry. These results suggest that the dephosphorylation step of gammaH2AX may be limiting and that the loss of foci is mediated not only by gammaH2AX dephosphorylation but also through its redistribution towards the chromatin.

Aminoglycosides↗

PubMed2005

Involvement of polynucleotide kinase in a poly(ADP-ribose) polymerase-1-dependent DNA double-strand breaks rejoining pathway.

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by the non-homologous end-joining pathway relying on DNA-PK activity, but other mechanisms may promote end-joining. We previously described a DSB repair pathway that requires synapsis of DNA ends by poly(ADP-ribose) polymerase-1 (PARP-1) and ligation by the XRCC1/DNA ligase III complex (XL). Here, the repair of non-ligatable DNA ends by this pathway was examined in human cell extracts. The phosphorylation of the 5'-terminal end was shown to represent a limiting step for the repair process. Polynucleotide kinase (hPNK) was identified as the 5'-DNA kinase associated with the PARP-1-dependent end-joining pathway because (i) hPNK was co-recruited to DNA ends together with PARP-1 and XL, (ii) ligation of 5'-OH terminal breaks was compromised in hPNK-depleted extracts and restored upon addition of recombinant hPNK, and (iii) recombinant hPNK was necessary for end-joining of 5'-OH terminal breaks reconstituted with the PARP-1/XL complex. Also, using an assay enabling us to follow the ligation kinetics of each strand of a DSB, we established that the two strands at the junction can be processed and joined independently, so that one strand can be ligated without a ligatable nick on the other strand at the DSB site. Taken together these results reveal functional parallels between the PARP-1 and DNA-PK-dependent end-joining processes.

DNA↗

PubMed2005

hMutS alpha is protected from ubiquitin-proteasome-dependent degradation by atypical protein kinase C zeta phosphorylation.

The hMutS alpha (hMSH2-hMSH6) protein heterodimer plays a critical role in the detection of DNA mispairs in the mismatch repair (MMR) process. We recently reported that hMutS alpha proteins were degraded by the ubiquitin-proteasome pathway in a cell-type-dependent manner, indicating that one or several regulator(s) may interfere with hMutS alpha protein ubiquitination and degradation. On the other hand, we and others have shown that protein kinase C (PKC) is involved as a positive regulator of MMR activity. Here, we provide evidence that the atypical PKC zeta regulates ubiquitination, degradation, and levels of hMutS alpha proteins. Using both PKC zeta-transfected U937 and PKC zeta siRNA-transfected MRC-5 cell lines, we found that PKC zeta protein expression was correlated with that of hMutS alpha as well as with MMR activity, but was inversely correlated with hMutS alpha protein ubiquitination and degradation. Interestingly, PKC zeta interacts with hMSH2 and hMSH6 proteins and phosphorylates both. Moreover, in an in vitro assay PKCzeta mediates phosphorylation events decreasing hMutS alpha protein degradation via the ubiquitin-proteasome pathway. Altogether, our results indicate that PKC zeta modulates hMutS alpha stability and protein levels, and suggest a role for PKC zeta in genome stability by regulating MMR activity.

Antimetabolites, Antineoplastic↗

PubMed2005

The double life of the Ku protein: facing the DNA breaks and the extracellular environment.

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double strand break recognition and repair. It has been shown, more than ten years ago, that Ku is also expressed at the cell surface of different cells types along with its intracellular pool within the nucleus and the cytoplasm but involvement of Ku in cell-cell and cell-extracellular matrix adhesion has been only recently demonstrated. In addition, we have shown that Ku may have a second and unexpected activity in cell/microenvironment interaction. Indeed, Ku appears to be involved in extracellular proteolytic processes through its specific interaction, on the cell surface, with the matrix metalloprotease 9. Taken together, these results suggest that Ku function at the cell surface is likely to be important in tumour invasion. Various fundamental questions arise from these observations. How Ku is expressed on the cell surface, why a protein with completely unrelated functions also serve as an integrin-like molecule once expressed at the cell surface and is this functional moonlighting of Ku related to cell transformation remain open issues that will be discussed here.

Animals↗

PubMed2005

Human chronic lymphocytic leukemia B cells can escape DNA damage-induced apoptosis through the nonhomologous end-joining DNA repair pathway.

Nonhomologous end-joining (NHEJ) DNA factors maintain genomic stability through their DNA double-strand break (DSB) repair and telomere-associated activities. Unrepaired or misrepaired DSBs can lead to apoptotic death or chromosomal damage. The B cells of some B-chronic lymphocytic leukemia (B-CLL) patients are resistant to radiation-induced apoptosis in vitro. We show here that the novel DNA-dependent protein kinase (DNA-PK) inhibitor, NU7026 (2-(morpholin-4-yl)-benzo[h]chomen-4-one), and the phosphatidylinositol 3 (PI-3) kinase inhibitor, wortmannin, restored sensitivity to DNA damage-induced apoptosis of otherwise resistant cells. These resistant malignant B cells also escaped DSB-induced apoptosis following exposure to etoposide or neocarzinostatin. We found that at 15 minutes after irradiation, the levels of NHEJ (as measured by an in vitro DSB end-ligation assay) and DNA-PK catalytic subunit (DNA-PKcs) activity were, respectively, 2-fold and 4-fold higher in radio-resistant than in radio-sensitive B-CLL cells or Epstein-Barr virus (EBV)-transformed B cells. Ku70/Ku80 heterodimer DNA end-binding activity was also 2- to 3-fold higher in the resistant B-CLL cell subset compared with the sensitive B-CLL cell subset. Our results provide the first evidence that overactivating the NHEJ DNA repair pathway impairs DNA damage-induced apoptosis in malignant B cells and that this may contribute to their resistance to current chemotherapy.

Androstadienes↗

PubMed2004

DNA-dependent protein kinase and XRCC4-DNA ligase IV mobilization in the cell in response to DNA double strand breaks.

Repair of DNA double strand breaks (DSBs) by the non-homologous end joining (NHEJ) pathway in mammals requires at least the DNA-dependent protein kinase (DNA-PK) and the DNA ligase IV-XRCC4 protein complexes. DNA-PK comprises the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs. Here we report the first description of the nuclear mobilization of endogenous NHEJ proteins after exposure of human cells to double strand-breaking agents. DSB infliction specifically induced a dose- and time-dependent mobilization of Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV proteins from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. XRCC4 recruitment was accompanied by its DNA-PK-dependent phosphorylation. The recruited proteins co-immunoprecipitated, indicating that they had assembled into complexes. However, DNA-PK was attached to chromatin, whereas XRCC4-ligase IV resisted solubilization by DNase I. The rates of appearance and dissolution of NHEJ proteins paralleled that of histone variant H2AX phosphorylation and dephosphorylation. We established that under conditions of genomic DSB infliction 1) Ku recruitment was not dependent on the co-recruitment of the other NHEJ proteins, 2) DNA-PKcs was physically required for the mobilization of the XRCC4-ligase IV complex, 3) DNA ligase IV was physically necessary for stable recruitment of XRCC4, and 4) phosphorylation of either H2AX or XRCC4 was unnecessary for DNA-PK or XRCC4-ligase IV recruitment. Altogether these results offer insights into the interplay between key NHEJ proteins during this repair process in the cell.

Antigens, Nuclear↗

PubMed2004

Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining.

The efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, the nonhomologous end-joining process that represents the predominant repair pathway relies on the DNA-dependent protein kinase (DNA-PK) and the XRCC4-DNA ligase IV complex. Nonetheless, several in vitro and in vivo results indicate that mammalian cells use more than a single end-joining mechanism. While searching for a DNA-PK-independent end-joining activity, we found that the pretreatment of DNA-PK-proficient and -deficient rodent cells with an inhibitor of the poly(ADP-ribose) polymerase-1 enzyme (PARP-1) led to increased cytotoxicity of the highly efficient DNA double-strand breaking compound calicheamicin gamma1. In addition, the repair kinetics of the DSBs induced by calicheamicin gamma1 was delayed both in PARP-1-proficient cells pretreated with the PARP-1 inhibitor and in PARP-1-deficient cells. In order to get new insights into the mechanism of an alternative route for DSBs repair, we have established a new synapsis and end-joining two-step assay in vitro, operating on DSBs with either nuclear protein extracts or recombinant proteins. We found an end-joining activity independent of the DNA-PK/XRCC4-ligase IV complex but that actually required a novel synapsis activity of PARP-1 and the ligation activity of the XRCC1-DNA ligase III complex, proteins otherwise involved in the base excision repair pathway. Taken together, these results strongly suggest that a PARP-1-dependent DSBs end-joining activity may exist in mammalian cells. We propose that this mechanism could act as an alternative route of DSBs repair that complements the DNA-PK/XRCC4/ligase IV-dependent nonhomologous end-joining.

Aminoglycosides↗

PubMed2004

The membrane form of the DNA repair protein Ku interacts at the cell surface with metalloproteinase 9.

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks repair. Ku is also expressed on the cell surface of different types of cells where its function remains poorly understood. From a yeast two-hybrid screen, we have identified a specific interaction between the core region of Ku80 and the hemopexin domain of metalloproteinase 9 (MMP-9), a key enzyme involved in the degradation of extracellular matrix (ECM) components. Ku associates with MMP-9 on the surface of leukemic cells as demonstrated by co-immunoprecipitation experiments in membrane extracts and double-label immunofluorescence studies. In normal and tumoral migratory cells, Ku80 and MMP-9 colocalize at the periphery of leading edge of cells and cellular invasion of collagen IV matrices was blocked by antibodies directed against Ku70 or Ku80 subunits as well as by Ku80-specific antisense oligonucleotides. Our results indicate that Ku and MMP-9 interact at the cell membrane of highly invasive hematopoietic cells of normal and tumoral origin and document the unexpected importance of the membrane-associated form of Ku in the regulation of ECM remodelling.

Antigens, Nuclear↗

PubMed2004

The Membrane-associated form of the DNA repair protein Ku is involved in cell adhesion to fibronectin.

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks recognition and repair. However, Ku is expressed also on the surface of different types of cells along with its intracellular pool within the nucleus and the cytoplasm. Participation of membrane-associated Ku in cell-cell interaction has been reported recently. Here, we describe a novel function of cell-surface Ku as an adhesion receptor for fibronectin (Fn). The role of Ku in cell adhesion was investigated by comparing the Ku80 deficient Chinese hamster ovary (CHO) cell line, xrs-6, with clones transfected stably with either the hamster or human Ku80 cDNA. Ku expression in transfectant cells resulted in a significant increased adhesion on Fn and type IV collagen as compared to control cells. The observed increase in cell adhesion relied on Ku cell-surface expression, since antibodies directed against Ku70 or Ku80 subunit inhibited adhesion on Fn of Ku80, but not control vector, transfected xrs-6 cells. In addition, both Ku70 and Ku80 present a structural relationship with integrin I (or A) domains and the A1 and A3 domains of von Willebrand factor, domains known to be involved in Fn binding. Both Ku70 and Ku80 exhibit a complete set of residues compatible in their position and chemical nature with the formation of a metal ion-dependent adhesion (MIDAS) site implicated in ligand binding and integrin activation. Taken together, these functional and structural approaches support a new role for Ku as an adhesion receptor for Fn.

Amino Acid Sequence↗

PubMed2004

Degadration of mismatch repair hMutSalpha heterodimer by the ubiquitin-proteasome pathway.

Mismatch repair plays a critical role in genome stability. This process requires several proteins including hMSH2/hMSH6 (hMutSalpha) heterodimer involved in the first stage of the process, the mispair recognition. We previously reported that in U937 and HL-60 cell lines, hMSH2 and hMSH6 protein expression was much lower than that in HeLa and KG1a cells. Here, we showed that the decreased expression of hMutSalpha results from differences in the degradation rate of both proteins by the ubiquitin-proteasome pathway. Our data suggest that in human cell lines, ubiquitin-proteasome could play an important role in the regulation of hMutSalpha protein expression, thereby regulating mismatch repair activity.

Base Pair Mismatch↗

PubMed2003

Possible anti-recombinogenic role of Bloom's syndrome helicase in double-strand break processing.

Bloom's syndrome (BS) which associates genetic instability and predisposition to cancer is caused by mutations in the BLM gene encoding a RecQ family 3'-5' DNA helicase. It has been proposed that the generation of genetic instability in BS cells could result from an aberrant non-homologous DNA end joining (NHEJ), one of the two main DNA double-strand break (DSB) repair pathways in mammalian cells, the second major pathway being homologous recombination (HR). Using cell extracts, we report first that Ku70/80 and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), key factors of the end-joining machinery, and BLM are located in close proximity on DNA and that BLM binds to DNA only in the absence of ATP. In the presence of ATP, BLM is phosphorylated and dissociates from DNA in a strictly DNA-PKcs-dependent manner. We also show that BS cells display, in vivo, an accurate joining of DSBs, reflecting thus a functional NHEJ pathway. In sharp contrast, a 5-fold increase of the HR-mediated DNA DSB repair in BS cells was observed. These results support a model in which NHEJ activation mediates BLM dissociation from DNA, whereas, under conditions where HR is favored, e.g. at the replication fork, BLM exhibits an anti-recombinogenic role.

Adenosine Triphosphatases↗

PubMed2003

hMSH2 expression is driven by AP1-dependent regulation through phorbol-ester exposure.

Mammalian mismatch repair (MMR) plays a prominent role in genomic stability and toxicity induced by some DNA damaging agents. Advance in the appreciation of regulation mechanisms of the key MMR protein hMSH2 would certainly lead to valuable information on its role and to a better understanding of MMR system dysfunctions with respect to their consequences in cells. We have previously reported that, in myeloid leukemic U937 cell line, the expression of hMSH2 MMR protein is regulated by protein kinase C (PKC) activity. Here we show that the increase of protein level following PKC activation by phorbol ester (TPA) treatment parallels that of hMSH2 mRNA. Our results support the view that the hMSH2 gene is prone to transcriptional regulation upon TPA induction, and that AP-1 is a factor implicated in the transactivation. When losing the AP-1-dependent hMSH2 promoter activity, either by mutating the AP-1 binding sites of the hMSH2 promoter or by using a dominant negative c-Jun factor, the hMSH2 overexpression induced by TPA is abolished both in vitro and in vivo. Thus the control of hMSH2 expression by PKC appears to be dependent, at least partially, on an up-regulation mediated by AP-1 transactivation.

Base Sequence↗

PubMed2003

Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment.

Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.

Antigens, Nuclear↗

PubMed2003

Long-patch DNA repair synthesis during base excision repair in mammalian cells.

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway.

Animals↗

PubMed2002

The radioprotective effect of the 24 kDa FGF-2 isoform in HeLa cells is related to an increased expression and activity of the DNA dependent protein kinase (DNA-PK) catalytic subunit.

We previously reported that overexpression of the 24 kDa basic fibroblast factor (or FGF-2) isoform provides protection from the cytotoxic effect of ionizing radiation (IR). DNA double-strand breaks (DSB), the IR-induced lethal lesions, are mainly repaired in human cells by non-homologous end joining system (NHEJ). NHEJ reaction is dependent on the DNA-PK holoenzyme (composed of a regulatory sub-unit, Ku, and a catalytic sub-unit, DNA-PKcs) that assembles at sites of DNA damage. We demonstrated here that the activity of DNA-PK was increased by twofold in two independent radioresistant cell lines, HeLa 3A and CAPAN A3, over expressing the 24 kDa FGF-2. This increase was associated with an overexpression of the DNA-PKcs without modification of Ku expression or activity. This overexpression was due to an up-regulation of the DNA-PKcs gene transcription by the 24 kDa FGF-2 isoform. Finally, HeLa 3A cells exhibited the hallmarks of phenotypic changes associated with the overexpression of an active DNA-PKcs. Indeed, a faster repair rate of DSB and sensitization to IR by wortmannin was observed in these cells. Our results represent the characterization of a new mechanism of control of DNA repair and radioresistance in human tumor cells dependent on the overproduction of the 24 kDa FGF-2 isoform.

Androstadienes↗

PubMed2002

Kit signaling inhibits the sphingomyelin-ceramide pathway through PLC gamma 1: implication in stem cell factor radioprotective effect.

Previous studies demonstrated that Kit activation confers radioprotection. However, the mechanism by which Kit signaling interferes with cellular response to ionizing radiation (IR) has not been firmly established. Based on the role of the sphingomyelin (SM) cycle apoptotic pathway in IR-induced apoptosis, we hypothesized that one of the Kit signaling components might inhibit IR-induced ceramide production or ceramide-induced apoptosis. Results show that, in both Ba/F3 and 32D murine cell lines transfected with wild-type c-kit, stem cell factor (SCF) stimulation resulted in a significant reduction of IR-induced apoptosis and cytotoxicity, whereas DNA repair remained unaffected. Moreover, SCF stimulation inhibited IR-induced neutral sphingomyelinase (N-SMase) stimulation and ceramide production. The SCF inhibitory effect on SM cycle was not influenced by wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor. The SCF protective effect was maintained in 32D-KitYF719 cells in which the PI3K/Akt signaling pathway is abolished due to mutation in Kit docking site for PI3K. In contrast, phospholipase C gamma (PLC gamma) inhibition by U73122 totally restored IR-induced N-SMase stimulation, ceramide production, and apoptosis in Kit-activated cells. Moreover, SCF did not protect 32D-KitYF728 cells (lacking a functional docking site for PLC gamma 1), from IR-induced SM cycle. Finally, SCF-induced radioprotection of human CD34(+) bone marrow cells was also inhibited by U73122. Altogether, these results suggest that SCF radioprotection is due to PLC gamma 1-dependent negative regulation of IR-induced N-SMase stimulation. Beyond the scope of Kit-expressing cells, it suggests that PLC gamma 1 status could greatly influence the post-DNA damage cellular response to IR, and perhaps, to other genotoxic agents.

Animals↗

PubMed2002

Implication of protein kinase C in the regulation of DNA mismatch repair protein expression and function.

The DNA mismatch repair (MMR) proteins are essential for the maintenance of genomic stability of human cells. Compared with hereditary or even sporadic carcinomas, MMR gene mutations are very uncommon in leukemia. However, genetic instability, attested by either loss of heterozygosity or microsatellite instability, has been extensively documented in chronic or acute malignant myeloid disorders. This observation suggests that in leukemia some internal or external signals may interfere with MMR protein expression and/or function. We investigated the effects of protein kinase C (PKC) stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA) on MMR protein expression and activity in human myeloid leukemia cell lines. First, we show here that unstimulated U937 cells displayed low level of PKC activity as well as MMR protein expression and activity compared with a panel of myeloid cell lines. Second, treatment of U937 cells with TPA significantly increased (3-5-fold) hMSH2 expression and, to a lesser extent, hMSH6 and hPMS2 expression, correlated to a restoration of MMR function. In addition, diacylglycerol, a physiological PKC agonist, induced a significant increase in hMSH2 expression, whereas chelerythrine or calphostin C, two PKC inhibitors, significantly decreased TPA-induced hMSH2 expression. Reciprocally, treatment of HEL and KG1a cells that exhibited a high level of PKC expression, with chelerythrine significantly decreased hMSH2 and hMSH6 expression. Moreover, the alteration of MMR protein expression paralleled the difference in microsatellite instability and cell sensitivity to 6-thioguanine. Our results suggest that PKC could play a role in regulating MMR protein expression and function in some myeloid leukemia cells.

Base Pair Mismatch↗