Methylation-induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1
2003; Springer Nature; Volume: 22; Issue: 9 Linguagem: Inglês
10.1093/emboj/cdg216
ISSN1460-2075
AutoresPetr Ćejka, Lovorka Stojic, Nina Mojaš, Anna Russell, Karl Heinimann, Elda Cannavò, Massimiliano di Pietro, Giancarlo Marra, Josef Jiricny,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle1 May 2003free access Methylation-induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1 Petr Cejka Petr Cejka Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Lovorka Stojic Lovorka Stojic Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Nina Mojas Nina Mojas Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Anna Marie Russell Anna Marie Russell Research Group Human Genetics, Departments of Research and Clinical-Biological Sciences, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland Search for more papers by this author Karl Heinimann Karl Heinimann Research Group Human Genetics, Departments of Research and Clinical-Biological Sciences, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland Search for more papers by this author Elda Cannavó Elda Cannavó Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Massimiliano di Pietro Massimiliano di Pietro Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Giancarlo Marra Giancarlo Marra Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Josef Jiricny Corresponding Author Josef Jiricny Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Petr Cejka Petr Cejka Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Lovorka Stojic Lovorka Stojic Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Nina Mojas Nina Mojas Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Anna Marie Russell Anna Marie Russell Research Group Human Genetics, Departments of Research and Clinical-Biological Sciences, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland Search for more papers by this author Karl Heinimann Karl Heinimann Research Group Human Genetics, Departments of Research and Clinical-Biological Sciences, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland Search for more papers by this author Elda Cannavó Elda Cannavó Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Massimiliano di Pietro Massimiliano di Pietro Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Giancarlo Marra Giancarlo Marra Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Josef Jiricny Corresponding Author Josef Jiricny Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland Search for more papers by this author Author Information Petr Cejka1, Lovorka Stojic1, Nina Mojas1, Anna Marie Russell2, Karl Heinimann2, Elda Cannavó1, Massimiliano di Pietro1, Giancarlo Marra1 and Josef Jiricny 1 1Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland 2Research Group Human Genetics, Departments of Research and Clinical-Biological Sciences, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2245-2254https://doi.org/10.1093/emboj/cdg216 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The mismatch repair (MMR) gene hMLH1 is mutated in ∼50% of hereditary non-polyposis colon cancers and transcriptionally silenced in ∼25% of sporadic tumours of the right colon. Cells lacking hMLH1 display microsatellite instability and resistance to killing by methylating agents. In an attempt to study the phenotypic effects of hMLH1 downregulation in greater detail, we designed an isogenic system, in which hMLH1 expression is regulated by doxycycline. We now report that human embryonic kidney 293T cells expressing high amounts of hMLH1 were MMR-proficient and arrested at the G2/M cell cycle checkpoint following treatment with the DNA methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), while cells not expressing hMLH1 displayed a MMR defect and failed to arrest upon MNNG treatment. Interestingly, MMR proficiency was restored even at low hMLH1 concentrations, while checkpoint activation required a full complement of hMLH1. In the MMR-proficient cells, activation of the MNNG-induced G2/M checkpoint was accompanied by phosphorylation of p53, but the cell death pathway was p53 independent, as the latter polypeptide is functionally inactivated in these cells by SV40 large T antigen. Introduction Mutations in mismatch repair (MMR) genes, predominantly hMSH2 and hMLH1, segregate with hereditary non-polyposis colon cancer (HNPCC). Inheritance of a single mutated allele of a MMR gene predisposes to precocious cancers of the colon, endometrium and ovary. Analysis of HNPCC tumour cells showed that repeated sequence elements (microsatellites) in their genomic DNA are frequently mutated (for a review see Peltomaki, 2001). As microsatellite instability (MSI) is a hallmark of defective MMR in all organisms tested to date, and has been shown to be present in all tumour cell lines that have lost both alleles of hMSH2 or hMLH1 (Boyer et al., 1995), it is assumed that the wild type alleles of the respective MMR genes in cells of HNPCC tumours have been lost or inactivated by mutation. But mutations in MMR genes are not an absolute prerequisite for MSI. In recent years, a number of sporadic colon tumours and tumour cell lines displaying MSI have been described that are MMR-deficient due to silencing of the hMLH1 promoter by hypermethylation (reviewed in Esteller, 2002). Once both MMR gene alleles have been inactivated, the cell's propensity towards acquiring mutations increases, especially in genes carrying microsatellite repeats. Should the mutated genes be involved in the control of cell proliferation, the mutator cell in, for example, the colonic epithelium would be able to divide in an uncontrolled manner and thus give rise to an adenomatous polyp. As the cells in this benign growth acquire further mutations with subsequent cell divisions, the adenoma would rapidly become transformed into a carcinoma. That such a path to transformation can be followed in vivo was demonstrated when numerous HNPCC colon cancers were shown to carry frameshift mutations in a run of 10 adenines within the coding sequence of the transforming growth factor β receptor type II (TGFβRII) gene, as well as in other genes involved in growth control or apoptosis (reviewed in Markowitz et al., 2002). Further support for this hypothesis comes from the finding that adenomas of HNPCC kindred transform to carcinomas with a much higher frequency than those associated with sporadic disease (Kinzler and Vogelstein, 1998), presumably due to a more rapid acquisition of transforming mutations. The above findings help explain how the loss of MMR might accelerate cellular transformation and tumour progression. What is unclear to date, however, is whether the transformation process begins only following the inactivation of both MMR gene alleles, or whether it commences already at the stage when only one allele is affected or when the expression of the given MMR gene is only attenuated, rather than shut off, such as might be the case in cells where the hMLH1 promoter is only partially methylated. The notion that a reduction in MMR protein levels might promote tumorigenesis originates in studies with Msh2+/− mice. Although the Msh2+/− embryonic stem cells were apparently normal in terms of their MMR capacity as measured by MSI (de Wind et al., 1995), the heterozygous animals were cancer prone, and presented with tumours that often still contained the wild-type Msh2 allele (de Wind et al., 1998). The propensity of the MMR heterozygous cells to transformation would thus appear to be linked to a process distinct from the correction of replication errors. What might the nature of these processes be? In recent years, MMR defects have been linked to several other phenomena, such as transcription-coupled repair and recombination—both mitotic and meiotic (reviewed in Harfe and Jinks-Robertson, 2000). In addition, the MMR system was implicated in activation of cell cycle checkpoints and apoptosis, as witnessed by the increased resistance of MMR-deficient cells to the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or cisplatin (reviewed in Bellacosa, 2001). Thus, while MMR+/− cells, or cells expressing low amounts of MMR proteins, may not display a mutator phenotype, they might have at least a partial defect in one of the above processes, specifically in the DNA damage signalling pathway, which we judged to be of the greatest relevance to cancer. We wanted to study these processes in detail, but we lacked isogenic cells expressing varying amounts of MMR proteins. Cells in which the MMR defect was corrected either by transfer of a chromosome carrying a single wild-type copy of the mutated MMR gene (Koi et al., 1994) or its cDNA (Risinger et al., 1998; Buermeyer et al., 1999; Lettieri et al., 1999; Claij and Te Riele, 2002) were unsuitable for our studies, because they express similar or even higher amounts of the complementing MMR proteins than MMR-proficient controls. Thus, in order to be able to study the phenotypic consequences associated with reduced levels of MMR proteins, we had to generate a new line, preferably of epithelial origin, in which the expression of a selected MMR gene could be regulated. We now describe the construction and characterization of a line in which the expression of hMLH1 can be tightly regulated by doxycycline with the help of the TetOff system. Results Construction of cells with inducible hMLH1 expression The human embryonic kidney cell line 293T is MMR deficient, because the hMLH1 gene in these cells is epigenetically silenced by promoter hypermethylation (Trojan et al., 2002). We set out to correct its MMR defect through the expression of exogenous hMLH1 using the TetOff expression system, which can be tightly regulated. We first generated the 293T-TetOff cell line by stable transfection of the 293T cells with a DNA vector encoding the tetracycline-controlled transactivator (tTA). In the second step, we stably transfected the 293T-TetOff cells with a vector carrying the hMLH1 cDNA under the control of the tetracycline response element (TRE), thus creating 293T Lα cells. In the absence of tetracycline, or its more stable analogue doxycycline (Dox), the tTA protein binds to the TRE and activates transcription of hMLH1; conversely, addition of the drug induces a conformational change in tTA, which loses its ability to bind DNA and the transcription of hMLH1 is thus turned off (Figure 1A). During the initial screening, we used Dox at a concentration of 2 μg/ml, as recommended by the manufacturer, but later we found that a concentration of 50 ng/ml was sufficient to turn off the expression of hMLH1 below the limit of detection by western blotting (see below). Figure 1.Inducible hMLH1 expression in 293T Lα cells. (A) In the Tet-Off system, hMLH1 is expressed in the absence of Dox, because the tTA factor binds to the promoter of the expression vector and thus activates transcription. Addition of Dox to the culture medium causes a conformational change in tTA, which leads to its dissociation from the promoter and thus to an inactivation of hMLH1 transcription. (B) Western blot analysis of cytoplasmic (CE) and nuclear (NE) extracts of cells cultured in the absence (−) or presence (+) of 50 ng/ml Dox. hMLH1 and hPMS2 were visualized using anti-hMLH1 or anti-hPMS2 antibodies as described in Materials and methods. Total extract (TE) of MMR proficient HeLa cells was used as a positive control. (C) Stability of hMutLα. The cells were cultured without Dox (−) to induce maximal hMLH1 expression. Following the addition of 50 ng/ml Dox (+), total cell extracts were isolated after 1, 2, 3, 4, 6 and 8 days. Western blot analysis was performed using anti-hMLH1 and anti-hPMS2 antibodies as in (B). Download figure Download PowerPoint In vivo, hMLH1 interacts with hPMS2 to form the heterodimer hMutLα, which is essential for MMR. Our previous studies have shown that hPMS2 is unstable in the absence of its cognate partner (Räschle et al., 1999). Indeed, no hMLH1 could be detected in the extracts of 293T cells, and hPMS2 was hardly detectable (Trojan et al., 2002). A similar situation also existed in our 293T Lα clone grown in the presence of Dox, i.e. under conditions where the hMLH1 promoter is shut off (Figure 1B). However, expression of hMLH1 brought about hPMS2 stabilization through the formation of hMutLα, such that the levels of the latter protein were comparable to those seen in extracts of MMR-proficient cell lines (Figure 1B). The expression of hMLH1 in the 293T Lα cells grown in the absence of Dox was substantially higher than in any MMR-proficient cell line tested by us to date (Figure 1B;data not shown). Interestingly, this overexpression did not appear to be toxic to the cells: we detected no increase in the rates of apoptosis, as described for cells microinjected with expression vectors encoding hMSH2 and hMLH1 (Zhang et al., 1999). Moreover, cells grown in the absence or presence of Dox divided roughly once every 24 h (data not shown), unlike HCT116 and SNU-1 cells, in which the stable expression of hMLH1 was reported to result in substantially slower growth rates (Shin et al., 1998). When the expression of the transgene was turned off by the addition of Dox, the hMLH1 and hPMS2 proteins were present in the cell extracts in a 1:1 ratio only 24 h later (Figure 1C) and decayed with similar kinetics. This experiment showed that hMutLα is extremely stable, as it was detectable in the extracts of 293T Lα cells even 6 days after the expression of hMLH1 was shut off. In the following text, cells grown in the presence of 50 ng/ml Dox that do not express hMLH1 and thus lack hMutLα will be referred to as 293T Lα− cells. Those grown in the absence of Dox, which express hMLH1 and thus contain functional hMutLα, will be referred to as 293T Lα+ cells. hMLH1 expression in 293T Lα cells restores MMR in vitro Extracts of the 293T Lα cells were tested for MMR activity in vitro using two different MMR assays (see Materials and methods). No MMR activity was detected in extracts of 293T Lα− cells, but as the defect could be complemented by the addition of the recombinant wild-type hMutLα, we concluded that this heterodimer was the only factor missing in these extracts (Figure 2). In contrast, extracts from 293T Lα+ cells were MMR proficient in both assays (Figure 2). Importantly, these results showed that the excess partnerless hMLH1 in the 293T Lα line does not inhibit MMR, at least not in our in vitro system. This differs from the situation in Saccharomyces cerevisiae, where overexpression of MLH1 gave rise to a mutator phenotype associated most likely with the inhibition of MMR through the homodimerization of this polypeptide (Shcherbakova and Kunkel, 1999; Shcherbakova et al., 2001). The MMR proficiency of the 293T Lα+ cells in our in vitro assay was similar irrespective of whether the extracts were prepared from cells grown in the absence of Dox, or 24 h after the addition of the drug (data not shown), at which time point the ratio of hMLH1 to hPMS2 was 1:1 (Figure 1C). Figure 2.MMR proficiency of 293T Lα cell extracts. (A) Repair efficiency of a G/T mismatch in the M13mp2 vector carrying a strand discrimination signal 3′ from the mispair, using cytoplasmic extracts of the 293T Lα+ and 293T Lα− cells, supplemented or not with recombinant hMutLα (see text for details). Error bars show standard errors. (B) Correction of a G/T mismatch within a BglII restriction site of a pGEM vector, following incubation with nuclear extracts of 293T Lα+ or 293T Lα− cells, supplemented or not with recombinant hMutLα. The strand discrimination signal in this heteroduplex substrate was 5′ from the mispair. Efficient repair resulted in the restoration of a BglII site and in the generation of two DNA fragments that co-migrate with those observed in the reference digest of the homoduplex molecule carrying a bona fide BglII site. Download figure Download PowerPoint Inducible hMLH1 expression restores sensitivity to alkylating agents In order to determine the effect of hMLH1 expression on the sensitivity of 293T Lα cells to MNNG, we used clonogenic assays to quantify the surviving fraction of 293T Lα− and 293T Lα+ cells following treatment with 5 μM MNNG. [Note that 293T Lα cells do not express MGMT, the enzyme responsible for the detoxification of methylation damage (G.Marra, unpublished data). For this reason, the experiments described below were carried out in the absence of the MGMT inhibitor O6-benzylguanine.] As shown in Figure 3A, 293T Lα+ cells were very sensitive to killing by MNNG, and the surviving fraction was indistinguishable from that obtained after MNNG treatment of the related MMR-proficient 293 cell line. In contrast, 293T Lα− cells were resistant to killing by MNNG, just like the parental, MMR-deficient 293T cells. The presence of Dox in the culture medium had no effect on the survival of any of the control cell lines used in this study (Figure 3A). Figure 3.Sensitivity of 293T Lα cells to MNNG. (A) Survival of 293T Lα+ and 293T Lα− cells following treatment with 5 μM MNNG. 293 and 293T cells were used as MMR-proficient and -deficient controls, respectively. The presence of Dox (+Dox) in the culture medium did not affect the control cells, but had a dramatic effect on the survival of the 293T Lα cell populations. (B) IC50 values of 293T Lα+ and 293T Lα− cells. Each value represents the mean ± SE. (C) Cell cycle profiles of 293T Lα+ and 293T Lα− cells treated with 0.2 μM MNNG. Shown are representative cytometrograms of cells expressing (293T Lα+) and not expressing (293T Lα−) hMLH1. G1, cell population in the G1 phase of the cell cycle with a 2n DNA content; G2, cells in the G2 and M stages of the cell cycle with a 4n DNA content; S, cells in various stages of DNA synthesis with a DNA content between 2n and 4n. Download figure Download PowerPoint The sensitivity of 293T Lα cells to MNNG was further examined using the MTT assay, which is based on the cleavage of the yellow tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by the action of mitochondrial dehydrogenases to form a violet formazan dye. As this reaction takes place only in living cells, these can be distinguished from non-viable cells in a simple colorimetric assay. As shown in Figure 3B, 293T Lα− cells were 125-fold more resistant to killing by MNNG than the same cells in a MMR-proficient mode (i.e. 293T Lα+ cells). Expression of hMLH1 in 293T Lα cells leads to activation of a methylation damage induced cell cycle arrest To determine whether the increased sensitivity of 293T Lα+ cells to MNNG resulted from induction of cell cycle arrest and cell death, the treated 293T Lα+ and 293T Lα− cell populations were analysed by flow cytometry. As shown in Figure 3C, 2 days after treatment with 0.2 μM MNNG, the 293T Lα+ cells were mostly arrested in the G2/M phase of the cell cycle. One day later, cells containing sub-G1 amounts of DNA became detectable, and this population increased with time. In contrast, no increase in the population of cells either arrested in G2/M or with a lower than 2n DNA content was detected in cultures of treated 293T Lα− cells. In order to further characterize the response of cells to MNNG, we analysed the phosphorylation status of Cdc2. As shown in Figure 4A, Cdc2 phosphorylated on Tyr15 accumulated exclusively in 293T Lα+ cells treated with 0.2 μM MNNG. This provides molecular evidence for a G2/M arrest, because so long as this kinase remains phosphorylated, entry into mitosis should be blocked. No difference in Cdc2 phosphorylation was observed in the extracts of MNNG-treated 293T Lα− cells (Figure 4A). Figure 4.Post-translational protein modification and strand break processing in MNNG-treated 293T Lα cells. (A) Phosphorylation status of p53 and cdc2 in 293T Lα+ and 293T Lα− cells 1–4 days after treatment with 0.2 μM MNNG. P-p53, P-cdc2, phosphorylated p53 and cdc2 proteins, respectively; C, untreated control cells; β-tubulin, internal standard used to ascertain equal gel loading. (B) γ-H2AX foci formation in MNNG-treated 293T Lα cells. In the control cell population, <10% of cells displayed H2AX foci. Following MNNG treatment, all cells contained foci until 24 h post-treatment. See text for details and Materials and methods for experimental procedures. Download figure Download PowerPoint The above results thus show that induction of hMLH1 expression in the 293T Lα cells was necessary and sufficient to endow them with a MMR-proficient status, which also enabled them to respond to DNA damage induced by MNNG. What is presently unclear is the role of the MMR system in this checkpoint activation. DNA damage signalling is known to be mediated via several protein phosphorylation cascades, which involve primarily the DNA-dependent protein kinase (DNA-PK), or the ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) kinases. The downstream target of the latter enzymes is the p53 tumour suppressor protein, the phosphorylation of which on Ser15 is known to lead to its stabilization and subsequent activation as a transcription factor (Tibbetts et al., 1999). Phosphorylation of p53 has indeed been shown to take place upon MNNG treatment, and was shown to be dependent on functional hMutSα and hMutLα (Duckett et al., 1999; Hickman and Samson, 1999; Adamson et al., 2002). However, as the latter experiments were carried out with drug concentrations 25- to 125-fold higher than those used in our study, we wanted to test whether Ser15 phosphorylation also took place in the 293T Lα cells treated with 0.2 μM MNNG. These cells overexpress the SV40 large T antigen and thus contain large amounts of stabilized p53 polypeptide. This system is ideally suited for the study of post-translational modification of p53, as the steady-state levels of the latter protein remain unaltered during the experiment (Tibbetts et al., 1999). As anticipated, the p53 steady-state levels in the 293T Lα cell extracts were high, irrespective of whether hMLH1 was expressed or not, or whether extracts of treated or untreated cells were examined (Figure 4A). However, following MNNG treatment, phosphorylation of p53 with a Ser15-specific antibody could be detected exclusively in the MMR-proficient 293T Lα+ cells. Notably, and in contrast to the study by Adamson et al. (2002), where the phosphorylation of p53 became detectable already just minutes after MNNG treatment, the MMR-dependent post-translational modification of p53 observed in our cells peaked at 48 h, i.e. at a time point where most cells were arrested at G2/M (Figure 3C). This difference is probably linked with the high concentration of MNNG (25 μM) used in the latter study, which would be expected to introduce numerous single- and double-strand breaks into DNA that arise through the spontaneous loss of methylated purines and the subsequent breakage of the sugar-phosphate DNA backbone by β-elimination at the resulting abasic sites (Loeb, 1985). DNA strand breaks rapidly activate the ATM/ATR kinases that subsequently phosphorylate a number of downstream targets, one of which is histone H2AX (Redon et al., 2002). This histone modification is thought to aid the recruitment of DNA repair factors to the sites of damage (Paull et al., 2000). H2AX is phosphorylated in the 293T Lα cells upon treatment with 0.2 μM MNNG, as witnessed by the formation of phospho-H2AX foci (Figure 4B). However, these foci arise in both 293T Lα+ and 293T Lα− cells soon after treatment. Thus, damage caused by direct modifications of DNA at low concentrations of MNNG does not trigger the G2/M checkpoint. The activation of the checkpoint machinery must take place after H2AX phosphorylation, in the second cell cycle post-treatment (Kaina et al., 1997), and must involve the MMR system, perhaps in conjunction with another pathway of DNA metabolism that remains to be identified. Thus, the lesions that trigger the checkpoint machinery are distinct from those that bring about phosphorylation of H2AX. MMR proficiency and response to MNNG treatment require different levels of hMLH1 expression The principal goal of this study was to investigate the phenotypic effects of reduced expression of MMR proteins, such as might be encountered when expression of the gene is attenuated by cytosine methylation. In order to achieve this goal, we attempted to modulate hMLH1 expression in the 293T Lα cells. This could be achieved by varying the Dox concentration in the culture media. Thus, cells grown in the presence of 0.1, 0.2, 0.4, 0.8 and 1.5 ng/ml Dox contained steadily decreasing amounts of hMLH1 and hPMS2, as compared with cells grown in the absence of the drug (Figure 5A). Figure 5.Mismatch correction efficiency and MNNG-induced G2/M arrest in cells expressing different amounts of hMLH1. (A) Dependence of hMLH1 expression on Dox concentration. hMLH1 and hPMS2 were visualized as described in Materials and methods. β-tubulin, internal standard used to ascertain equal loading. (B) MMR efficiency of a G/T mispair in an M13mp2 substrate carrying a strand-discrimination signal 3′ from the mispair. Error bars show standard errors. (C) Variation in doubling times of 293T Lα cells grown in the indicated Dox concentrations following treatment with 5 μM MNNG. (D) FACS analysis of 293T Lα cell populations grown in the indicated Dox concentrations, either untreated (Control), or 72 h after treatment with 0.2 μM MNNG (see also Figure 3C). (E) Phosphorylation of p53 and cdc2 48 h after treatment of cells (grown in the indicated Dox concentrations) with 0.2 μM MNNG. β-tubulin, internal standard used to ascertain equal loading. Download figure Download PowerPoint When we tested how this variation in the amount of hMutLα affected MMR efficiency, we found that extracts of cells expressing as little as 10% of the amounts found in cells grown in the absence of Dox were still proficient in the in vitro MMR assays. Cells cultivated with 0.1 and 0.2 ng/ml Dox showed MMR activities comparable to those of the MMR-positive 293T Lα+ cells grown in the absence of Dox, and even extracts of cells cultivated with 0.4 ng/ml Dox were still able to repair mismatches in vitro, albeit with lower efficiency (Figure 5B). MMR proficiency was lost only in cell extracts in which the hMLH1 and hPMS2 proteins became difficult to detect by western blotting (Figure 5A). To test whether the results of the in vitro MMR assays were reflected also in the MSI phenotype of the cells, we analysed the BAT26 microsatellite marker, which contains a repeat of 26 deoxyadenosines, and which is considered to be a reliable indicator of MSI. Because the 293T Lα cells are hypo-triploid, and because this cell line was MMR deficient for many generations prior to our intervention, the BAT26 locus was found to be highly heterogeneous. The product of PCR amplification had on average eight peaks, and we therefore applied the HNPCC criteria of MSI (Loukola et al., 2001), whereby only PCR products that differed by three or more peaks at this locus were considered to be a sign of MSI. By these criteria, the BAT26 instability in the cells propagated for 35 generations in 0 or 0.2 ng/ml Dox was ∼1%, whereas cells grown with 50 ng/ml Dox displayed MSI that was ∼5-fold higher (Table I). How ever, closer inspection of the data revealed that cells propagated in 0 or 0.2 ng/ml Dox displayed no alleles (0/211) that differed by more than 4 bp from the median. In contrast, two such alleles (two out of 73; 2.7%) were found in the cells grown with 50 ng/ml Dox (Table I, numbers in parentheses). This suggests that MSI at the BAT26 locus in the 293T Lα− cells is substantially greater than in cells expressing hMLH1, and thus that expression of even low amounts of hMutLα are sufficient to correct the MMR defect in these cells, both in vitro (Figure 2) and in vivo (Table I). Table 1. Instability of the BAT26 chromosomal locus in 293T Lα cells expressing varying amounts of hMLH1 Dox (ng/ml) MSI+/total % MSI 0 2 (0)/131 1.5 0.2 1 (0)/80 1.3 50 4 (2)/73 5.5 (2.7) MSI+ clones were defined as those displaying more than three extra peaks in the sequence of the PCR product. Numbers in parentheses refer to clones with more than four extra peaks. We were interested to determine whether the low amounts of the hMLH1/hPMS2 heterodimer that wer
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