C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency

Promoter methylation of MLH1, PMS2, MSH2 and p16 is a phenomenon of advanced-stage HCCs

Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion. We investigated promoter methylation pattern of the MMR genes MLH1, MSH2 and PMS2 as well as the cyclin-dependent kinase inhibitor 2A gene (p16) in 61 well characterized patients with HCCs associated with HCV, Hepatitis B virus infection or alcoholic liver disease. DNA was isolated from formalin-fixed, paraffin-embedded tumour and non-tumour adjacent tissue and analysed by methylation-specific PCR. Moreover, microsatellite analysis was performed in tissues showing methylation in MMR gene promoters. Our data demonstrated that promoter methylation of MLH1, MSH2, PMS2 and p16 is present among all considered HCCs. Hereby, promoter silencing was detectable more frequently in advanced-stage HCCs than in low-stage ones. However, there was no significant correlation between aberrant DNA methylation of MMR genes or p16 and HCV infection in related HCC specimens. In summary, we show that promoter methylation of essential MMR genes and p16 is detectable in HCCs most dominantly in pT3 stage tumour cases. Since loss of MMR proteins was previously described to be not only responsible for tumour development but also for chemotherapy resistance, the knowledge of mechanisms jointly responsible for HCC progression might enable significant improvement of individual HCC therapy in the future

Influence of fluorescent tags on MMR activity of MutLα.

<p>HEK293T cells were transiently cotransfected with various labeled or unlabeld MutLα constructs and 48 h post transfection MMR activity of different MutLαs were assessed in vitro in parallel with unlabeled MutLα by quantifying the 3′-nick-directed correction of a G-T mismatch in a restriction site of a plasmid substrate as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#s4" target="_blank">Materials and Methods</a>” and as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#pone.0031863-Brieger3" target="_blank">[10]</a>. <i>In vitro</i> repair was scored on a 2-kb circular DNA substrate that contains an <i>EcoRV</i> site which is destroyed by a G-T mismatch. Upon repair of the G-T mismatch to an A-T base pair the intact <i>EcoRV</i> site together with an <i>AseI</i> site gives rise to a 0.8- and a 1.2-kb fragment, whereas unrepaired DNA is only linearized by <i>AseI</i> to a 2-kb fragment. Repair efficiency was assessed by measuring the signal intensities of linearized and digested vector with Bio-Rad Quantity One software using the “rolling ball” baseline correction. The signal intensity of the repair bands was divided by the intensity of all three bands. Repair efficiency of unlabeled MutLα was set at 100 percent and repair of fluorescent tagged MutLα was determined in relation to the wild-type sample that was expressed, processed and tested in parallel. Average repair values and standard deviations (±) were determined from four independent experiments. Single PMS2 tagged MutLαs, single MLH1-GFP-N tagged MutLαs as well as MLH1-GFP-N coexpressed with PMS2-Red-N or MLH1-Red-N coexpressed with PMS2-GFP-N were MMR proficient while all other tagged variants showed MMR deficiency. 1: mock control (untransfected). 2: MLH1/PMS2 unlabeled (positive control); 3: MLH1/PMS2-GFP-N; 4: MLH1/PMS2-GFP-C; 5: MLH1/PMS2-Red-N; 6: MLH1/PMS2-Red-C; 7: MLH1-GFP-N/PMS2; 8: MLH1-GFP-C/PMS2; 9: MLH1-Red-N/PMS2; 10: MLH1-Red-C/PMS2; 11: MLH1-GFP-N/PMS2-Red-N; 12: MLH1-GFP-N/PMS2-Red-C; 13: MLH1-GFP-C/PMS2-Red-N; 14: MLH1-GFP-C/PMS2-Red-C; 15: MLH1-Red-N/PMS2-GFP-N; 16: MLH1-Red-N/PMS2-GFP-C; 17: MLH1-Red-C/PMS2-GFP-N; 18: MLH1-Red-C/PMS2-GFP-C. Symbols see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#pone-0031863-g001" target="_blank">Figure 1</a>.</p

Putative three-dimensional structure models of fluorescent labeled MutLα.

<p>Using PyMol (Warren DeLano, <a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>), GFP or Red fluorescent proteins were attached to (A) N-termini of MLH1 and PMS2 or (B) C-termini of MutLα. C-terminal tags seem to hide the C-terminal region of MutLα and consequently might avoid DNA interaction. (C) Corresponding amino acid sequences of linker regions are shown. The dashed line between PMS2 and the N-terminal fluorescent tag illustrates a putative α-helix (unknown structure) of the first thirty amino acids of PMS2.</p

Influence of fluorescent labeling of MutLα on protein expression levels.

<p>To analyze the effect of fluorescent dyes on protein expression levels, HEK293T cells were transiently cotransfected with different MutLα constructs (see below) and (A) Western blot analysis was carried out after 48 h using anti-MLH1 or anti-PMS2, respectively, controlled by β-actin detection. (B) Amounts of expressed proteins were assessed by measuring the signal intensities of protein bands with Multi Gauge V3.2 software. Graphs indicate the results (mean ±S.D.) of at least four independent experiments in which the proportion of protein expression using an unbiased method were presented. 1: negative control (untransfected). 2: MLH1/PMS2 unlabeled; 3: MLH1/PMS2-GFP-N; 4: MLH1/PMS2-GFP-C; 5: MLH1/PMS2-Red-N; 6: MLH1/PMS2-Red-C; 7: MLH1-GFP-N/PMS2; 8: MLH1-GFP-C/PMS2; 9: MLH1-Red-N/PMS2; 10: MLH1-Red-C/PMS2; 11: MLH1-GFP-N/PMS2-Red-N; 12: MLH1-GFP-N/PMS2-Red-C; 13: MLH1-GFP-C/PMS2-Red-N; 14: MLH1-GFP-C/PMS2-Red-C; 15: MLH1-Red-N/PMS2-GFP-N; 16: MLH1-Red-N/PMS2-GFP-C; 17: MLH1-Red-C/PMS2-GFP-N; 18: MLH1-Red-C/PMS2-GFP-C. Symbols see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031863#pone-0031863-g001" target="_blank">Figure 1</a>.</p

Dye tags influence single expression of MLH1 and PMS2.

<p>To determine the influence of fluorescent tags on single expressed MLH1 or PMS2 variants, HEK293T cells were transfected with different (A) MLH1 or (B) PMS2 constructs. Amounts of expressed proteins were assessed after Western blotting by measuring the signal intensities of protein bands with Multi Gauge V3.2 software. Graphs indicate the results (mean ±S.D.) of at least four independent experiments in which the proportion of protein expression using an unbiased method were presented. 1: MLH1 unlabeled; 2: MLH1-GFP-N; 3: MLH1-GFP-C; 4: MLH1-Red-N; 5: MLH1-Red-C; 6: PMS2 unlabeled; 7: PMS2-GFP-N; 8: PMS2-GFP-C; 9: PMS2-Red-N; 10: PMS2-Red-C.</p

Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1

Introduction: Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated. Methods: Nine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively. Results: MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability. Conclusions: These data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1

MSP analysis.

<p>Representative examples of MSP analysis for MLH1, MSH2, PMS2 and p16 methylation in HCC and non-tumour adjacent tissues were shown. Bisulfite-modified DNA was amplified using MSP primers specific to a CpG-rich region of each gene promoter. PCR-amplified products were resolved by 2% agarose gel electrophoresis. (U) Lanes represent amplification of unmethylated alleles, and (M) lanes contain only methylated alleles.</p

Frequency of promoter methylation.

<p>Frequency of promoter methylation.</p

Primer sequences and annealing temperatures used in MSP.

<p>Primer sequences and annealing temperatures used in MSP.</p