Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines

Abstract

The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an indicator of apoptosis, showed that MLH1 translocation only occurred in MMR proficient (SW480) cells upon induction of apoptosis further suggested a MSH2 dependent role of MLH1 in apoptosis. These data suggest a role of MLH1 in mediation of apoptosis in a MSH2-dependent manner. Taken together, our data supported an interdependence of mismatch repair proteins, particularly MLH1 and MSH2, in the mediation of apoptosis in human colorectal carcinoma cell lines.

5-Fluorouracil upregulates cell surface B7-H1 (PD-L1) expression in gastrointestinal cancers

“…Differences between western and flow analysis may reflect differences in surface vs intracellular expression or may be influenced by dead or dying cells, which can be excluded from analysis using flow. HCT 116 cells have functional MSH2 , but non-functional MSH1 and are therefore classified as a microsatellite instable cell line (MSI) [ 26 , 27 ]. MSI tumors have been shown to upregulate multiple immune checkpoints including PD-1 and B7-H1, with microsatellite stable tumors being less responsive and therefore our HCT 116 cells could be expected to be ideal for studying B7-H1 induction [ 28 , 29 ].…”

Cytotoxicity of Manganese (III) Complex in Human Breast Adenocarcinoma Cell Line Is Mediated by the Generation of Reactive Oxygen Species Followed by Mitochondrial Damage

“…40 However, the Yo-Pro-1 appears to have more sensitivity and wide application, particularly in multiplexing systems of flow cytometry. 38 Our laboratory has also used this apoptotic assay kit in a number of studies [37][38][39] to determine whether the cell death was due to apoptosis or necrosis.…”

“…37 Yo-Pro-1 has been used as a marker to detect early and late apoptotic events by many investigators including our laboratory. [37][38][39][40] Yo-Pro-1-based flow cytometry detects changes in plasma membrane phospholipid orientation, similar to annexin V-based flow cytometry, and both methods provide comparable results. 40…”

C. tropicalis promotes chemotherapy resistance in colon cancer through increasing lactate production to regulate the mismatch repair system

“…However, oxaliplatin treatment had less inhibitory effect in mice bearing HCT116 tumor cells ( Figure S2 A-C). Several studies have shown that HCT116 is an MMR-deficient cell line while SW480 is an MMR-proficient cell line, and that loss of MMR leads to increased adaptive variability and chemoresistance in CRC 10 , 16 - 18 . Therefore, we detected the protein and mRNA expression of functional proteins in MMR.…”