Knockdown of Minichromosome Maintenance Proteins Inhibits Foci Forming of Mediator of DNA-Damage Checkpoint 1 in Response to DNA Damage in Human Esophageal Squamous Cell Carcinoma TE-1 Cells.

Abstract

Esophageal squamous cell carcinoma (ESCC) has a high morbidity in China and its treatment depends greatly on adjuvant chemotherapy. However, DNA damage repair in cancer cells severely affects the outcome of treatment. This study investigated the potential mechanism regarding mediator of DNA-damage checkpoint 1 (MDC1) and minichromosome maintenance proteins (MCMs) during DNA damage in ESCC. Recombinant vectors of MDC1 and MCMs with tags were constructed and transfected into human ESCC cell line TE-1. Immunoprecipitation and mass spectrometry were performed to screen the MCMs interacting with MDC1, and direct interaction was confirmed by glutathione S-transferase (GST) pull-down assay in vitro. MCM2 and MCM6 were knocked down by shRNAs, after which chromatin fraction and foci forming of MDC1 upon bleomycin-induced DNA damage were examined. The results showed that MCM2/3/5/6 were immunoprecipitated by antibodies against the tag of MDC1 in TE-1 nuclei, and the GST pull-down assay indicated the direct interaction. Knockdown of MCM2 or MCM6 reduced the chromatin fraction of MDC1 according to Western blot results. Moreover, knockdown of MCM2 or MCM6 could significantly inhibit foci forming of MDC1 in TE-1 nuclei in response to bleomycin-induced DNA damage (p < 0.001). This study indicates the direct interaction between MDC1 and MCMs in TE-1 nuclei. Downregulation of MCMs can inhibit chromatin fraction and foci forming of MDC1 in TE-1 cells upon DNA damage, which suggests MCMs and MDC1 as potential targets to improve the outcome of chemotherapy in ESCC.