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) pulldown 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.
Similar content being viewed by others
References
Wu, C., Hu, Z., He, Z., Jia, W., Wang, F., Zhou, Y., Liu, Z., Zhan, Q., Liu, Y., Yu, D., Zhai, K., Chang, J., Qiao, Y., Jin, G., Liu, Z., Shen, Y., Guo, C., Fu, J., Miao, X., Tan, W., Shen, H., Ke, Y., Zeng, Y., Wu, T., and Lin, D. (2011) Genome-wide association study identifies three new susceptibility loci for esophageal squamous-cell carcinoma in Chinese populations, Nat. Genet., 43, 679–684.
Torre, L. A., Bray, F., Siegel, R. L., Ferlay, J., LortetTieulent, J., and Jemal, A. (2015) Global cancer statistics, 2012, Cancer J. Clin., 65, 87–108.
Van Heijl, M., Van Lanschot, J. J., Koppert, L. B., Van Berge Henegouwen, M. I., Muller, K., Steyerberg, E. W., Van Dekken, H., Wijnhoven, B. P., Tilanus, H. W., Richel, D. J., Busch, O. R., Bartelsman, J. F., Koning, C. C., Offerhaus, G. J., and Van der Gaast, A. (2008) Neoadjuvant chemoradiation followed by surgery versus surgery alone for patients with adenocarcinoma or squamous cell carcinoma of the esophagus (CROSS), BMC Surg., 8, 21.
Roth, J. A., Pass, H. I., Flanagan, M. M., Graeber, G. M., Rosenberg, J. C., and Steinberg, S. (1988) Randomized clinical trial of preoperative and postoperative adjuvant chemotherapy with cisplatin, vindesine, and bleomycin for carcinoma of the esophagus, J. Thorac. Cardiovasc. Surg., 96, 242–248.
Liu, M., Hales, B. F., and Robaire, B. (2014) Effects of four chemotherapeutic agents, bleomycin, etoposide, cisplatin, and cyclophosphamide, on DNA damage and telomeres in a mouse spermatogonial cell line, Biol. Reprod., 90, 72.
Coster, G., and Goldberg, M. (2010) The cellular response to DNA damage: a focus on MDC1 and its interacting proteins, Nucleus, 1, 166–178.
Solier, S., and Pommier, Y. (2011) MDC1 cleavage by caspase-3: a novel mechanism for inactivating the DNA damage response during apoptosis, Cancer Res., 71, 906–913.
Mendez, J., and Stillman, B. (2000) Chromatin association of human origin recognition complex, Cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis, Mol. Cell. Biol., 20, 8602–8612.
Han, X., Aslanian, A., Fu, K., Tsuji, T., and Zhang, Y. (2014) The interaction between checkpoint kinase 1 (Chk1) and the minichromosome maintenance (MCM) complex is required for DNA damage-induced Chk1 phosphorylation, J. Biol. Chem., 289, 24716–24723.
Costantino, L., Sotiriou, S. K., Rantala, J. K., Magin, S., Mladenov, E., Helleday, T., Haber, J. E., Iliakis, G., Kallioniemi, O. P., and Halazonetis, T. D. (2014) Breakinduced replication repair of damaged forks induces genomic duplications in human cells, Science, 343, 88–91.
Okumura, H., Natsugoe, S., Matsumoto, M., Mataki, Y., Takatori, H., Ishigami, S., Takao, S., and Aikou, T. (2005) The predictive value of p53, p53R2, and p21 for the effect of chemoradiation therapy on oesophageal squamous cell carcinoma, Br. J. Cancer, 92, 284–289.
Okumura, H., Uchikado, Y., Setoyama, T., Matsumoto, M., Owaki, T., Ishigami, S., and Natsugoe, S. (2014) Biomarkers for predicting the response of esophageal squamous cell carcinoma to neoadjuvant chemoradiation therapy, Surg. Today, 44, 421–428.
McDonald, W. H., Tabb, D. L., Sadygov, R. G., MacCoss, M. J., Venable, J., Graumann, J., Johnson, J. R., Cociorva, D., and Yates, J. R. (2004) MS1, MS2, and SQT-three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications, Rapid Commun. Mass Spectrom., 18, 2162–2168.
Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J., and Gygi, S. P. (2003) Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome, J. Proteome Res., 2, 43–50.
Schwacha, A., and Bell, S. P. (2001) Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication, Mol. Cell, 8, 1093–1104.
Takahashi, T. S., Wigley, D. B., and Walter, J. C. (2005) Pumps, paradoxes and ploughshares: mechanism of the MCM2-7 DNA helicase, Trends Biochem. Sci., 30, 437–444.
Li, N., Zhai, Y., Zhang, Y., Li, W., Yang, M., Lei, J., Tye, B. K., and Gao, N. (2015) Structure of the eukaryotic MCM complex at 3.8 Å, Nature, 524, 186–191.
Bleichert, F., Botchan, M. R., and Berger, J. M. (2015) Crystal structure of the eukaryotic origin recognition complex, Nature, 519, 321–326.
Lee, K. Y., Bae, J. S., Yoon, S., and Hwang, D. S. (2014) Dephosphorylation of Orc2 by protein phosphatase 1 promotes the binding of the origin recognition complex to chromatin, Biochem. Biophys. Res. Commun., 448, 385–389.
Celeste, A., Difilippantonio, S., Difilippantonio, M. J., Fernandez-Capetillo, O., Pilch, D. R., Sedelnikova, O. A., Eckhaus, M., Ried, T., Bonner, W. M., and Nussenzweig, A. (2003) H2AX haploinsufficiency modifies genomic stability and tumor susceptibility, Cell, 114, 371–383.
Stucki, M., Clapperton, J. A., Mohammad, D., Yaffe, M. B., Smerdon, S. J., and Jackson, S. P. (2005) MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks, Cell, 123, 1213–1226.
Shi, H., Zhu, S., Liu, Z., and Su, J. (2013) The impact to MDC1 and 53BP1 after silence H2AX in esophageal carcinoma ECA109, Basic Clin. Med., 33, 808–813.
Crevel, G., Hashimoto, R., Vass, S., Sherkow, J., Yamaquchi, M., Heck, M. M. S., and Cotterill, S. (2007) Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells, PLoS One, 2, e833.
Zhang, X., Teng, Y., Yang, F., Wang, M., Hong, X., Ye, L. G., Gao, Y. N., and Chen, G. Y. (2015) MCM2 is a therapeutic target of lovastatin in human non-small cell lung carcinomas, Oncol. Rep., 33, 2599–2605.
Olive, P. L., and Banath, J. P. (1993) Detection of DNA double-strand breaks through the cell cycle after exposure to X-rays, bleomycin, etoposide and 125IdUrd, Int. J. Radiat. Biol., 64, 349–358.
Lou, Z., and Chen, J. (2004) Use of siRNA to study the function of MDC1 in DNA damage responses, Methods Mol. Biol., 281, 179–187.
Wang, Z., Zeng, Q., Chen, T., Liao, K., Bu, Y., Hong, S., and Hu, G. (2015) Silencing NFBD1/MDC1 enhances the radiosensitivity of human nasopharyngeal cancer CNE1 cells and results in tumor growth inhibition, Cell Death Dis., 6, e1849.
Liu, Z. K., Zhu, S. C., Su, J. W., Wang, Y. X., Yang, J. J., Li, J., and Shen, W. B. (2010) Short hairpin RNA-mediated MDC1 gene silencing enhances the radiosensitivity of esophageal squamous cell carcinoma cell line ECA109, J. South Med. Univ., 30, 1830–1834.
Yuan, C., Bu, Y., Wang, C., Yi, F., Yang, Z., Huang, X., Cheng, L., Liu, G., Wang, Y., and Song, F. (2012) NFBD1/MDC1 is a protein of oncogenic potential in human cervical cancer, Mol. Cell. Biochem., 359, 333–346.
Toyokawa, G., Masuda, K., Daigo, Y., Cho, H. S., Yoshimatsu, M., Takawa, M., Hayami, S., Maejima, K., Chino, M., Field, H. I., Neal, D. E., Tsuchiya, E., Ponder, B. A., Maehara, Y., Nakamura, Y., and Hamamoto, R. (2011) Minichromosome Maintenance Protein 7 is a potential therapeutic target in human cancer and a novel prognostic marker of non-small cell lung cancer, Mol. Cancer, 10, 65.
Ha, S. A., Shin, S. M., Namkoong, H., Lee, H., Chio, G. W., Hur, S. Y., Kim, T. E., and Kim, J. W. (2004) Cancerassociated expression of minichromosome maintenance 3 gene in several human cancers and its involvement in tumorigenesis, Clin. Cancer Res., 10, 8386–8295.
Kato, H., Miyazaki, T., Fukai, Y., Nakajima, M., Sohda, M., Takita, J., Masuda, N., Fukuchi, M., Manda, R., Ojima, H., Tsukada, K., Asao, T., and Kuwano, H. (2003) A new proliferation marker, minichromosome maintenance protein 2, is associated with tumor aggressiveness in esophageal squamous cell carcinoma, J. Surg. Oncol., 84, 24–30.
Huang, B., Hu, B., Su, M., Tian, D., Guo, Y., Lian, S., Liu, Z., Wu, X., Li, Q., Zheng, R., and Gao, Y. (2011) Potential role of minichromosome maintenance protein 2 as a screening biomarker in esophageal cancer high-risk population in China, Hum. Pathol., 42, 808–816.
Goldberg, M., Stucki, M., Falck, J., D’ Amours, D., Rahman, D., Pappin, D., Bartek, J., and Jackson, S. P. (2003) MDC1 is required for the intra-S-phase DNA damage checkpoint, Nature, 421, 952–956.
Author information
Authors and Affiliations
Corresponding author
Additional information
These authors contributed equally to this work.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM16-122, September 12, 2016.
Rights and permissions
About this article
Cite this article
Yu, J., Wang, R., Wu, J. et al. 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. Biochemistry Moscow 81, 1221–1228 (2016). https://doi.org/10.1134/S0006297916100205
Received:
Revised:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0006297916100205