Mammalian MutY homolog (MYH or MUTYH) protects cells from oxidative DNA damage.

MutY DNA glycosylase homologs (MYH or MUTYH) reduce G:C to T:A mutations by removing misincorporated adenines or 2-hydroxyadenines paired with guanine or 8-oxo-7,8-dihydroguanine (8-oxo-G). Mutations in the human MYH (hMYH) gene are associated with the colorectal cancer predisposition syndrome MYH-associated polyposis. To examine the function of MYH in human cells, we regulated MYH gene expression by knockdown or overproduction. MYH knockdown human HeLa cells are more sensitive to the killing effects of H2O2 than the control cells. In addition, hMYH knockdown cells have altered cell morphology, display enhanced susceptibility to apoptosis, and have altered DNA signaling activation in response to oxidative stress. The cell cycle progression of hMYH knockdown cells is also different from that of the control cells following oxidative stress. Moreover, hMYH knockdown cells contain higher levels of 8-oxo-G lesions than the control cells following H2O2 treatment. Although MYH does not directly remove 8-oxo-G, MYH may generate favorable substrates for other repair enzymes. Overexpression of mouse Myh (mMyh) in human mismatch repair defective HCT15 cells makes the cells more resistant to killing and refractory to apoptosis by oxidative stress than the cells transfected with vector. In conclusion, MYH is a vital DNA repair enzyme that protects cells from oxidative DNA damage and is critical for a proper cellular response to DNA damage.

The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase.

Human (h) DNA repair enzyme thymine DNA glycosylase (hTDG) is a key DNA glycosylase in the base excision repair (BER) pathway that repairs deaminated cytosines and 5-methyl-cytosines. The cell cycle checkpoint protein Rad9-Rad1-Hus1 (the 9-1-1 complex) is the surveillance machinery involved in the preservation of genome stability. In this study, we show that hTDG interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. The hHus1 interacting domain is mapped to residues 67-110 of hTDG, and Val74 of hTDG plays an important role in the TDG-Hus1 interaction. In contrast to the core domain of hTDG (residues 110-308), hTDG(67-308) removes U and T from U/G and T/G mispairs, respectively, with similar rates as native hTDG. Human TDG activity is significantly stimulated by hHus1, hRad1, hRad9 separately, and by the 9-1-1 complex. Interestingly, the interaction between hRad9 and hTDG, as detected by co-immunoprecipitation (Co-IP), is enhanced following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. A significant fraction of the hTDG nuclear foci co-localize with hRad9 foci in cells treated with methylating agents. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of the BER.

The role of adenovirus-mediated retinoblastoma 94 in the treatment of head and neck cancer.

A truncated retinoblastoma (RB) protein of approximately 94 kDa (RB94), lacking the NH2 -terminal 112 amino acid residues of the full-length RB, has been found to have great efficacy in tumor suppression. This study investigated the role of adenovirus-mediated RB94 (Ad-RB94) gene therapy for human head and neck squamous cell carcinoma (HNSCC) and explored the cellular and molecular mechanism of tumor inhibition after Ad-RB94 gene transfer. Randomized controlled studies in vitro and in vivo were performed to assess antitumor responses of Ad-RB94 gene transfer against human HNSCC. Human HNSCC cell lines, JHU006 and JHU012, were used in this study. Tumors originated from the HNSCC cell lines were propagated as xenografts in nude mice. Ad-RB94 gene transfer was performed both in vitro and in vivo with replication-defective virus (DL312) and no treatment as controls. Transgene expression, cell viability, and tumor growth were evaluated in transfected cells and tumor implants. To determine the mechanism behind the observed antitumor action, cell cycle analysis was performed, and telomerase activity was examined. Tumors were evaluated for RB94-induced apoptosis. Transgene expression of RB94 was detected by Western blot analysis, real-time quantification reverse transcription-PCR, and immunohistochemistry. RB94 expression led to flattening of cell growth curves and caused tumor regression. Animals treated with Ad-RB94 were seen to have a significant reduction in tumor size when compared with DL312 (P = 0.02, both cell lines) and to no treatment groups (P = 0.01, both cell lines). Cell cycle arrest in the G(2)-M phase and increased levels of apoptosis occurred in tumor cells treated with Ad-RB94. In addition, telomerase activity decreased significantly and specifically after Ad-RB94 treatment. This study demonstrates that Ad-RB94 gene transfer effectively inhibits HNSCC tumor cell growth in vitro and in vivo. The unique property of Ad-RB94 gene transfer to arrest HNSCC tumor cells in the G2-M phase of the cell cycle makes it a good candidate for adjuvant therapy with radiation or chemotherapy, as tumor cells are most sensitive to radiation or cytotoxic drug in this cell cycle phase.