Cloning, Mapping, Expression, Function, and Mutation Analyses of the Human Ortholog of the Hamster Putative Tumor Suppressor Gene doc-1

Abstract

doc-1 is a putative tumor suppressor gene isolated and identified from the hamster oral cancer model. Here, we report the molecular cloning and the functional characterization of the human ortholog of the hamster doc-1 gene. Human doc-1 cDNA is 1.6 kilobase pairs in length and encodes for a 115-amino acid polypeptide (12.4 kDa, pI 9.53). Sequence analysis showed 98% identity between human and hamster doc-1 protein sequences. DOC-1 is expressed in all normal human tissues examined. In oral keratinocytes, expression of DOC-1 is restricted to normal oral keratinocytes. By immunostaining of normal human mucosa, DOC-1 is detected in both the cytoplasm and nuclei of basal oral keratinocytes; while in suprabasilar cells, it is primarily found in the nuclei. Human oral cancers in vivo did not exhibit immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates with DNA polymerase ␣/primase and mediates the phosphorylation of the large p180 catalytic subunit, suggesting it may be a potential regulator of DNA replication in the S phase of the cell cycle. Using a human doc-1 cosmid as a probe, human doc-1 is mapped to chromosome 12q24. We identified four exons in the entire human doc-1 gene and determined the intron-exon boundaries. By polymerase chain reaction and direct sequencing, we examined premalignant oral lesion and oral cancer cell lines and found no intragenic mutations. Squamous cell carcinoma (SCC)1 of the oral cavity is newly diagnosed in 38,000 Americans each year and in 350,000 people worldwide (1, 2). Approximately half of the patients afflicted die within 5 years of diagnosis, while surviving patients may be left with severe cosmetic and/or functional compromise (1-3). Survival curves of oral cancer patients have plateaued over the past 2 decades and remained among the worst of all cancer sites.The hamster oral cancer model is an excellent model to study the molecular event during oral carcinogenesis (4 -7). doc-1 is a putative tumor suppressor gene identified and isolated from the carcinogen-induced hamster oral cancer model (8). DOC-1 is predicted to be a 114-amino acid peptide with a molecular mass of 12.4 kDa. Transfection of doc-1 into malignant oral keratinocytes led to the reversion of transformation phenotypes including anchorage independence, doubling time, and morphology. The genetic sequence of doc-1 matched to a tumor necrosis factor-␣-induced early-response murine transcript, TU-166 (9), suggesting that doc-1 may be a downstream event in the tumor necrosis factor-␣ signaling pathway. We have recently cloned the full-length mouse doc-1 cDNA (GenBank TM number AF011644); its DNA sequence in the open reading frame is 94% identical to that of the hamster. The predicted amino acid peptides encoded by the mouse and hamster doc-1 open reading frames are identical. Each open reading frame encodes for a 114-amino acid peptide that has a predicted molecular mass of 12.4 kDa and a pI of 9.53.The highly conserved nature of the rodent doc-1 genes prompted us to clone and examine the role of doc-1 in...

Modulation of CDK2-AP1 (p12DOC−1) expression in human colorectal cancer

“…Importantly, after transient transfection of pcDNA3-CDK2-AP1 into CDK2-AP1 deficient cell lines (RKO and SW48), FACS analysis demonstrated a significant decrease in S phase and a significant increase in apoptosis. These results are consistent with previous reports of CDK2-AP1 interaction with DNA polymerase alpha/primase, CDK2, and pRB, genetic elements associated with cell cycle regulation and apoptosis (Tsuji et al, 1998;Shintani et al, 2000). The observations described above, specifically, CDK2-AP1 modulation of apoptosis and S phase in the setting of both microsatellite stable and unstable cell systems, suggests that CDK2-AP1 is a true mediator of cell cycle kinetics and apoptosis independent of microsatellite status.…”

Preneoplasia and carcinogenesis of the oral cavity

“…Oncogenes expressed in the resulting hamster pouch carcinomas have been found to include c-erb-B [50], c-Ha-ras [51,52], Ki-ras [50] and mutant p53 [53]. A new putative oral cancer suppressor gene (doc-1) was found in a study of hamster buccal pouch carcinogenesis, and a human counterpart of this gene was subsequently found, cloned and mapped [54]. Thus, studies on the molecular biology and molecular genetics in the experimental pathology of oral cancer in the hamster buccal pouch have led to an increased understanding of the molecular mechanisms underlying the development of human oral cancer [55,56].…”

Molecular mechanisms of head and neck cancer

“…Although an exact mechanism is not known, it is hypothesized that CDK2AP1 inhibits DNA polymerase-α/primase activity either through direct interaction or through its interaction with CDK2. Reduced or absent expression of CDK2AP1 has been demonstrated in about 60% of human oral cancer cell lines and tumor samples [101][102][103]. CDK2AP1 is also a downstream target for the TGF-β pathway.…”