Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12 DOC-1 is a growth suppressor isolated from normal keratinocytes. We report that p12 DOC-1 associates with CDK2. More specifically, p12 DOC-1 associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12 DOC-1 resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12 DOC-1 transfectants (1 G 1 and 2 S). The p12 DOC-1 -mediated decrease of CDK2 was prevented if the p12 DOC-1 transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin -lactone, suggesting that p12 DOC-1 may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12 DOC-1 -mediated, CDK2-associated cell cycle phenotypes. These data support p12 DOC-1 as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2. Cell cycle inhibitors of the p16INK4a and p21families exert their effects by negatively regulating cyclin and cyclin-dependent kinase (CDK) complex formation and kinase activities (10,14). While the p16 INK4a family is specific for CDK4 and CDK6, and the p21 WAF1/CIP1/CAP20 family of CDK inhibitors is universal for CDKs, there is no known specific inhibitor for CDK2. CDK2, when complexed with cyclins E and A, is implicated in G 1 /S transition, DNA replication, and progression through the DNA synthesis phase (6, 7, 9). p12 DOC-1 is a growth suppressor identified and isolated from normal keratinocytes (12). It is a highly conserved cellular gene. Our laboratory (12, 13) and others (4, 5) have cloned p12 DOC-1 cDNA from human, mouse, and hamster. The fulllength human and mouse p12 DOC-1 cDNAs are 1.6 kb and 1.2 kb, respectively. Human p12 DOC-1 has one additional amino acid at residue 19, which corresponds to an alanine, and differs from the mouse and hamster p12 DOC-1 at only two other amino acid residues (Ala 3 Thr at residue 8 and Gly 3 Ser at residue 100). Human and rodent p12 DOC-1 polypeptides have 97% identity, and the mouse and hamster p12 DOC-1 protein sequences are identical. Human p12 DOC-1 is a 115-amino-acid peptide with a molecular mass of 12.4 kDa (pI, 9.62).Ectopic expression of p12 DOC-1 in keratinocytes is associated with increased doubling time, suggestive of a growth suppressor function (11). These observations prompted us to examine if p12 DOC-1 interacts with regulatory proteins in the cell division cycle. We report that p12 DOC-1 associates with CDK2. Data are presented to support the role of p12 DOC-1 as a specifically CDK2-associated protein, which, when overexpressed, negatively regulates CDK2-associated kinase activities and cell cycle phenotypes. MATERIALS AND METHODSCell culture and transfections. Transfection of human 293 cells was performed using Lipofectamine Plus (Life...
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...
Various kinds of diseases may be found in the oral and maxillofacial regions and various modalities may be applied for their diagnosis, including intra-oral radiography, panoramic radiography, ultrasonography, computed tomography, magnetic resonance imaging, and nuclear medicine methods such as positron emission tomography. Of these modalities, ultrasound imaging is easy to use for the detection of noninvasive and soft tissue-related diseases. Doppler ultrasound images taken in the B-mode can provide vascular information associated with the morphology of soft tissues. Thus, ultrasound imaging plays an important role in confirming the diagnosis of many kinds of diseases in such oral and maxillofacial regions as the tongue, lymph nodes, salivary glands, and masticatory muscles. In the present article, we introduce three new applications of ultrasonography: guided fine-needle aspiration, measurement of tongue cancer thickness, and diagnosis of metastasis to cervical lymph nodes.
Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12 DOC-1 is a growth suppressor isolated from normal keratinocytes. We report that p12 DOC-1 associates with CDK2. More specifically, p12 DOC-1 associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12 DOC-1 resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12 DOC-1 transfectants (1 G 1 and 2 S). The p12 DOC-1 -mediated decrease of CDK2 was prevented if the p12 DOC-1 transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin -lactone, suggesting that p12 DOC-1 may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12 DOC-1 -mediated, CDK2-associated cell cycle phenotypes. These data support p12 DOC-1 as a specific CDK2associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.Cell cycle inhibitors of the p16 INK4a and p21 WAF1/CIP1/CAP20 families exert their effects by negatively regulating cyclin and cyclin-dependent kinase (CDK) complex formation and kinase activities (10,14). While the p16 INK4a family is specific for CDK4 and CDK6, and the p21 WAF1/CIP1/CAP20 family of CDK inhibitors is universal for CDKs, there is no known specific inhibitor for CDK2. CDK2, when complexed with cyclins E and A, is implicated in G 1 /S transition, DNA replication, and progression through the DNA synthesis phase (6,7,9). p12 DOC-1 is a growth suppressor identified and isolated from normal keratinocytes (12). It is a highly conserved cellular gene. Our laboratory (12, 13) and others (4, 5) have cloned p12 DOC-1 cDNA from human, mouse, and hamster. The fulllength human and mouse p12 DOC-1 cDNAs are 1.6 kb and 1.2 kb, respectively. Human p12 DOC-1 has one additional amino acid at residue 19, which corresponds to an alanine, and differs from the mouse and hamster p12 DOC-1 at only two other amino acid residues (Ala 3 Thr at residue 8 and Gly 3 Ser at residue 100). Human and rodent p12 DOC-1 polypeptides have 97% identity, and the mouse and hamster p12 DOC-1 protein sequences are identical. Human p12 DOC-1 is a 115-amino-acid peptide with a molecular mass of 12.4 kDa (pI, 9.62).Ectopic expression of p12 DOC-1 in keratinocytes is associated with increased doubling time, suggestive of a growth suppressor function (11). These observations prompted us to examine if p12 DOC-1 interacts with regulatory proteins in the cell division cycle. We report that p12 DOC-1 associates with CDK2. Data are presented to support the role of p12 DOC-1 as a specifically CDK2-associated protein, which, when overexpressed, negatively regulates CDK2-associated kinase activities and cell cycle phenotypes.
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