Hiroe Ohyama

Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis.

Large scale gene expression profiling was carried out on laser capture microdissected (LCM) tumor and normal oral epithelial cells and analysed on high-density oligonucleotide microarrays. About 600 genes were found to be oral cancer associated. These oral cancer associated genes include oncogenes, tumor suppressors, transcription factors, xenobiotic enzymes, metastatic proteins, differentiation markers, and genes that have not been implicated in oral cancer. The database created provides a verifiable global profile of gene expression during oral carcinogenesis, revealing the potential role of known genes as well as genes that have not been previously implicated in oral cancer.

p12(DOC-1) is a novel cyclin-dependent kinase 2-associated protein.

ABSTRACT Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12DOC-1 is a growth suppressor isolated from normal keratinocytes. We report that p12DOC-1 associates with CDK2. More specifically, p12DOC-1 associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12DOC-1 resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12DOC-1transfectants (↑ G1 and ↓ S). The p12DOC-1-mediated decrease of CDK2 was prevented if the p12DOC-1 transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin β-lactone, suggesting that p12DOC-1 may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12DOC-1-mediated, CDK2-associated cell cycle phenotypes. These data support p12DOC-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.

p12DOC-1, a growth suppressor, associates with DNA polymerase α/primase

p12DOC‐1 is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12DOC1 in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12DOC1 associates with DNA polymerase α/primase (pol‐α: primase) in vitro and in cells. The pol‐α:primase binding domain in p12DOC‐1 is mapped to the amino‐terminal six amino acid (MSYKPN). The biological effect of p12DOC1 on pol‐α:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12DOC‐1 suppresses DNA rep lication, leveling at —50%. Similar results were obtained using the M13 single‐stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12DOC1 affects the initiation step, not the elongation phase. The p12DOC1 suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol‐α:primase or by its effect on cyclin‐dependent kinase 2 (CDK2), a recently identified p12DOC‐1‐associated protein known to stimulate DNA replication by phosphorylating pol‐α:primase. p12DOC1 suppresses CDK2‐mediated phosphorylation of pol‐α:primase. These data support a role of p12DOC1 as a regulator of DNA replication by direct inhibition of pol‐α:primase or by negatively regulating the CDK2‐mediated phosphorylation of pol‐α:primase.—Matsuo, K., Shintani, S., Tsuji, T., Nagata, E., Lerman, M., McBride, J., Nakahara, Y., Ohyama, H., Todd, R., Wong, D. T. W. p12DOC‐1, a growth suppressor, associates with DNA polymerase α/primase. FASEB J. 14, 1318–1324 (2000)

IL-1β and IL-6 in mouse parotid acinar cells: characterization of synthesis, storage, and release

Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1β (IL-1β) and the anti-inflammatory cytokine IL-6 have not been established in normal exocrine gland secretory cells. Parotid glands and isolated acinar cells prepared from BALB/c mice were homogenized for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). IL-1β and IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on supernatants prepared from mouse parotid acinar cell (MPAC) preparations unstimulated or stimulated between 0 and 10 min with 10-5 M norepinephrine at 37°C. MPACs were fixed in paraformaldehyde, frozen sectioned for light and electron microscopy, and labeled with antibodies to IL-1β and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6 (614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant increase in IL-1β ( P < 0.03) and IL-6 ( P < 0.01) release into supernatants by 10 min that paralleled the time course of amylase release. In situ hybridization showed the presence of transcripts for IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1β and IL-6 were significantly higher ( P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1β and IL-6 and release these cytokines from their granules after α- and β-adrenergic stimulation.

Odontogenic carcinoma: a functional genomic comparison with oral mucosal squamous cell carcinoma

Intraosseous squamous cell carcinomas of the mandible arise de novo or secondary to a tumor or transformed cyst epithelium. Current diagnostic tests frequently fail to distinguish between these tumors, leading to confusing classification schemes. We report the functional genomic analysis of a mandibular odontogenic carcinoma. Malignant keratinocytes from the lesion were isolated using laser capture microdissection. Target sample generated from the total RNA of the LCM-procured cells was used to hybridize high-density oligonucleotide arrays. Functional genomic analysis of the odontogenic carcinoma database compared with four oral mucosal squamous cell carcinoma gene expression databases was performed. Preliminary results suggest a small subset of genes distinguish this odontogenic carcinoma from oral mucosal epidermoid carcinomas.

Mutation of Cys105 Inhibits Dimerization of p12CDK2-AP1 and Its Growth Suppressor Effect

p12CDK2-AP1 (p12) is a CDK2-associated protein that negatively regulates its kinase activity. Growth arrest of normal diploid cells by contact inhibition resulted in an induction of p27kip1 and reduction of CDK2 levels. Interestingly, we observed concomitantly in growth-arrested cells, there was a reduction of nuclear p12 and the appearance of a nuclear 25-kDa molecule (p25) recognized by anti-p12 polyclonal antibody. Biochemical analysis showed that bacterial His-tagged p12 could be converted into a dimeric p25 in a reducing agent-dependent manner, and mutating the only cysteine residue of p12 (Cys105 → Ala105) abolished the dimerization. Transient transfection of wild type p12 into U2OS cells showed a reducing agent-sensitive dimerization that was also abolished by the C105A mutation. Furthermore, reduction of p12 expression by a short interfering RNA resulted in a parallel reduction of p25. These data supports the possibility that p25 is a homodimeric form of p12 through the cysteine residue. More interestingly, transient transfection of p12 (C105A) into the normal diploid lung fibroblast CCD18LU cells resulted in a reduction of the growth-inhibitory effect of p12 and abolished the inhibitory effect of p12 on CDK2 kinase activity. In addition, we found that the C105A mutation did not alter nuclear localization of p12, but it prevented association with CDK2. Taken together, our data suggest that p12 forms a nuclear homodimers in contact inhibited normal diploid cells and dimerization of p12 is a necessary process for the growth inhibition effect by p12.

Optimized conditions for gene transfection into the human eosinophilic cell line EoL-1 by electroporation

Eosinophils are emerging as an increasingly important cell in the immunoregulatory network of normal and pathological processes. No studies has yet described optimized experimental strategies to transfect DNA into human eosinophils. Using a frequently employed in vitro model of human eosinophil, the EoL-1 cells, we now described the optimal transfection of DNA into these cells by electroporation. Our results indicate that electroporation can efficiently and reproducibly transfect DNA into EoL-1 cells. Optimal electroporation conditions consist of the use of 1 X RPMI medium 1640 with 10% FBS, voltage setting at 275 V, 1150 microF capacitance, 40 mg of DNA and 4.0 X 10(7) cells/ml per electroporation in a total volume of 0.5 ml in 0.4 cm gap cuvettes. These conditions may be a useful protocol for transfecting eosinophil cell lines.

Dissecting oral cancer through large-scale gene expression profiling of laser capture microdissection samples

Dissecting oral cancer through large-scale gene expression profiling of laser capture microdissection samples

The Role of Digital 3D Scanned Models in Dental Students’ Self-Assessments in Preclinical Operative Dentistry

The aim of this study was to determine how dental student self-assessment and faculty assessment of operative preparations compared for conventional visual assessment versus assessment of scanned digital 3D models. In 2016, all third-year students in the Class of 2018 (N=35) at Harvard School of Dental Medicine performed preclinical exams of Class II amalgam preparations (C2AP) and Class III composite preparations (C3CP) and completed self-assessment forms; in 2017, all third-year students in the Class of 2019 (N=34) performed the same exams. Afterwards, the prepared typodont teeth were digitally scanned. Students self-assessed their preparations digitally, and four faculty members graded the preparations conventionally and digitally. The results showed that, overall, the students assessed their preparations higher than the faculty assessments. The mean student-faculty gaps for C2AP and C3CP in the conventional assessments were 11% and 5%, respectively. The mean digital student-faculty gap for C2AP and C3CP were 8% and 2%, respectively. In the conventional assessments, preclinical performance was negatively correlated with the student-faculty gap (r=-0.47, p<0.001). The correlations were not statistically significant with the digital assessments (p=0.39, p=0.26). Students in the bottom quartile significantly improved their self-assessment accuracy using digital self-assessments over conventional assessments (C2AP 10% vs. 17% and C3CP 3% vs. 10%, respectively). These results suggest that digital assessments offered a significant learning opportunity for students to critically self-assess themselves in operative preclinical dentistry. The lower performing students benefitted the most, improving their assessment ability to the level of the rest of the class.