Randy Todd

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.

The Molecular Biology of Oral Carcinogenesis: Toward a Tumor Progression Model

PURPOSE An understanding of the molecular basis of oral carcinogenesis will alter our clinical approach to oral cancer. The nomenclature and major themes of molecular oral tumor biology are reviewed, beginning with the regulation events governing normal cellular physiology. In carcinogenesis, chromosomal or cytogenetic alterations lead to deregulation of tightly controlled stimulatory and inhibitory pathways, growth-promoting proto-oncogenes are mutated into overactive oncogenes, and growth-suppressing or tumor suppressor genes are inactivated. Recent advances in defining these fundamental mechanisms of tumor biology may allow prevention, diagnosis, and treatment of oral cancer to be approached at the molecular level.

Deleted in oral cancer-1 (doc-1), a novel oral tumor suppressor gene.

We have identified, isolated, and par‐tially characterized doc‐1, a novel cDNA sequence whose activity is consistent with a suppressor of hamster oral carcinogenesis. Doc‐1 is an evolutionarily conserved gene exhibiting loss of heterozygosity and marked reduction in expression in malignant hamster oral keratinocytes. The full‐length doc‐1 cDNA encodes an 87 amino acid product that shows a significant homology to one of the seven novel genes induced in mouse fibroblasts by TNF‐α. Transfection of the full‐length doc‐1 cDNA into malignant hamster oral keratinocytes alters the behavior of the recipients in terms of morphology, growth rate, and anchorage‐independent growth, suggesting reversion of transformation phenotypes. We propose that doc‐1 is a novel tumor suppressor gene in oral cancer development.—Todd, R., McBride, J., Tsuji, T., Donoff, R. B., Nagai, M., Chou, M. Y., Chiang, T., Wong, D. T. W. Deleted in oral cancer‐1 (doc‐1), a novel oral tumor suppressor gene. FASEB J. 9, 1362‐1370(1995)

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.

Molecular Biology of Human Oral Cancer

The application of molecular biological tools to the study of cancer has significantly advanced the field of human cancer research. Such study has demonstrated the involvement of two classes of highly conserved cellular genes in the malignant transformation process: oncogenes and tumor suppressor genes. Despite these advances in the molecular biology of human cancers, our understanding of human oral cancer lags behind that of cancer of other body sites. This review attempts to assess the current status of the molecular biology of human oral cancer.

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)

Simultaneous maxillary and mandibular distraction osteogenesis with a semiburied device

Distraction osteogenesis is a technique utilizing natural healing mechanisms to generate new bone; it is commonly used to lengthen the hypoplastic mandible. Distraction of the maxilla and mandible as a unit is an obvious extension of the technique. We describe the application of a semiburied distractor to simultaneously lengthen the mandible and maxilla and level a canted occlusal plane in three cases. The indications for bimaxillary distraction are reviewed, including its advantages, disadvantages and limitations.

Apoptosis, proliferation and p12doc-1 profiles in normal, dysplastic and malignant squamous epithelium of the Syrian hamster cheek pouch model

Disruption of the homeostatic balance between proliferation and apoptosis is widely believed to contribute to human oral carcinogenesis. Using the Syrian hamster oral cancer model, we examined normal, hyperplastic, dysplastic and malignant oral epithelium for the fraction of apoptotic, proliferating and p12(doc-1) expressing keratinocytes using the TUNEL assay, as well as PCNA and p12(doc-1) immunostaining, respectively. The percentage of TUNEL positive cells progressively increased from normal to dysplastic epithelium (P<0.0019), but returned to normal keratinocyte levels in the malignant epithelium (P<0.20). However, PCNA positive cells increased progressively through hamster oral malignant progression (P<0.0012). The overall ratio of apoptotic to proliferating keratinocytes remains similar until the transition between dysplastic and malignant epithelium, where the ratio is markedly reduced (P<0.05). p12(doc-1) labeling demonstrated a similar expression pattern (P<0.008). This study demonstrates that apoptosis, proliferation and the expression of p12(doc-1) reflects alterations reported during human oral carcinogenesis and supports the use of the Syrian hamster model for the further examination of these pathways.

Induction of epithelial differentiation and DNA demethylation in hamster malignant oral keratinocyte by ornithine decarboxylase antizyme.

The hamster ornithine decarboxylase antizyme (ODC-Az) cDNA was transfected into the hamster malignant oral keratinocyte cell line, HCPC-1. Ectopic expression of ODC-Az resulted in the reversion of malignant phenotypes and alteration of DNA methylation status of CCGG sites. The phenotypes examined include ODC enzymatic activity, doubling time, morphological change, anchorage dependent growth, tumorigenicity in nude mice, induction of epithelial differentiation marker protein (involucrin), and change of cell cycle position. Comparison of CCGG DNA methylation status of the ODC-Az and control vector transfectants revealed a significant increase in demethylation of 5-methyl cytosines (m5C) of CCGG sites in the ODC-Az transfectants. Ectopic expression of ODC-Az gene in hamster malignant oral keratinocytes led to reduce ODC activity and the subsequent demethylation of 5-methyl cytosines, presumably via the ODC/ polyamines/ decarboxylated S-adenosylmethionine (dc-AdoMet) pathways. Our data suggest that ODC-Az shared the same pathway of polyamines/ dc-AdoMet/DNA methyltransferase (DNA MTase). We propose that ODC-Az mediates a novel mechanism in tumor suppression by DNA demethylation and presumably re-activation of key cellular genes silenced by DNA hypermethylation during cancer development.

Eosinophil-derived transforming growth factors (TGF-α and TGF-β1) in human periradicular lesions

Inflammatory mediators of periradicular lesions are poorly understood. Transforming growth factors-α and -β 1 (TGF-α and TGF-β 1 ) have been linked with the cellular processes for both soft and hard tissue wound healing. The purpose of this study is to demonstrate the cellular sources of TGF-α and TGF-β 1 mRNA and protein in periapical lesions by in situ hybridization and immunohistochemistry. Nine periapical granulomas and nine periapical cysts were examined. TGF-α mRNA and protein were not detectable in the granulomas examined. However, eosinophils surrounding the periapical cysts demonstrated both TGF-α mRNA and protein. The vast majority of eosinophils present in the periapical granulomas and cysts also demonstrated TGF-β 1 mRNA and protein. Other cells producing TGF-β 1 were lymphocytes, fibroblasts, and monocytes. The presence of wound repair cytokines, such as TGF-α and TGF-β 1 , suggests a mechanism by which the host inflammatory response may participate in the repair and remodeling of periapical tissues.