Abstract.The structure and function of chromatin can be altered by modifications to histone. Histone acetylation is a reversible process governed by histone acetyltransferases and histone deacetylases (HDACs). HDAC6 is a subtype of the HDAC family that deacetylates ·-tubulin and increases cell motility. We investigated the expression levels of HDAC6 mRNA and protein expression in oral squamous cell carcinoma (OSCC)-derived cell lines and human primary OSCCs to elucidate the potential involvement of HDAC6 in OSCC. Using quantitative real-time reverse transcription polymerase chain reaction and Western blots on nine OSCC-derived cell lines and normal oral keratinocytes (NOKs), HDAC6 mRNA and protein expression were commonly up-regulated in all cell lines compared with the NOKs. Immunofluorescence analysis detected HDAC6 protein in the cytoplasm of OSCC cell lines. Similar to OSCC cell lines, high frequencies of HDAC6 up-regulation were evident in both mRNA (74%) and protein (51%) levels of primary tumors. Among the clinical variables analyzed, the clinical tumor stage was found to be associated with the HDAC6 expression states. The analysis indicated a significant difference in the HDAC6 expression level between the early stage (stage I and II) and advancedstage (stage III and IV) tumors (P=0.014). These results suggest that HDAC6 expression may be correlated with tumor aggressiveness and offer clues to the planning of new treatments.

Collagens represent a large family of structurally related extracellular matrix proteins containing unique triple helical structure. One of the characteristics of this structural protein is its extensive post-translational modifications that have major effects on molecular assembly, stability, and metabolism. Hydroxylation of specific lysine residues is one of such unique modifications found in collagen, and the pattern/extent of this modification influences fibrillogenesis, cross-linking, and matrix mineralization. The formation of covalent intermolecular cross-linking is the final modification in collagen biosynthesis and is critical for the stability of collagen. The process of cross-linking is dynamic and the pathways vary depending on the tissues and tissue's physiological state. This tissue specificity of cross-linking pattern may in part be the results of differential expression of various isoforms of lysyl hydroxylases and lysyl oxidases that have been recently identified and partially characterized. This chapter concentrates on recent research progress on these two modifications and the methods for analysis we have developed.

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-S receptor (IL-5R) consists of two distinct membrane proteins, a and P. The a chain (IL-5Ra) is specific to IL-5.The chain is the common ,1 chain (Ic) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both a and I1 chains are essential for signal transduction. In this study, we generated cDNAs of IL-5Ra having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membraneproximal proline-rich sequence of the cytoplasmic domain of IL-5Ra, which is conserved among the a chains of IL-SR, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5Ra and the cytoplasmic domain of Dc suggested that dimerization of the cytoplasmic domain of ,c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.Interleukin-5 (IL-5) is a cytokine that regulates the production and function of B cells, eosinophils, and basophils (40). The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, ao and P. IL-SRa alone specifically binds IL-5 but with low affinity (27,39). While the P chain does not bind IL-5 by itself, it does form a high-affinity IL-SR in combination with IL-5Rox (6, 37, 38,41). The P chain is the common P3 chain (,c) of receptors for IL-3 and granulocyte-macrophage colonystimulating factor (GM-CSF) (23). 1c forms high-affinity receptors for IL-3 and GM-CSF, the ligands of which bind specifically with IL-3Ra and the a chain of GM-CSF receptor (GM-CSFRa), respectively (7,9,10,16,29). Pc is not only required for high-affinity binding but also essential for signal transduction. IL-5, IL-3, and GM-CSF have several overlapping functions, especially in eosinophils (23,33,40). The ,c shared by the receptors of these three cytokines provides a molecular basis for the functional redundancy of these cytokines.The extracellular domains of the a chains and ,c are similar among members of the cytokine receptor superfamily. The a. chains have a short cytoplasmic domain (-55 amino acid residues) with a short amino acid sequence which is conserved among these a chains (16,39). In contrast, ,3c has a relatively large cytoplasmic domain (-440 amino acid residues). Although the cytoplasmic domains of the a chains and ,Bc have no homology with signaling molecules such as kinases, phosphatases, nucleotide-binding proteins, and src homology domains, it has been well established that IL-5, IL-3, and GM-CSF induce rapid tyrosine phosphorylation of cellular proteins (13,24,28) as well as transcription of nuclear proto-oncogenes (3). ...

Mice with a heterozygous deletion of the Atp2a2 gene (Atp2a2 ؉/-) encoding SERCA2 spontaneously develop SCCs of the skin and upper digestive tract, including the oral cavity. To elucidate the contribution of ATP2A2 to human oral carcinogenesis, we analyzed genetic and epigenetic changes as well as mRNA and protein expression in primary OSCCs and OPLs. With the exception of one OSCC-derived cell line showing a 12 bp deletion of ATP2A2, we found no mutations in the coding sequence of the gene in primary OSCCs (n ‫؍‬ 52), OPLs (n ‫؍‬ 32) and cell lines (n ‫؍‬ 8). In immunohistochemistry, however, high frequencies of ATP2A2 downregulation were evident not only in primary OSCCs (42%, 42/100) but also in OPLs (31%, 10/32). Real-time quantitative RT-PCR data were consistent with the protein expression status. Aberrant DNA methylation within ATP2A2 also was detected in 9 of 30 ATP2A2-downregulated OSCCs. Moreover, restoration or elevated expression of the ATP2A2 protein was induced in most of the cell lines showing ATP2A2 methylation after treatment with 5-aza-2 -dC, a DNA demethylating agent. These results suggest that inactivation of the ATP2A2 gene is a frequent and early event during oral carcinogenesis and that loss of expression may be regulated partly by an epigenetic mechanism.

This study was designed to identify specific gene expression changes in tongue squamous cell carcinomas (TSCCs) compared with normal tissues using in-house cDNA microarray that comprised of 2304 full-length cDNAs from a cDNA library prepared from normal oral tissues, primary oral cancers, and oral cancer cell lines. The genes identified by our microarray system were further analysed at the mRNA or protein expression level in a series of clinical samples by real-time quantitative reverse transcriptasepolymerase chain reaction (qRT -PCR) analysis and imuunohositochemistry. The microarray analysis identified a total of 16 genes that were significantly upregulated in common among four TSCC specimens. Consistent with the results of the microarray, increased mRNA levels of selected genes with known molecular functions were found in the four TSCCs. Among genes identified, Rab1a, a member of the Ras oncogene family, was further analysed for its protein expression in 54 TSCCs and 13 premalignant lesions. We found a high prevalence of Rab1A-overexpression not only in TSCCs (98%) but also in premalignant lesions (93%). Thus, our results suggest that rapid characterisation of the target gene(s) for TSCCs can be accomplished using our in-house cDNA microarray analysis combined with the qRT -PCR and immunohistochemistry, and that the Rab1A is a potential biomarker of tongue carcinogenesis.