Large scale gene expression pro®ling 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, di erentiation markers, and genes that have not been implicated in oral cancer. The database created provides a veri®able global pro®le 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. Oncogene (2001) 20, 6196 ± 6204.

Current advances in biomolecular technology allow precise genetic fingerprinting of specific cells responsible for the pathogenesis of human diseases. This study demonstrates the feasibility of generating target samples from laser capture microdissection (LCM) tissues suitable for hybridization of high-density oligonucleotide arrays for gene expression profiling. RNA was successfully isolated by LCM from three paired specimens of oral cancer and linearly amplified using T7 RNA polymerase. Evaluation of the cDNA revealed that five of five cellular maintenance transcripts are detected. Biotinylated cRNA was generated and hybridized to the human Test 1 GeneChip probe arrays, which demonstrated that the RNA is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the samples to the HuGenFL GeneChip probe arrays revealed that 26.5%-33.0% of the approximately 7000 represented genes are expressed in each of the six samples. These results demonstrate that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.

IntroductionMechanical force is an important biological regulator of cellular functions, affecting cellular morphology and adhesiveness, especially in smooth-muscle cells (SM cells), which surround contractile organs and are always exposed to mechanical stress. Transmembrane receptors of integrins consist of α and β subunits, and are considered to play a key role in SM-cell mechanotransduction (Schmidt et al., 1998;Shyy and Chien, 1997;Wang et al., 1993;Wang and Ingber, 1994). The α subunit of integrins mainly determines binding specificity for the extracellular matrix (ECM), whereas the β subunit initiates intracellular signaling. In cultured cells, the attachment of integrins to the ECM is known to induce the aggregation of integrins to form focal adhesions, which contain actin fibers as well as cytoskeleton-associated proteins such as FAK, vinculin, paxillin and talin (Turner, 2000a;Turner, 2000b). The sites presumably transmit force between the intracellular contractile apparatus and the ECM, and the focaladhesion proteins are good candidates for the mediators of mechanical signals to the cytoskeleton. In contrast to cultured cells, the molecular machinery that mediates the attachment of integrins to the ECM in vivo is poorly understood, whereas, in SM cells, transmembrane-associated dense plaques are structurally related to the focal adhesions of cultured cells (Burridge and Chrzanowska-Wodnicka, 1996).

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...