P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.

BackgroundBacterial infections have been linked to malignancies due to their ability to induce chronic inflammation. We investigated the association of oral bacteria in oral squamous cell carcinoma (OSCC/tumor) tissues and compared with adjacent non-tumor mucosa sampled 5 cm distant from the same patient (n = 10). By using culture-independent 16S rRNA approaches, denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing, we assessed the total bacterial diversity in these clinical samples.ResultsDGGE fingerprints showed variations in the band intensity profiles within non-tumor and tumor tissues of the same patient and among the two groups. The clonal analysis indicated that from a total of 1200 sequences characterized, 80 bacterial species/phylotypes were detected representing six phyla, Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria and uncultivated TM7 in non-tumor and tumor libraries. In combined library, 12 classes, 16 order, 26 families and 40 genera were observed. Bacterial species, Streptococcus sp. oral taxon 058, Peptostreptococcus stomatis, Streptococcus salivarius, Streptococcus gordonii, Gemella haemolysans, Gemella morbillorum, Johnsonella ignava and Streptococcus parasanguinis I were highly associated with tumor site where as Granulicatella adiacens was prevalent at non-tumor site. Streptococcus intermedius was present in 70% of both non-tumor and tumor sites.ConclusionsThe underlying changes in the bacterial diversity in the oral mucosal tissues from non-tumor and tumor sites of OSCC subjects indicated a shift in bacterial colonization. These most prevalent or unique bacterial species/phylotypes present in tumor tissues may be associated with OSCC and needs to be further investigated with a larger sample size.

BackgroundThe present study is aimed at identifying potential candidate genes as prognostic markers in human oral tongue squamous cell carcinoma (SCC) by large scale gene expression profiling.MethodsThe gene expression profile of patients (n=37) with oral tongue SCC were analyzed using Affymetrix HG_U95Av2 high-density oligonucleotide arrays. Patients (n=20) from which there were available tumor and matched normal mucosa were grouped into stage (early vs. late) and nodal disease (node positive vs. node negative) subgroups and genes differentially expressed in tumor vs. normal and between the subgroups were identified. Three genes, GLUT3, HSAL2, and PACE4, were selected for their potential biological significance in a larger cohort of 49 patients via quantitative real-time RT-PCR.ResultsHierarchical clustering analyses failed to show significant segregation of patients. In patients (n=20) with available tumor and matched normal mucosa, 77 genes were found to be differentially expressed (P< 0.05) in the tongue tumor samples compared to their matched normal controls. Among the 45 over-expressed genes, MMP-1 encoding interstitial collagenase showed the highest level of increase (average: 34.18 folds). Using the criterion of two-fold or greater as overexpression, 30.6%, 24.5% and 26.5% of patients showed high levels of GLUT3, HSAL2 and PACE4, respectively. Univariate analyses demonstrated that GLUT3 over-expression correlated with depth of invasion (P<0.0001), tumor size (P=0.024), pathological stage (P=0.009) and recurrence (P=0.038). HSAL2 was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only GLUT3 showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). PACE4 mRNA expression failed to show correlation with any of the relevant parameters. ConclusionThe characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by real-time RT-PCR offers a powerful strategy for identification of novel targets for prognostication and treatment of oral tongue carcinoma.

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO 4 . This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.The three cyclin-dependent kinases, CDK7, CDK8, and CDK9, 1 have an established connection with transcription machinery and are regulated by constitutively expressed cyclins. These CDKs can hyperphosphorylate the CTD of the large subunit of RNA polymerase II (pol II) and therefore are CTD kinases (1). The CTD of mammalian pol II consists of 52 heptad repeats with the consensus sequence 1 YSPTSPS 7 , which is especially well-conserved in the first half (N-terminal) of the CTD. Of the phosphorylatable amino acid residues, serine 5 is the most conserved, with only one threonine replacement, indicating that it is likely to have a crucial role in the function of the CTD. Pol II CTD is highly phosphorylated in vivo, mostly at serine (positions 2 and 5) but also at threonine and tyrosine (2). Hyperphosphorylated CTD is correlated with transcription initiation and elongation, whereas the holoenzyme and preinitiation complexes contain pol II with hypophosphorylated CTD (3). In yeast cells, replacing either serine 2 or serine 5 with alanine in each of this organism's 26 CTD heptad repeats causes lethality (4).The functions of these kinases correlate with different stages of transcription. CDK7 is a subunit of the general transcription factor TFIIH and is the kinase that phosphorylates pol II CTD after the preinitiation complex is formed (5). CDK7 is the catalytic member of a three-subunit complex called CAK (CDKactivating kinase) composed of CDK7/cyclin H and MAT-1 (6, 7). CAK is also a subassembly of TFIIH (8). Comparison between the kinase activity of CAK and TFIIH revealed differences in substrate specificity: CAK shows a strong preference for CDK2, consistent with its function in the cell cycle, but cannot hyperphosphorylate pol II; TFIIH hyperphosphorylates pol II, but CDK2 i...

Chromosomal amplification at 3q is common to multiple human cancers, but has a specific predilection for squamous cell carcinomas (SCC) of mucosal origin. We identified and characterized a novel oncogene, SCC-related oncogene (SCCRO), which is amplified along the 3q26.3 region in human SCC. Amplification and overexpression of SCCRO in these tumors correlate with poor clinical outcome. The importance of SCCRO amplification in malignant transformation is established by the apoptotic response to short hairpin RNA against SCCRO, exclusively in cancer cell lines carrying SCCRO amplification. The oncogenic potential of SCCRO is underscored by its ability to transform fibroblasts (NIH-3T3 cells) in vitro and in vivo. We show that SCCRO regulates Gli1-a key regulator of the hedgehog (HH) pathway. Collectively, these data suggest that SCCRO is a novel component of the HH signaling pathway involved in the malignant transformation of squamous cell lineage.