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.

Covalent modification of cullins by the ubiquitin-like protein NEDD8 (neddylation) regulates protein ubiquitination by promoting the assembly of cullin-RING ligase E3 complexes. Like ubiquitination, neddylation results from an enzymatic cascade involving the sequential activity of a dedicated E1 (APPBP1/ Uba3), E2 (Ubc12), and an ill-defined E3. We show that SCCRO (also known as DCUN1D1) binds to the components of the neddylation pathway (Cullin-ROC1, Ubc12, and CAND1) and augments but is not required for cullin neddylation in reactions using purified recombinant proteins. We also show that SCCRO recruits Ubc12ϳNEDD8 to the CAND1-Cul1-ROC1 complex but that this is not sufficient to dissociate or overcome the inhibitory effects of CAND1 on cullin neddylation in purified protein assays. In contrast to findings in cellular systems where no binding is seen, we show that SCCRO and CAND1 can bind to the neddylated Cul1-ROC1 complex in assays using purified recombinant proteins. Although neddylated (not unneddylated) Cul1-ROC1 is released from CAND1 upon incubation with testis lysate from SCCRO ؉/؉ mice, the addition of recombinant SCCRO is required to achieve the same results in lysate from SCCRO ؊/؊ mice. Combined, these results suggest that SCCRO is an important component of the neddylation E3 complex that functions to recruit charged E2 and is involved in the release of inhibitory effects of CAND1 on cullin-RING ligase E3 complex assembly and activity.Post-translational modification of proteins by ubiquitin (Ub) 4 regulates diverse cellular functions including protein turnover, differentiation, apoptosis, cell cycle, and transcription (1-5). Given its essential role, ubiquitination is a highly regulated process that involves the sequential action of three enzymes termed as E1, E2, and E3. In this enzymatic cascade, E1 initiates the process by forming a high energy thioester bond with Ub in an ATP-coupled reaction. The Ub is then transferred to E2 as a thioester intermediate. Finally, E3s serve as the targeting arm in the ubiquitination process, mediating the transfer of Ub from E2 to the target protein to create an isopeptide bond between the C-terminal glycine in Ub and a lysine residue on the substrate protein. Once attached, the Ub itself can be modified to generate polyubiquitin chains on the target protein (6). The functional effects of ubiquitination are influenced by the chain length and the residue on the Ub to which the chain is attached. Polyubiquitination promotes translocation to the 26 S proteasome for degradation. Other functional effects of mono-and polyubiquitination include protein translocation, interaction, and activation.Although there is only one known E1 (except in plants) and relatively few E2s, E3s exist in multiple forms to allow for specific protein targeting (6). In general, E3s are modular multiprotein complexes that can be divided into two broad categories based on the presence of either a HECT (homologous to E6-AP C terminus) or RING (Really Interesting New Gene)-finger domain-containi...

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO ؊/؊ mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO ؊/؊ mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.The cullin family of proteins anchor Cullin RING finger-type E3 ubiquitination complexes (CRL) 4 that regulate the degradation and activity of proteins involved in a wide range of cellular processes (1, 2). Several reports have shown that the activity of CRL complexes is primarily regulated by neddylation, a process mechanistically analogous to ubiquitination, where the cullin family of proteins are covalently modified by ubiquitin (Ub)-like protein Nedd8 (3-11). All cullin proteins in humans (Cul1, Cul2, Cul3, Cul4a, Cul4b, Cul5, and PARC) are subject to neddylation, with the exception of Cul7 (12, 13). Lethality resulting from knocking out core components in all organisms studied (except budding yeast) emphasizes the indispensable role of neddylation in normal cellular function (14 -20).Like ubiquitination, neddylation involves a sequential, tripartite enzymatic cascade (21, 22). The vertebrate enzymes for neddylation are the heterodimeric complex APP-BP1/Uba3 (E1) and Ubc12 or Ube2f (E2) (21,(23)(24)(25). The presence of enzymatic activity in the RING domain of Roc1 combined with its requirement and sufficiency to promote Nedd8 conjugation in vitro supports a role for Roc1 as the E3 for neddylation (26). Recent work identified a novel protein SCCRO/DCUN1D1/ DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1, domain containing 1/defective in cullin neddylation) that binds to components of the E3 complex for neddylation (Ubc12 and Cullin-Roc1) and increases neddylation efficiency in vitro (27)(28)(29)(30). Biochemical studies and structural modeling suggest that DCN1 functions as an E3 promoting neddylation by reducing nonspecific Rub1 (Nedd8 in mammals) discharge and directing...

Originally identified as an oncogene activated by amplification in squamous cell carcinomas, several lines of evidence now suggest that squamous cell carcinoma-related oncogene (SCCRO; aka DCUN1D1) may play a role in the pathogenesis of a wide range of human cancers including gliomas. SCCRO's oncogenic function is substantiated by its ectopic expression, resulting in transformation of cells in culture and xenograft formation in nude mice. The aim of this study was to assess the in vivo oncogenicity of SCCRO in a murine model. Ubiquitous expression of SCCRO resulted in early embryonic lethality. Because SCCRO overexpression was detected in human gliomas, its in vivo oncogenic activity was assessed in an established murine glioma model. Conditional expression of SCCRO using a replication-competent ASLV long terminal repeat with splice acceptor/nestin-(tumor virus-A) tv-a model system was not sufficient to induce tumor formation in a wild-type genetic background, but tumors formed with increasing frequency and decreasing latency in facilitated background containing Ink4a deletion alone or in combination with PTEN loss. Ectopic expression of SCCRO in glial progenitor cells resulted in lower-grade gliomas in Ink4a(-/-) mice, whereas its expression in Ink4a(-/-)/PTEN(-/-) background produced high-grade glioblastoma-like lesions that were indistinguishable from human tumors. Expression of SCCRO with platelet-derived growth factor-beta (PDGF-beta) resulted in an increased proportion of mice forming glioblastoma-like tumors compared with those induced by PDGF-beta alone. This work substantiates SCCRO's function as an oncogene by showing its ability to facilitate malignant transformation and carcinogenic progression in vivo and supports a role for SCCRO in the pathogenesis of gliomas and other human cancers.