Brd4 is a mammalian bromodomain protein that binds to acetylated chromatin. Proteomic analysis revealed that Brd4 interacts with cyclinT1 and Cdk9 that constitutes core positive transcription elongation factor b (P-TEFb). Brd4 interacted with P-TEFb in the living nucleus through its bromodomain. About half of P-TEFb was bound to the inhibitory subunit and functionally inactive. Brd4 interacted with P-TEFb that was free of the inhibitory subunit. An increase in Brd4 expression led to increased P-TEFb-dependent phosphorylation of RNA polymerase II (RNAPII) CTD and stimulation of transcription from promoters in vivo. Conversely, a reduction in Brd4 expression by siRNA reduced CTD phosphorylation and transcription, revealing that Brd4 is a positive regulatory component of P-TEFb. In chromatin immunoprecipitation (ChIP) assays, the recruitment of P-TEFb to a promoter was dependent on Brd4 and was enhanced by an increase in chromatin acetylation. Together, P-TEFb alternately interacts with Brd4 and the inhibitory subunit to maintain functional equilibrium in the cell.

Interleukin (IL)-21 is the most recently recognized of the cytokines that share the common cytokine receptor γ chain (γc), which is mutated in humans with X-linked severe combined immunodeficiency. We now report that IL-21 synergistically acts with IL-15 to potently promote the proliferation of both memory (CD44high) and naive (CD44low) phenotype CD8+ T cells and augment interferon-γ production in vitro. IL-21 also cooperated, albeit more weakly, with IL-7, but not with IL-2. Correspondingly, the expansion and cytotoxicity of CD8+ T cells were impaired in IL-21R−/− mice. Moreover, in vivo administration of IL-21 in combination with IL-15 boosted antigen-specific CD8+ T cell numbers and resulted in a cooperative effect on tumor regression, with apparent cures of large, established B16 melanomas. Thus, our studies reveal that IL-21 potently regulates CD8+ T cell expansion and effector function, primarily in a synergistic context with IL-15.

Basic fibroblast growth factor (bFGF) and human immunodeficiency virus type 1 (HIV-1) Tat protein synergize in inducing angiogenic Kaposi's sarcoma-like lesions in mice. Synergy is due to Tat, which enhances endothelial cell growth and type-IV collagenase expression in response to bFGF mimicking extracellular matrix proteins. The bFGF, extracellular Tat and Tat receptors are present in HIV-1-associated KS, which may explain the higher frequency and aggressiveness of this form compared to classical Kaposi's sarcoma where only bFGF is present.

Despite the existence of morphologically indistinguishable disease, patients with advanced ovarian tumors display a broad range of survival end points. We hypothesize that gene expression profiling can identify a prognostic signature accounting for these distinct clinical outcomes. To resolve survival-associated loci, gene expression profiling was completed for an extensive set of 185 (90 optimal/95 suboptimal) primary ovarian tumors using the Affymetrix human U133A microarray. Cox regression analysis identified probe sets associated with survival in optimally and suboptimally debulked tumor sets at a P value of <0.01. Leave-one-out cross-validation was applied to each tumor cohort and confirmed by a permutation test. External validation was conducted by applying the gene signature to a publicly available array database of expression profiles of advanced stage suboptimally debulked tumors. The prognostic signature successfully classified the tumors according to survival for suboptimally (P = 0.0179) but not optimally debulked (P = 0.144) patients. The suboptimal gene signature was validated using the independent set of tumors (odds ratio, 8.75; P = 0.0146).

p53 exerts its cell cycle regulatory effects through its ability to function as a sequence-specific DNA binding transcription factor. CREB-binding protein (CBP)/p300, through its interaction with the N terminus of p53, acts as a coactivator for p53 and increases the sequencespecific DNA-binding activity of p53 by acetylating its C terminus. The same N-terminal domain of p53 has recently been shown to be phosphorylated at Ser 15 in response to ␥-irradiation. Remarkably, we now demonstrate that phosphorylation of p53 at Ser 15 increases its ability to recruit CBP/p300. The increase in CBP/p300 binding was followed by an increase in the overall level of acetylation of the C terminus of p53. These results provide a mechanism for the activation of p53-regulated genes following DNA damage, through a signaling pathway linking p53 N-terminal kinase and C-terminal acetyltransferase activities.

Hepatic injury induced by various toxic agents, including acetaminophen (APAP), has been attributed, in part, to the production of proinflammatory cytokines and other mediators by resident Kupffer cells within the liver. However, recent evidence from our laboratory has demonstrated that hepato-protective factors, such as interleukin (IL)-10 and cyclooxygenase-derived mediators, are also upregulated in response to hepatic damage to help protect against exacerbated injury, and Kupffer cells have been suggested to be a source of these modulatory factors. In other models, Kupffer cells also serve important regulatory functions in pathophysiological states of the liver. Therefore, we reevaluated the role of Kupffer cells in a murine model of APAP-induced liver injury using liposome-entrapped clodronate (liposome/clodronate) as an effective Kupffer cell-depleting agent. We show that in contrast to pretreatment of mice with a widely used macrophage inhibitor, gadolinium chloride, which did not deplete Kupffer cells but moderately protected against APAP-induced hepatotoxicity as reported previously, the intravenous injection of liposome/clodronate caused nearly complete elimination of Kupffer cells and significantly increased susceptibility to APAP-induced liver injury as compared with mice pretreated with empty liposomes. This increased susceptibility was apparently unrelated to the metabolism of APAP since liposome/clodronate pretreatment did not alter APAP-protein adduct levels. Instead, Kupffer cell depletion by liposome/clodronate led to significant decreases in the levels of hepatic mRNA expression of several hepato-regulatory cytokines and mediators, including IL-6, IL-10, IL-18 binding protein and complement 1q, suggesting that Kupffer cells are a significant source for production of these mediators in this model. Our findings indicate that, in addition to their protoxicant activities, Kupffer cells can also have an important protective function in the liver through the production of a variety of modulatory factors which may counteract inflammatory responses and/or stimulate liver regeneration.

Papillary serous low malignant potential (LMP) tumors are characterized by malignant features and metastatic potential yet display a benign clinical course. The role of LMP tumors in the development of invasive epithelial cancer of the ovary is not clearly defined. The aim of this study is to determine the relationships among LMP tumors and invasive ovarian cancers and identify genes contributing to their phenotypes. Affymetrix U133 Plus 2.0 microarrays (Santa Clara, CA) were used to interrogate 80 microdissected serous LMP tumors and invasive ovarian malignancies along with 10 ovarian surface epithelium (OSE) brushings. Gene expression profiles for each tumor class were used to complete unsupervised hierarchical clustering analyses and identify differentially expressed genes contributing to these associations. Unsupervised hierarchical clustering analysis revealed a distinct separation between clusters containing borderline and high-grade lesions. The majority of low-grade tumors clustered with LMP tumors. Comparing OSE with high-grade and LMP expression profiles revealed enhanced expression of genes linked to cell proliferation, chromosomal instability, and epigenetic silencing in high-grade cancers, whereas LMP tumors displayed activated p53 signaling. The expression profiles of LMP, low-grade, and high-grade papillary serous ovarian carcinomas suggest that LMP tumors are distinct from high-grade cancers; however, they are remarkably similar to low-grade cancers. Prominent expression of p53 pathway members may play an important role in the LMP tumor phenotype. (Cancer Res 2005; 65(22): 10602-12)

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of carboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay, we have analyzed the effect of Tat on kinase activity during the initiation and elongation phases of HIV-1 transcription. Our results demonstrate that cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 preinitiation complex. Hyperphosphorylation of the RNA polymerase II (RNAP II) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5. Analysis of preinitiation complexes formed in immunodepleted extracts suggests that CDK9 phosphorylates serine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the substrate specificity of CDK9 is altered, such that the kinase phosphorylates both serine 2 and serine 5. Tat-induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole, an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is released from the transcription complex between positions ؉14 and ؉36, prior to the synthesis of transactivation response (TAR) RNA. In contrast, CDK9 stays associated with the complex through ؉79. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the preinitiation complex and the other between ؉36 and ؉79. The second phase of CTD phosphorylation is Tatdependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex.Human immunodeficiency virus type 1 (HIV-1) encodes a transactivator protein, Tat, which stimulates transcription elongation through interaction with the transactivation response (TAR) RNA element located at the 5Ј end of nascent transcripts (12,28,68,75). In view of the observations that hyperphosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II) correlates with the formation of processive elongation complexes (11) and that Tat transactivation requires the CTD (6,44,46,79), it has been proposed that a critical step in Tat transactivation is mediated through a cellular kinase(s) (68, 80). Two cyclin-dependent kinase (CDK)-cyclin pairs, present in two distinct transcription factor complexes, have been implicated as Tat cofactors which could phosphorylate the CTD (54, 68). TFIIH, a general transcription factor which contains nine polypeptides (ERCC3/XPB, ERCC2/XPD, p62, p54, p44, CDK7 [MO15], cyclin H, MAT1, and p34) (13, 24), possesses CTD kinase activity (14, 37). The kinase activity of TFIIH resides in the CDK7 subunit (15,58,61,62). In association with cyclin H and Mat1, CDK7 forms the CDK-activating kinase (CAK) complex that phosphorylates CDKs involved in the regulation of the cell cycle (19,42,43,53,67). The assoc...