Cyclin T1 Expression Is Regulated by Multiple Signaling Pathways and Mechanisms during Activation of Human Peripheral Blood Lymphocytes

Marshall, Renée M.; Salerno, Dominic; Garriga, Judit; Graña, Xavier

doi:10.4049/jimmunol.175.10.6402

Cited by 49 publications

(57 citation statements)

References 64 publications

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“…As the expression of cyclin T1 and CDK9 is low in unstimulated PBLs and increases following mitogenic stimulation, we have suggested that the activity of these complexes might be important for the increased transcriptional needs associated with the process of T cell activation (Marshall et al, 2005). In contrast, in transformed T cells and other cell lines, cyclin T1 levels are comparable to those seen in activated PBLs.…”

Section: Fvp Inhibits Mitogen-induced T Cell Activation In Primary Humentioning

confidence: 84%

“…pCPP-HA-CDK9 and pCPP-HA-dnCDK9 vectors were generated by subcloning EcoRI digested cDNAs into the EcoRI site of the pCPP vector. Lentiviral particles were generated by transient transfection in 293T cells by CaCl 2 method as described in (Marshall et al, 2005) with some significant modifications. Briefly, 15 μg of the transfer vector (directing expression of the gene of interest), 10 μg of pCMVΔ8.2 (packaging construct) and 10 μg of the pCMV-VSV-G (envelope) vector (Sena-Esteves et al, 2004) were cotransfected into 293T cells.…”

Section: Lentiviral Constructs Recombinant Viral Production and Cellmentioning

confidence: 99%

“…Hence, the importance of performing these experiments using PBLs. Thus, to begin examining the effects of inhibiting CDK9 activity during T cell activation we stimulated human PBLs purified from healthy donors with PMA (Phorbol 12-Myristate, 13-Acetate) and PHA (Phytohemagglutinin) as previously described (Garriga et al, 1998;Marshall et al, 2005) in the presence of the indicated concentrations of FVP and measured markers of T cell activation by flow cytometric and Western blot analyses. To allow comparison with transduced PBLs (see below), PBLs used in this experiment were transduced with control lentiviruses expressing EGFP 12 hrs prior to PMA/PHA stimulation.…”

Section: Fvp Inhibits Mitogen-induced T Cell Activation In Primary Humentioning

confidence: 99%

“…Upregulation of cyclin T1 in PBLs correlates with phosphorylation of RNAPII and HIV-1 replication (Garriga et al, 1998) and is coordinated with the expression of other transcriptional regulators (Marshall et al, 2005). T cell activation involves a complex reprogramming of gene expression that leads to the expression of genes required for T cell effector functions, as well as cell cycle entry and proliferation (Huang and Wange, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Direct inhibition of CDK9 blocks HIV-1 replication without preventing T-cell activation in primary human peripheral blood lymphocytes

Salerno

Hasham

Marshall

et al. 2007

Gene

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HIV-1 transcription is essential for the virus replication cycle. HIV-1 Tat is a viral transactivator that strongly stimulates the processivity of RNA polymerase II (RNAPII) via recruitment of the cyclin T1/CDK9 positive transcription elongation factor, which phosphorylates the C-terminal domain (CTD) of RNAPII. Consistently, HIV-1 replication in transformed cells is very sensitive to direct CDK9 inhibition. Thus, CDK9 could be a potential target for anti-HIV-1 therapy. A clearer understanding of the requirements for CDK9 activity in primary human T cells is needed to assess whether the CDK9-dependent step in HIV-1 transcription can be targeted clinically. We have investigated the effects of limiting CDK9 activity with recombinant lentiviruses expressing a dominant negative form of CDK9 (HA-dnCDK9) in Peripheral Blood Lymphocytes (PBLs) and other cells. Our results show that direct inhibition of CDK9 potently inhibits HIV-1 replication in single-round infection assays with little to undetectable effects on RNAPII transcription, RNA synthesis, proliferation and viability. In PBLs purified from multiple donors, direct inhibition of CDK9 activity blocks HIV-1 replication/transcription but does not prevent T cell activation, as determined via measurement of cell surface and cell cycle entry and progression markers, and DNA synthesis. We have also compared the effects of HA-dnCDK9 to flavopiridol (FVP), a general CDK inhibitor that potently inhibits CDK9. In contrast to HA-dnCDK9, FVP interferes with key cellular processes at concentrations that inhibit HIV-1 replication with potency similar to HA-dnCDK9. In particular, FVP inhibits several T cell activation markers and DNA synthesis in primary PBLs at the minimal concentrations required to inhibit HIV-1 replication. Our results imply that small pharmacological compounds targeting CDK9 with enhanced selectivity could be developed into effective anti-HIV-1 therapeutic drugs.

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Section: Fvp Inhibits Mitogen-induced T Cell Activation In Primary Humentioning

confidence: 84%

Section: Lentiviral Constructs Recombinant Viral Production and Cellmentioning

confidence: 99%

Section: Fvp Inhibits Mitogen-induced T Cell Activation In Primary Humentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Direct inhibition of CDK9 blocks HIV-1 replication without preventing T-cell activation in primary human peripheral blood lymphocytes

Salerno

Hasham

Marshall

et al. 2007

Gene

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“…Hexim1 not only inhibits MHC class II transcription but also is important for the differentiation of neural and hematopoietic cells (24)(25)(26). Because HMBA induces Hexim1 (20,27), it also differentiates a variety of cells (28,29).…”

Section: Discussionmentioning

confidence: 99%

Hexim1 sequesters positive transcription elongation factor b from the class II transactivator on MHC class II promoters

Kohoutek

Blažek

Peterlin

2006

Proc. Natl. Acad. Sci. U.S.A.

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The class II transactivator (CIITA) is the master integrator of expression of MHC class II genes. It interacts with variety of basal transcription factors to initiate and elongate transcription of these genes. Among others, it recruits positive transcription elongation factor b (P-TEFb) to MHC class II promoters. In cells, P-TEFb is found in small active or large inactive complexes. The large complex is composed of P-TEFb, 7SK small nuclear RNA, and hexamethylene bisacetamide-inducible protein 1 (Hexim1). The present study identifies Hexim1 as a potent inhibitor of CIITA-mediated transcription. Not only the exogenously expressed but also IFN-␥-induced CIITA was inhibited by Hexim1. This inhibition did not result from an association between Hexim1 and CIITA but depended on the intact Cyclin T1-binding domain in Hexim1. Importantly, Hexim1 sequestered P-TEFb from CIITA, as documented by binding competition and ChIP assays. Conversely, the depletion of Hexim1 from cells by siRNA increased CIITA-mediated transcription. Thus, modulating ratios between active and inactive P-TEFb complexes is an additional mechanism of regulating transcriptional activators such as CIITA.7SK small nuclear RNA

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Selective control of gene expression by CDK9 in human cells

Garriga¹,

Xie²,

Obradović³

et al. 2009

Journal Cellular Physiology

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CDK9 associates with T-type cyclins and positively regulates transcriptional elongation by phosphorylating RNA polymerase II (RNAPII) and Negative Elongation Factors. However, it is unclear whether CDK9 is required for transcription of most genes by RNAPII or alternatively plays a role regulating the expression of restricted subsets of genes. We have investigated the direct effects of inhibiting cellular CDK9 activity in global gene expression in human cells by using a dominant negative form of CDK9 (dnCDK9). We have also compared direct inhibition of cellular CDK9 activity to pharmacological inhibition with flavopiridol (FVP), a CDK inhibitor that potently inhibits CDK9 and cellular transcription. Because of its presumed selectivity for CDK9, FVP has been previously used as a tool to infer the role of CDK9 on global gene expression. DNA microarray analyses described here show that inhibition of gene expression by FVP is consistent with global inhibition of transcription. However, specific inhibition of CDK9 activity with dnCDK9 leads to a distinctive pattern of changes in gene expression, with more genes being specifically upregulated (122) than downregulated (84). Indeed, the expression of many short-lived transcripts downregulated by FVP is not modulated by dnCDK9. Nevertheless, consistently with FVP inhibiting CDK9 activity, a significant number of the genes downregulated/upregulated by dnCDK9 are modulated with a similar trend by FVP. Our data suggests that the potent effects of FVP on transcription are likely to involve inhibition of CTD kinases in addition to CDK9. Our data also suggest complex and gene–specific modulation of gene expression by CDK9.

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Cyclin T1 Expression Is Regulated by Multiple Signaling Pathways and Mechanisms during Activation of Human Peripheral Blood Lymphocytes

Cited by 49 publications

References 64 publications

Direct inhibition of CDK9 blocks HIV-1 replication without preventing T-cell activation in primary human peripheral blood lymphocytes

Direct inhibition of CDK9 blocks HIV-1 replication without preventing T-cell activation in primary human peripheral blood lymphocytes

Hexim1 sequesters positive transcription elongation factor b from the class II transactivator on MHC class II promoters

Selective control of gene expression by CDK9 in human cells

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