Luigi Bagella

Activation and function of cyclin T-Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy

Hypertrophic growth is a risk factor for mortality in heart diseases. Mechanisms are lacking for this global increase in RNA and protein per cell, which underlies hypertrophy. Hypertrophic signals cause phosphorylation of the RNA polymerase II C-terminal domain, required for transcript elongation. RNA polymerase II kinases include cyclin-dependent kinases-7 (Cdk7) and Cdk9, components of two basal transcription factors. We report activation of Cdk7 and -9 in hypertrophy triggered by signaling proteins (Gαq, calcineurin) or chronic mechanical stress. Only Cdk9 was activated by acute load or, in culture, by endothelin. A preferential role for Cdk9 was shown in RNA polymerase II phosphorylation and growth induced by endothelin, using pharmacological and dominant-negative inhibitors. All four hypertrophic signals dissociated 7SK small nuclear RNA, an endogenous inhibitor, from cyclin T–Cdk9. Cdk9 was limiting for cardiac growth, shown by suppressing its inhibitor (7SK) in culture and preventing downregulation of its activator (cyclin T1) in mouse myocardium.Note: In the AOP version of this article, the numbering of the author affiliations was incorrect. This has now been fixed, and the affiliations appear correctly online and in print.

From G0 to S phase: A view of the roles played by the retinoblastoma (Rb) family members in the Rb-E2F pathway

Tumor suppressor pRb/p105, pRb/p107, and pRb2/p130 genes belong to the retinoblastoma (Rb) gene family. The members of the Rb gene family and the transcription factor E2F play an essential role in regulating cell cycle and, consequently, cell proliferation. This mini‐review describes the mechanisms by which Rb family members and E2F regulate cell cycle progression. J. Cell. Biochem. 102: 1400–1404, 2007.

Histone deacetylase inhibitors in the treatment of hematological malignancies and solid tumors.

The human genome is epigenetically organized through a series of modifications to the histone proteins that interact with the DNA. In cancer, many of the proteins that regulate these modifications can be altered in both function and expression. One example of this is the family of histone deacetylases (HDACs), which as their name implies remove acetyl groups from the histone proteins, allowing for more condensed nucleosomal structure. HDACs have increased expression in cancer and are also believed to promote carcinogenesis through the acetylation and interaction with key transcriptional regulators. Given this, small molecule histone deacetylases inhibitors have been identified and developed, which not only inhibit HDACs, but can also lead to growth arrest, differentiation, and/or apoptosis in tumors both in vitro and in vivo. Here, we will discuss some of the recent developments in clinical trials utilizing HDACs inhibitors for the treatment of both hematological malignancies as well as solid tumors.

Activation of MyoD-dependent transcription by cdk9/cyclin T2.

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-specific gene expression at least in part by down-regulating the activity of these cdks. Unlike the other cdks, cdk9 is not serum- or cell cycle-regulated and is instead involved in the regulation of transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. While ectopic expression of cdk2 together with its regulatory subunits (cyclins E and A) inhibits myogenic transcription, overproduction of cdk9 and its associated cyclin (cyclin T2a) strengthens MyoD-dependent transcription and stimulates myogenic differentiation in both MyoD-converted fibroblasts and C2C12 muscle cells. Conversely, inhibition of cdk9 activity by a dominant negative form (cdk9-dn) represses the myogenic program. Cdk9, cyclinT2 and MyoD can be detected in a multimeric complex in C2C12 cells, with the minimal cdk9-binding region of MyoD mapping within 101–161 aa of the bHLH region. Finally, cdk9 can phosphorylate MyoD in vitro, suggesting the possibility that cdk9/cycT2a regulation of muscle differentiation includes the direct enzymatic activity of the kinase on MyoD.

A UNIQUE DOMAIN OF PRB2/P130 ACTS AS AN INHIBITOR OF CDK2 KINASE ACTIVITY

The Cdk2 kinase has long been known to be involved in the progression of mammalian cells past the G1 phase restriction point and through DNA replication in the cell cycle. The Rb family of proteins, consisting of pRb, p107, and pRb2/p130, has also been shown to monitor progression of G1 phase, mostly through their interaction with E2F family members. p107 is able to inhibit Cdk2 kinase activity through this interaction via a p21-related domain present in the C terminus of the protein. We show here that pRb2/p130 also possesses this activity, but through a separate domain. Moreover, we correlate the increased expression of pRb2/p130 during various cellular processes with the decreased kinase activity of Cdk2. We hypothesize that pRb2/p130 may act not only to bind and modify E2F activity, but also to inhibit Cdk2 kinase activity in concert with p21 in a manner different from p107.

Ezh2 reduces the ability of HDAC1-dependent pRb2/p130 transcriptional repression of cyclin A

The polycomb group (PcG) proteins are known to be involved in maintaining the silenced state of several developmentally regulated genes. Enhancer of zeste homolog 2 (Ezh2), a member of this large protein family, has also been shown to be deregulated in different tumor types and its role, both as a potential primary effector and as a mediator of tumorigenesis, has become a subject of increased interest. We observed that Ezh2 binds to pRb2/p130, a member of the retinoblastoma family; as such, we were led to consider the possible ability of Ezh2 to modulate cell cycle progression. Both Ezh2 and pRb2/p130 repress gene expression by recruiting histone deacetylase (HDAC1), which decreases DNA accessibility for activating transcription factors. Additionally, we observed that Ezh2 interacts with the C-terminal region of pRb2/p130, essential for interaction with HDAC1. We show that Ezh2 is able to reverse pRb2/p130-HDAC1-mediated repression of the cyclin A promoter. This indicates a functional role of this complex in regulating cyclin A expression, known to be crucial in mediating cell cycle advancement. We also detected a significant decrease in the retention of HDAC1 activity associated with pRb2/p130 when Ezh2 was overexpressed. Finally, electromobility shift assays (EMSA) demonstrated that overexpression of Ezh2 caused the abrogation of the pRb2/p130–HDAC1 complex on the cyclin A promoter. These data, taken together, suggest that Ezh2 competes with HDAC1 in binding to pRb2/p130, disrupting their occupancy on the cyclin A promoter. In this study, we propose a new mechanism for the functional inactivation of pRb2/p130 that ultimately contributes to cell cycle progression and malignant transformation.

Importance of Ezh2 polycomb protein in tumorigenesis process interfering with the pathway of growth suppressive key elements.

An understanding of the mechanisms that uncover the dynamic changes in the distribution of the chromatin modifying enzymes and regulatory proteins on their target loci could provide further insight into the phenomenon of malignant transformation. Based on the current available data, it seems more and more clear that an abnormal expression of Ezh2, a member of the Polycomb group (PcG) protein, may be involved in the tumorigenesis process, in addition, different studies identify Ezh2 as a potential marker that distinguish aggressive prostate and breast cancer from indolent one. Recent investigation show that ectopic expression of Ezh2 provides proliferative advantage to primary cells through interaction with the pathways of key elements that control cell growth arrest and differentiation, like members of the retinoblastoma (Rb) family. Here, we outline how these pathways converge and we review the recent advances on the molecular mechanisms that promote cell cycle progression through deregulation of Ezh2 protein level, providing novel links between cancer progression and chromatin remodeling machineries. J. Cell. Physiol. 214: 295–300, 2008.

Physical interaction between pRb and cdk9/cyclinT2 complex

Cyclin-dependent kinase 9 (cdk9) is a multifunctional kinase with roles in different cellular pathways such as transcriptional elongation, differentiation and apoptosis. Cdk9/cyclin T differs functionally from other cdk/cyclin complexes that regulate cell cycle progression, but maintains structural affinity with those complexes. In addition, previous reports have demonstrated that the cdk9 complex is able to phosphorylate p56/pRb in vitro. In this report we show in vitro and in vivo interaction between cdk9/cyclinT2 and the protein product of the retinoblastoma gene (pRb) in human cell lines. The interaction involves the region composed of residues 129–195 of cdk9, cyclinT2 (1–642 aa) and the C-terminal domain of pRb (835–928 aa). We located the minimal region of cdk9 phosphorylation on the C-terminus of pRb, by identifying the residues between 793 and 834. This region contains at least three proline-directed serines (sp), S795, S807 and S811, which have been reported to be phosphorylated in vivo and which could be targeted by the cdk9 complex. These data suggest that, in logarithmically growing cells, cdk9/cyclin T2 and pRb are located in a nuclear multiprotein complex probably involved in transduction of cellular signals to the basal transcription machinery and that one of these signals could be the cdk9 phosphorylation of pRb.

Cloning of murine CDK9/PITALRE and its tissue-specific expression in development.

The cdc2‐family of serine/threonine kinases and their binding partners recently were implicated in developmental roles. We previously cloned a cdc2‐related kinase, cdk9/PITALRE, that is able to phosphorylate the retinoblastoma protein in vitro. We describe here the cloning and the characterization of the mouse homolog of cdk9/PITALRE. The murine cDNA is 98% identical with humans and is expressed at high levels in brain and kidney tissues. The kinase activity and protein expression of cdk9/PITALRE were highest in terminally differentiated tissues such as the muscle and brain. In situ immunohistology and immunofluorescence detected cdk9/PITALRE protein not only within terminally differentiated cells such as muscle and neuronal cells, but also in proliferating cells. C2C12 and P19 cells induced to differentiate along muscle and neural lineages peaked in cdk9/PITALRE kinase activity at the end of differentiation. These results suggest that, among other roles, cdk9/PITALRE plays a role not unlike cdk5 in the differentiation of certain cell types. J. Cell. Physiol. 177:206–213, 1998.

CDK9/CYCLIN T1 expression during normal lymphoid differentiation and malignant transformation

CDK9 is a member of the CDC2‐like family of kinases. Its cyclin partners are members of the CYCLIN T family (T1, T2a, and T2b) and CYCLIN K. The CDK9/CYCLIN T1 complex is very important in the differentiation programme of several cell types, controlling specific differentiation pathways. Limited data are available regarding the expression of CDK9/CYCLIN T1 in haematopoietic and lymphoid tissues. The aim of this study was to analyse the expression of the CDK9/CYCLIN T1 complex in lymphoid tissue, in order to assess its role in B‐ and T‐cell differentiation and lymphomagenesis. CDK9/CYCLIN T1 expression was found by immunohistochemistry in precursor B and T cells. In peripheral lymphoid tissues, germinal centre cells and scattered B‐ and T‐cell blasts in interfollicular areas expressed CDK9/CYCLIN T1, while mantle cells, plasma cells, and small resting T‐lymphocytes displayed no expression of either molecule. CDK9/CYCLIN T1 expression therefore appears to be related to particular stages of lymphoid differentiation/activation. CDK9 and CYCLIN T1 were highly expressed in lymphomas derived from precursor B and T cells, from germinal centre cells, such as follicular lymphomas, and from activated T cells (ie anaplastic large cell lymphomas). Hodgkin and Reed–Sternberg cells of classical Hodgkins lymphoma also showed strong nuclear staining. Diffuse large B‐cell, Burkitts lymphomas, and peripheral T‐cell lymphomas, among T‐cell lymphoproliferative disorders, showed a wide range of values. No expression of CDK9 or CYCLIN T1 was detected in mantle cell and marginal zone lymphomas. However, at the mRNA level, an imbalance in the CDK9/CYCLIN T1 ratio was found in follicular lymphoma and diffuse large B‐cell lymphomas with germinal centre phenotype, and in the cell lines of classical Hodgkins lymphomas, Burkitts lymphomas, and anaplastic large cell lymphoma, in comparison with reactive lymph nodes. These results suggest that the CDK9/CYCLIN T1 complex may affect the activation and differentiation programme of lymphoid cells. The molecular mechanism through which the CDK9/CYCLIN T1 complex is altered in malignant transformation needs to be elucidated. Copyright