Members of the cell division cycle 2 (CDC2) family of kinases play a pivotal role in the regulation of the eukaryotic cell cyde. In this communication, we report the isolation of a cDNA that encodes a CDC2-related human protein kinase temporarily designated PITALRE for the characteristic Pro-Ile-Thr-Ala-Leu-Arg-Glu motif. Its deduced amino acid sequence is 47% identical to that of the human cholinesterase-related cell division controller (CHED) kinase, which is required during hematopoiesis, and 42% identical to the Saccharomyces cerewasle SGV1 gene product, a putative kinase involved in the response to pheromone via its guanine nucleotide-binding protein a subunit. PITALRE expression is ubiquitous, but its expression levels are different in various human tissues. PITALRE is an =43-kDa protein that associates with three cellular polypeptides of 80, 95, and 155 kDa. PITALRE is lad primarily to the nucleus. In addition, we have identified a retinoblastoma protein kinase activity associated with PITALRE Immunocomplexes that cannot phosphorylate histone Hi, suggesting that the target phosphorylation site of PITALRE differs from that of CDC2 kinase. Interestingly, the retinoblastoma kinase activity associated with PITALRE does not oscillate during the cell cycle.The cell cycle in eukaryotes is regulated by a sequence of restriction points. In yeast, the first restriction point occurs during the G1 phase prior to the DNA synthesis and the second occurs before the initiation of mitosis. In Saccharomyces cerevisiae, the cell division cycle 28 (CDC28) kinase controls both restriction points through association with the CLN cyclins in G1 and with CLB cyclins in G2/M (1). In vertebrate cells, the regulatory mechanisms involved in cell cycle progression are more complex. CDC2 kinase, in association with cyclin B, appears to be a universal regulator of the eukaryotic entry into mitosis. However, in G1, just before the onset of DNA synthesis, cyclin-dependent kinase 2 (CDK2), but not CDC2, is required (2, 3). Additional mammalian CDC2-related kinases have been isolated that share >40% identity at the amino acid level (4)(5)(6)(7)(8)(9)(10)(11) This indicates that CDK4-cyclin D complexes possess a different phosphorylation specificity than the CDC2 kinase. Nevertheless, no kinase activity has been detected in CDK4 immunocomplexes (12). The association of CDK5 with cyclins D1/D3 and with proliferating cell nuclear antigen (PCNA) suggests a role for this kinase in the cell cycle (13). However, the high levels of expression of cdk5 found in neurons, cells no longer dividing, indicate a role for CDK5 in terminally differentiated cells (11). The study of CDC2 and CDC2-related kinases over the past few years has revealed a key role for these kinases in the regulation of the cell cycle. Most recently, an involvement in differentiation processes has also been proposed (8,11).With the aim of isolating additional putative controllers of the mammalian cell cycle, we performed a combination of PCR amplification and low-stringency ...

Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line. Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice. A polyclonal anti-HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined. These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.

The secreted proteoglycan decorin has been implicated in the negative control of cell proliferation primarily by virtue of its ability to block transforming growth factor-␤. Moreover, decorin expression is markedly up-regulated during quiescence but suppressed upon viral transformation, whereas de novo decorin expression in colon carcinoma cells abrogates the malignant phenotype by arresting the cells in the G 1 phase of the cell cycle. Here we show that this decorin-induced growth arrest is associated with up-regulation of p21 mRNA and protein in a transforming growth factor-␤-and p53-independent pathway. The augmented p21 protein is present as a multimeric complex with various cyclins and cyclin-dependent kinases in the nuclei of decorin-expressing cells, thereby leading to suppression of cyclin-dependent kinase activity and block of cell division. Through the usage of decorin-specific antisense oligodeoxynucleotide treatment, we demonstrate that the expression of decorin is closely linked to that of p21 and that abrogation of decorin leads to suppression of p21 and restoration of cell division. Collectively, our results provide a plausible mechanism by which decorin may contribute to retard and suppress the growth of tumor cells in vivo.The genes that regulate the transition from a proliferative to a quiescent state are beginning to be understood at the molecular level. It has been recognized, however, that several key molecules in cancer development are proteins that operate outside the nucleus and often interact at the cell-matrix boundaries either by detecting changes in the extracellular environment or by relaying messages from receptor-transducer proteins to their control machineries. Under the appropriate circumstances, the latter genes may act as tumor suppressor genes. Several lines of evidence indicate that decorin

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-speci®c 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 di erentiation in both MyoD-converted ®broblasts 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 di erentiation includes the direct enzymatic activity of the kinase on MyoD.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.Histone deacetylase (HDAC) inhibitors are promising new epi-drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II-specific HDAC inhibitor MC1568, we separated class I-and class II-dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4-HDAC3-MEF2D complex, and (iii) paradoxically, by inhibiting differentiation-induced MEF2D acetylation. In vivo MC1568shows an apparent tissue-selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2-HDAC complexes in a repressed state. Our results suggest that HDAC class II-selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases.

The oncoprotein of simian virus-40, SV40 large T-antigen (Tag), is reported to target and to inactivate growth suppressive proteins such as the retinoblastoma family and p53 (ref. 4, 5), leading to transformation of human cell lines in vitro, tumor production in rodents, and detection of Tag in several human cancers including mesotheliomas. The retinoblastoma family contains three members, pRb, p107 and pRb2/p130 (ref. 9), that are phosphorylated in a cell cycle-dependent manner, have cell growth suppressive properties and bind to specific members of the E2F family and various cyclins. Even though mesotheliomas are among the most aggressive human cancers, alterations of important cell-cycle "controllers," such as the Rb family genes, have never been reported in these tumors. We found the presence of SV40-like sequences in 86% of 35 archival specimens of mesothelioma. We also demonstrated that SV40 Tag, isolated from frozen biopsies of human mesothelioma, binds each of the retinoblastoma family proteins, pRb, p107 and pRb2/p130, in four of four specimens. We propose that the tumorigenic potential of SV40 Tag in some human mesotheliomas may arise from its ability to interact with and thereby inactivate several tumor and/or growth suppressive proteins.

Background: Neurovascular coupling (NVC) is the mechanism whereby an increase in neuronal activity (NA) leads to local elevation in cerebral blood flow (CBF) to match the metabolic requirements of firing neurons. Following synaptic activity, an increase in neuronal and/or astrocyte Ca2+ concentration leads to the synthesis of multiple vasoactive messengers. Curiously, the role of endothelial Ca2+ signaling in NVC has been rather neglected, although endothelial cells are known to control the vascular tone in a Ca2+-dependent manner throughout peripheral vasculature. Methods: We analyzed the literature in search of the most recent updates on the potential role of endothelial Ca2+ signaling in NVC. Results: We found that several neurotransmitters (i.e., glutamate and acetylcholine) and neuromodulators (e.g., ATP) can induce dilation of cerebral vessels by inducing an increase in endothelial Ca2+ concentration. This, in turn, results in nitric oxide or prostaglandin E2 release or activate intermediate and small-conductance Ca2+-activated K+ channels, which are responsible for endothelial-dependent hyperpolarization (EDH). In addition, brain endothelial cells express multiple transient receptor potential (TRP) channels (i.e., TRPC3, TRPV3, TRPV4, TRPA1), which induce vasodilation by activating EDH. Conclusions: It is possible to conclude that endothelial Ca2+ signaling is an emerging pathway in the control of NVC.

Notch proteins are a transmembrane receptor family that is structurally and functionally conserved from worms to humans. The mammalian family of Notch proteins consists of several genes encoding Notch receptors and related Notch ligands. Notch signaling is involved in different aspects of the cell-fate decision tree: differentiation, proliferation, and apoptosis. These three processes are finely regulated in human placenta in order to allow a successful pregnancy and correct fetal growth. Notch and its ligands also participate in vascular remodeling and stabilization. Vasculogenesis and blood regulation are of importance in the human placenta for normal fetal development and growth; any disorder of these systems leads to preeclampsia. Drawing on this background, we have investigated the expression of Notch-1, Notch-4, and Jagged-1, together with two members related to the Notch pathway in angiogenesis: VEGF and p21. Normal and preeclamptic human placentas have been evaluated by immunohistochemistry. In preeclamptic samples, a down-regulation of Notch pathway members occurs with a weak/moderate expression of the Notch protein members in all components of placenta compared with physiological placentas that, at term, exhibit the strong expression of Jagged-1 and a moderate expression of both Notch-1 and Notch-4 in all compartments of the placental villi. Moreover, preeclamptic samples also reveal a down-regulation of VEGF expression, together with a moderate nuclear expression of p21(Cip1) in the syncytiotrophoblast, cytotrophoblast, and endothelial cells. This down-regulation of VEGF in preeclamptic placentas, in turn, probably decreases Notch protein expression in placental compartments and in endothelial cells and could offer an ethiopathogenetic explanation for the onset of this pathology.