Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To examine the p53-mediated biological activities and signalling pathways, we generated stable transfectants of the p53-null IW32 murine erythroleukemia cells expressing the temperature-sensitive p53 mutant DNA, tsp53(val135). Two clones with different levels of p53 protein expression were selected for further characterization. At permissive temperature, clone 1-5 cells differentiated along the erythroid pathway, and clone 3-2 cells that produced greater levels (3.5-fold) of p53 underwent apoptosis. Apoptosis of 3-2 cells was accompanied by mitochondrial cytochrome c release and caspase activation as well as by cleavage of caspase substrates. Bax protein was induced to a similar extent in these clones by wild-type p53; expression of p21(Cip1/Waf1) and p27(Kip1) proteins was also increased. However, significantly lesser extent of induction for both CDK inhibitors was detected in the apoptotic 3-2 clone. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.fmk) blocked the p53-induced apoptosis in 3-2 cells, with a concomitant elevation of p27(Kip1), suggesting that p27(Kip1) protein underwent caspase-dependent proteolysis in the apoptotic 3-2 cells. Together these results linked a pathway involving cytochrome c release, caspase activation and p27(Kip1) degradation to the p53-induced apoptosis in IW32 erythroleukemia cells.
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PMID:Modulation of caspase activation and p27(Kip1) degradation in the p53-induced apoptosis in IW32 erythroleukemia cells. 1222 Jun 22

Epidemiological observations indicate that resveratrol, a natural antioxidant stilbene, exerts cardioprotective and chemopreventive effects. Moreover, the molecule induces in vitro cell growth inhibition and differentiation. Using human erythroleukemic K562 cells as model system, we demonstrated that resveratrol induces a remarkable gamma-globin synthesis, the erythroid differentiation being linked to impairment of cell proliferation, increased p21Cip1 expression and inhibition of cdk2 activity. The up-regulation of p21Cip1 transcription is prevented by cycloheximide, indicating the requirement of intermediate protein(s), which, in turn, regulate gene expression. The quantitative analysis of some transcription factors involved in the erythroid lineage, namely GATA-1, GATA-2, and Egr1, indicated that resveratrol selectively up-regulates Egr1 by an Erk1/2-dependent mechanism. The presence of an Egr1 consensus sequence in the p21Cip1 promoter suggested the hypothesis that this transcription factor directly regulates the expression of the cdk inhibitor. Transfection studies with deleted gene promoter constructs, as well as EMSA, pull-down, and chromatin immunoprecipitation experiments substantiated this view, demonstrating that Egr1 binds in vitro and in vivo to the identified consensus sequence of the p21Cip1 promoter. Moreover, an Egr1 phosphorothioate antisense hinders p21Cip1 accumulation and the antiproliferative effects of resveratrol. In conclusion, this is the first demonstration that Egr1 controls p21Cip1 expression by directly interacting with a specific sequence on its gene promoter. The identified regulatory mechanism also contributes to the clarification of the complex chemopreventive and antiproliferative properties of resveratrol.
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PMID:p21Cip1 gene expression is modulated by Egr1: a novel regulatory mechanism involved in the resveratrol antiproliferative effect. 1269 Jan 10

Tumor necrosis factor alpha (TNFalpha) potently inhibits the in vitro growth of highly purified human d-6 erythroid colony forming cells (ECFC). Unlike the inhibitory effect of TNFalpha on other cells, including more immature ECFC, this antiproliferative effect of TNFalpha is not related to apoptosis because the d-6 cell descendants were morphologically normal, without apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling assay and without caspase activation by Western blots after TNFalpha treatment. TNFalpha did not appear to affect the cell cycle distribution, but the cell cycle duration was significantly longer in TNFalpha-treated cells. DNA synthesis was also significantly reduced by TNFalpha. Studies of various proteins that regulate the cell cycle showed that cyclin-dependent kinase 6 (CDK6) protein and mRNA levels were concomitantly decreased in the presence of TNFalpha, suggesting that inhibition of cell growth was related to reduced CDK6. To evaluate this, the CDK6 gene was transferred into ECFC using green fluorescence protein-retrovirus-mediated gene transfer. The results showed that the level of cell growth produced by TNFalpha was increased by 30% when the cells were transfected with CDK6. Therefore, the modification of cell cycle progression in the presence of TNFalpha through a reduction of CDK6 is an important mechanism in the TNFalpha inhibition of human ECFC expansion.
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PMID:Reduction of cell cycle progression in human erythroid progenitor cells treated with tumour necrosis factor alpha occurs with reduced CDK6 and is partially reversed by CDK6 transduction. 1278 4

Butyrate, a non-toxic short-chain fatty acid (SCFA) and inhibitor of histone deacetylase (HDAC), has potential as an anti-tumor agent because it imposes a reversible G1 block in normal cells yet induces apoptosis in tumor lines. As a potent reactivator of fetal globin transcription, butyrate is used clinically in the treatment of hemoglobinopathies. The anti-proliferative effect of butyrate and its derivatives on in vivo erythroid cell maturation, however, has limited their utility. The molecular mechanisms underlying the G1 arrest induced by butyrate and related SCFAs remain unclear. One model, drawing on tumor cell data, proposes that HDAC inhibition and subsequent transcriptional induction of cyclin-dependent kinase inhibitor (CKI) p21CIP are required. However, because of potentially confounding genetic mutations present in tumor models, we examined SCFA effects on CKIs in a non-transformed growth control model. Using murine 3T3 fibroblasts, we find p27KIP1 is also strongly induced. Unlike previously described effects of butyrate and HDAC inhibition on p21CIP, p27KIP1 induction did not occur at the transcriptional level; instead, the stability of the p27KIP1 protein increased. Other structurally unrelated HDAC inhibitors, including trichostatin A (TSA), induced p27KIP1 similarly. p27KIP1 was found in cyclin E/Cdk2 complexes, concomitant with suppression of cdk2 activity. Elevation of p27KIP1 is required for the observed G1 blockade, as p27KIP1-deficient fibroblasts were resistant to HDAC inhibition-induced arrest. These data suggest a novel activity for HDAC inhibitors and demonstrate a critical role for p27KIP1 in mediating G1 arrest in response to these drugs.
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PMID:Histone deacetylase inhibition-mediated post-translational elevation of p27KIP1 protein levels is required for G1 arrest in fibroblasts. 1538 42

The cyclin-dependent kinase (Cdk) inhibitors p21(Cip1) and p27(Kip1) have been proposed to exert redundant functions in cell cycle progression and differentiation programs, although nonoverlapping functions have also been described. To gain further insights into the relevant mechanisms and to detect possible functional differences between both proteins, we conditionally expressed p21(Cip1) and p27(Kip1) in K562, a multipotent human leukemia cell line. Temporal ectopic expression of either p21(Cip1) or p27(Kip1) arrested proliferation, inhibited Cdk2 and Cdk4 activities, and suppressed retinoblastoma phosphorylation. However, whereas p21(Cip1) arrested cells in both G(1) and G(2) cell cycle phases, p27(Kip1) blocked the G(1)/S-phase transition. Furthermore, although both p21(Cip1) and p27(Kip1) associated with Cdk6, only p27(Kip1) significantly inhibited its activity. Most importantly, each protein promoted differentiation along a distinct pathway; p21(Cip1) triggered megakaryocytic maturation, whereas p27(Kip1) resulted in the expression of erythroid markers. Consistently, p21(Cip1) and p27(Kip1) were rapid and transiently up-regulated when K562 cells are differentiated into megakaryocytic and erythroid lineages, respectively. These findings demonstrate distinct functions of p21(Cip1) and p27(Kip1) in cell cycle regulation and differentiation and indicate that these two highly related proteins possess unique biological activities and are not functionally interchangeable.
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PMID:p21Cip1 and p27Kip1 induce distinct cell cycle effects and differentiation programs in myeloid leukemia cells. 1574 92

Over 10 years ago, cdk6 was identified as a new member in a family of vertebrate cdc-2 related kinases. This novel kinase was found to partner with the D-type cyclins and to possess pRb kinase activity in vitro and has since been understood to function solely as a pRb kinase in the regulation of the G(1) phase of the cell cycle. In the past 2 years, several independent studies in multiple cell types have indicated a novel role for cdk6 in differentiation. For example, cdk6 expression must be reduced to allow proper osteoblast and osteoclast differentiation, forced cdk6 expression blocked differentiation of mouse erythroid leukemia cells and cdk6 expression in primary astrocytes favors the expression of progenitor cell markers. Since exit from the cell cycle is a necessary step in terminal differentiation, down-regulation of a mitogenic factor may be expected in this process, however it is surprising that this association has not been previously uncovered and that it is apparently not shared with cdk4, long understood to be a functional homolog of cdk6. The mechanism of cdk6 function in differentiation is not understood, but it may extend beyond the established role of cdk6 as a pRb kinase. As this story unfolds it will be important to discover if the function of cdk6 in differentiation is pRb-dependent or pRb-independent, since pRb has long been established as a key factor in initiating and maintaining cell cycle exit during differentiation.
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PMID:Beyond the cell cycle: a new role for Cdk6 in differentiation. 1629 22

Roscovitine is a specific inhibitor of cyclin-dependent kinases (cdks) cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35. The studies on the enzyme inhibitory properties and cellular effects of roscovitine revealed that it arrests cells in G(2)/M and G(1)/S phase, inhibits the proliferation of mammalian cells and induces cell death. However, the characteristics of cell death and exact mechanism by which this cdk inhibitor kills transformed cells are unknown. We previously investigated that the roscovitine induces apoptotic death of mitotic PC12 cells. The present study was to identify whether the roscovitine-induced death is related with the specific elements of caspases in pathway of apoptosis. The morphological data of caspase-3 immunofluorocytochemistry double staining with hoechst 33342 indicated that apoptotic nuclei were identified as nuclei with chromatin condensation and nuclear fragmentation, and that caspase-3 active p17 subunit co-existed in PC12 cells treated with roscovitine 50 micromol/L for 4 h. The number of the caspase-3 positive cells increased significantly to about 42%, as compared with the normal control (P<0.001). The data of MTT assay showed that the number of viable cells treated by roscovitine (50 micromol/L) alone for 12 h was 29.03%, of the untreated controls. Both a broad-spectrum caspase inhibitor Z-VAD-FMK (50 mumol/L) and a specific caspase-3 inhibitor Z-DEVD-FMK (100 micromol/L) increased viable PC12 cells to 45.16%, (Z-DEVD-FMK) and 58.06%, (Z-VAD-FMK), respectively, in the presence of roscovitine. Non-erythroid a-spectrin is a cytoskeleted protein that is a substrate of caspase-3 cysteine proteases. To confirm the activity of caspase-3 that produced in roscovitine (50 micromol/L for 12 h)-induced PC12 cell death, activated caspase-3 specific 120 kDa spectrin breakdown products (SBDP) were detected by Western bloting using the mouse anti-non-erythroid a-spectrin monoclonal antibody. The mean relative density of bands corresponding to caspase-3 specific SBDP levels were significantly increased in the cytosolic fractions treated with roscovitine, as compared to the normal control (P<0.001). These results indicate that caspase signals, especially caspase-3 signal are necessary for the progression of proliferating PC12 cell apoptotic death evoked by roscovintine.
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PMID:[Caspase-3 plays a required role in PC12 cell apoptotic death induced by roscovitine]. 1634 2

To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1-/-; p130-/- mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1-/- counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1-/-; p130-/- animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1-/- splenocytes. The finding that the p27Kip1-/-; p130-/- splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.
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PMID:p27Kip1 and p130 cooperate to regulate hematopoietic cell proliferation in vivo. 1688 May 27

In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.
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PMID:Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia. 2909 81

Oxidative stress leads to perturbation of a variety of cellular processes resulting in inhibition of cell proliferation. This study has determined the effect of oxidative stress on protein synthesis in human K562 cells using a hydrophilic peroxyl radical initiator, AAPH and H(2)O(2). The results indicated that oxidative stress leads to a significant decrease in the rate of protein synthesis caused due to induced activation as well as expression of the erythroid cell-specific eIF-2alpha kinase, called the Heme Regulated Inhibitor (HRI). Elevated levels of HRI expression and activity were accompanied by increased lipid peroxidation and decreased cell proliferation. Further, oxidative stress also caused inactivation of p34(cdc2) kinase, thereby arresting cell division leading to apoptosis. Thus, the data provides the mechanism of inhibition of protein synthesis and perturbation of a cell cycle regulatory protein leading to inhibition of cell proliferation in K562 cells during oxidative stress.
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PMID:Oxidative stress perturbs cell proliferation in human K562 cells by modulating protein synthesis and cell cycle. 1966 95


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