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

We present evidence of cell death by apoptosis during the development of bone-like tissue formation in vitro. Fetal rat calvaria-derived osteoblasts differentiate in vitro, progressing through three stages of maturation: a proliferation period, a matrix maturation period when growth is downregulated and expression of the bone cell phenotype is induced, and a third mineralization stage marked by the expression of bone-specific genes. Here we show for the first time that cells differentiating to the mature bone cell phenotype undergo programmed cell death and express genes regulating apoptosis. Culture conditions that modify expression of the osteoblast phenotype simultaneously modify the incidence of apoptosis. Cell death by apoptosis is directly demonstrated by visualization of degraded DNA into oligonucleosomal fragments after gel electrophoresis. Bcl-XL, an inhibitor of apoptosis, and Bax, which can accelerate apoptosis, are expressed at maximal levels 24 h after initial isolation of the cells and again after day 25 in heavily mineralized bone tissue nodules. Bcl-2 is expressed in a reciprocal manner to its related gene product Bcl-XL with the highest levels observed during the early post-proliferative stages of osteoblast maturation. Expression of p53, c-fos, and the interferon regulatory factors IRF-1 and IRF-2, but not cdc2 or cdk, were also induced in mineralized bone nodules. The upregulation of Msx-2 in association with apoptosis is consistent with its in vivo expression during embryogenesis in areas that will undergo programmed cell death. We propose that cell death by apoptosis is a fundamental component of osteoblast differentiation that contributes to maintaining tissue organization.
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PMID:Apoptosis during bone-like tissue development in vitro. 940 12

p21(Cip1/WAF1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21(Cip1/WAF1) is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21(Cip1/WAF1) in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21(Cip1/WAF1), and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21(Cip1/WAF1) demonstrated G1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21(Cip1/WAF1), concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21(Cip1/WAF1) forms a complex with the apoptosis signal-regulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21(Cip1/WAF1) lacking the nuclear localization signal (DeltaNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21(Cip1/WAF1) itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21(Cip1/WAF1), which are determined by its intracellular distribution and are dependent on the stage of differentiation.
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PMID:Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation. 1006 89

The interface between apoptosis (programmed cell death) and the cell cycle is essential to preserve homeostasis and genomic integrity. Here, we show that survivin, an inhibitor of apoptosis over-expressed in cancer, physically associates with the cyclin-dependent kinase p34(cdc2) on the mitotic apparatus, and is phosphorylated on Thr(34) by p34(cdc2)-cyclin B1, in vitro and in vivo. Loss of phosphorylation on Thr(34) resulted in dissociation of a survivin-caspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data identify survivin as a mitotic substrate of p34(cdc2)-cyclin B1 and suggest that survivin phosphorylation on Thr(34) may be required to preserve cell viability at cell division. Manipulation of this pathway may facilitate the elimination of cancer cells at mitosis.
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PMID:Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. 1106 2

Fumonisin B(1) (FB(1)) is a mycotoxin produced by the phytopathogenic fungus Fusarium moniliforme, which structurally resembles sphingoid bases. FB(1) perturbs sphingolipid synthesis by inhibiting the activity of ceramide synthase. Depending on the host, ingestion of FB(1) causes equine leukoencephalomalacia or porcine pulmonary edema. It is also carcinogenic to rats and may play a role in certain human cancers. Previous studies showed that FB(1) repressed specific isoforms of protein kinase C and cyclin-dependent kinase 2 (CDK2) activity. Conversely, FB(1) induced expression of CDK inhibitors, p21(Waf1/Cip1), p27(Kip1), and p57(Kip2) in monkey kidney cells (CV-1). Consequently, FB(1) treatment of CV-1 cells leads to cell-cycle arrest and apoptosis. The baculovirus IAP gene (inhibitor of apoptosis), which blocks tumor necrosis factor (TNF)-induced apoptosis, protects several fibroblast cell types from apoptosis, suggesting the TNF pathway is important for FB(1)-induced apoptosis. To identify genes that are induced by FB(1), we used a PCR-based subtraction approach. Eight genes that showed high similarity (> 90%) to known mammalian genes were identified. These genes included: tumor necrosis factor type 1 receptor associated protein 2 (TRAP2), human leukemia virus receptor (GLVR1), human Scaffold attachment factor A (SAF-A) also called heterogeneous nuclear ribonucleoprotein U (hnRNP-U), human protein kinase C-binding protein (RACK7), human oligosaccharyl transferase STT3 subunit, mouse WW-domain binding protein 2 (WBP2), human fibronectin, and an unknown human clone. The ability of FB(1) to alter gene expression and signal transduction pathways may be necessary for its carcinogenic and toxic effects.
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PMID:Identification of differentially expressed genes following treatment of monkey kidney cells with the mycotoxin fumonisin B(1). 1125 50

Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala(3)-Ile(19) exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence, nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys(57)-Trp(67) identified a cytosolic pool of survivin, which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala(3)-Ile(19), Met(38)-Thr(48), Pro(47)-Phe(58) and Cys(57)-Trp(67) identified both survivin pools within the same mitotic cell. A ratio of approximately 1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34(cdc2), and was phosphorylated by p34(cdc2) on Thr(34), in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34(cdc2) and was not phosphorylated on Thr(34). Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.
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PMID:Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function. 1186 64

Survivin is a member of the inhibitor of apoptosis gene family that is expressed in most human cancers and may facilitate evasion from apoptosis and aberrant mitotic progression. Here, exposure of breast carcinoma MCF-7 or cervical carcinoma HeLa cells to anticancer agents, including Adriamycin, Taxol, or UVB resulted in a 4-5-fold increased survivin expression. Changes in survivin levels after anticancer treatment did not involve modulation of survivin mRNA expression and were independent of de novo gene transcription. Conversely, inhibition of survivin phosphorylation on Thr(34) by the cyclin-dependent kinase inhibitor flavopiridol resulted in loss of survivin expression, and nonphosphorylatable survivin Thr(34)-->Ala exhibited accelerated clearance as compared with wild-type survivin. Sequential ablation of survivin phosphorylation on Thr(34) enhanced tumor cell apoptosis induced by anticancer agents independently of p53 and suppressed tumor growth without toxicity in a breast cancer xenograft model in vivo. These data suggest that Thr(34) phosphorylation critically regulates survivin levels in tumor cells and that sequential ablation of p34(cdc2) kinase activity may remove the survivin viability checkpoint and enhance apoptosis in tumor cells.
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PMID:Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis. 1251 2

The impact of disruption of the PI3K (phosphatidylinositol 3-kinase) pathway on the response of human leukemia cells to pharmacological cyclin-dependent kinase (CDK) inhibitors has been examined. Exposure of U937 monocytic leukemia cells to minimally toxic concentrations of flavopiridol (FP), roscovitine, or CGP74514A for 3 h in conjunction with the PI3K inhibitor LY294002 (abbreviated LY in the article) resulted in a marked decrease in Akt phosphorylation. Coexposure of cells to LY and CDK inhibitors also resulted in an early (i.e., within 3 h) and striking increase in mitochondrial damage [e.g., cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis (IAP)-binding protein with low isoelectric point (Smac/DIABLO), and apoptosis-initiating factor (AIF) release], caspase activation, and apoptosis. Similar interactions were observed in a variety of other leukemia cell types (e.g., HL-60, Jurkat, Raji, and NB4). Apoptosis, induced by FP/LY, was substantially blocked by ectopic expression of Bcl-2, but to a considerably lesser extent by dominant-negative caspase-8. FP-induced apoptosis was not enhanced by agents that inhibited protein kinase (PK) A (H89), PKC (GFX), mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK1/2; U0126), p38 MAP kinase (MAPK; SB202190), m-target of rapamycin (TOR; rapamycin), or ataxia-telangiectasia mutation (ATM; caffeine), whereas the PI3K inhibitor wortmannin exerted effects similar to those of LY. The dramatic potentiation of CDK inhibitor-induced apoptosis by LY was accompanied by diminished Bad phosphorylation, induction of Bcl-2 cleavage, and down-regulation of X-linked IAP (XIAP) and Mcl-1. Cells exposed to CDK inhibitors + LY also exhibited reduced phosphorylation of glycogen synthase kinase (GSK)-3, forkhead transcription factor (FKHR), p70(S6K), and ERK, but increased activation of p34(cdc2) and p38 MAPK. LY/CDK inhibitor-treated cells also displayed diminished pRb dephosphorylation on CDK2- and CDK4-specific sites, retinoblastoma protein cleavage, and down-regulation of cyclin D(1). Inducible expression of constitutively active (myristolated) Akt significantly, albeit partially, attenuated apoptosis in Jurkat leukemia cells treated with either FP alone or the combination of FP and LY. Finally, cotreatment with LY and FP resulted in a dramatic increase in apoptosis in primary leukemic blasts obtained from a patient with acute myeloblastic leukemia. Together, these findings suggest that the PI3K/Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to pharmacological CDK inhibitors and raise the possibility that combined interruption of CDK- and PI3K-related pathways may represent a novel therapeutic strategy in hematological malignancies.
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PMID:The lethal effects of pharmacological cyclin-dependent kinase inhibitors in human leukemia cells proceed through a phosphatidylinositol 3-kinase/Akt-dependent process. 1270 69

All-trans retinoic acid (ATRA) can down regulate the anti-apoptotic protein Bcl-2 and the cell cycle proteins cyclin D1 and cdk2 in estrogen receptor-positive breast cancer cells. We show here that retinoids can also reduce expression of the inhibitor of apoptosis protein, survivin. Here we have compared the regulation of these proteins in MCF-7 and ZR-75 breast cancer cells by natural and synthetic retinoids selective for the RA receptors (RARs) alpha, beta, and gamma then correlated these with growth inhibition, induction of apoptosis and chemosensitization to Taxol. In both cell lines ATRA and 9-cis RA induced the most profound decreases in cyclin D1 and cdk2 expression and also mediated the largest growth inhibition. The RARalpha agonist, Ro 40-6055 also strongly downregulated these proteins although did not produce an equivalent decrease in S-phase cells. Only ATRA induced RARbeta expression. ATRA, 9-cis RA and 4-HPR initiated the highest level of apoptosis as determined by mitochondrial Bax translocation, while only ATRA and 9-cis RA strongly reduced Bcl-2 and survivin protein expression. Enumeration of dead cells over 96 h correlated well with downregulation of both survivin and Bcl-2. Simultaneous retinoid-mediated reduction of both these proteins also predicted optimal Taxol sensitization. 4-HPR was much weaker than the natural retinoids with respect to Taxol sensitization, consistent with the proposed requirement for reduced Bcl-2 in this synergy. Neither the extent of cell cycle protein regulation nor AP-1 inhibition fully predicted the antiproliferative effect of the synthetic retinoids suggesting that growth inhibition requires regulation of a spectrum of RAR-regulated gene products in addition even to pivotal cell cycle proteins.
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PMID:Differential regulation of protein expression, growth and apoptosis by natural and synthetic retinoids. 1458 26

To explore the molecular mechanisms underlying the actions of Taxol and the functionally related molecule epothilone B (EpoB), we have analyzed the gene expression profiles in A549 cells in response to increasing concentrations of these microtubule-stabilizing drugs. An almost identical expression pattern was observed in cells treated with either Taxol or EpoB. Low concentrations of the drugs induced aberrant mitosis including asymmetric and multipolar cell divisions. At drug concentrations that triggered G(2)-M arrest, cells escaped from a prolonged mitotic arrest without cell division, resulting in tetraploid G(1) cells. This mitotic slippage is correlated with diminished expression of cdc2 kinase, topoisomerase IIalpha, BUB3, and BUB2-like protein 1, as well as with an increased expression of 14-3-3-sigma. Poly(ADP-ribose) polymerase cleavage, an early indicator of apoptosis, occurred in cells undergoing mitotic slippage and in aneuploid cells resulting from aberrant mitosis. In contrast, cells arrested in mitosis demonstrated no signal for apoptosis but had an increased expression of survivin, an inhibitor of apoptosis. Induction of aneuploid or tetraploid G(1) cells was accompanied by increased expression of CD95, p21, and BTG2 that may contribute to cell death because their expression was diminished in an EpoB-resistant cell line. In contrast, expression of GADD45 and PTGF-beta could promote cell survival. We conclude that abnormal mitotic exit is required for apoptotic cell death induced by microtubule-stabilizing drugs.
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PMID:Gene expression and mitotic exit induced by microtubule-stabilizing drugs. 1463 18

The Saccharomyces cerevisiae inhibitor of apoptosis (IAP) repeat protein Bir1 localizes as a chromosomal passenger. A deletion analysis of Bir1 identified two regions important for function. The C-terminal region is essential for growth, binds Sli15, and is necessary and sufficient for the localization of Bir1 as a chromosomal passenger. The middle region is not essential but is required to localize the inner kinetochore protein Ndc10 to the spindle during anaphase and to the midzone at telophase. In contrast, precise deletion of the highly conserved IAP repeats conferred no phenotype and did not alter the cell cycle delay caused by loss of cohesin. Bir1 is phosphorylated in a cell cycle-dependent manner. Mutation of all nine CDK consensus sites in the middle region of Bir1 significantly decreased the level of phosphorylation and blocked localization of Ndc10 to the spindle at anaphase. Moreover, immunoprecipitation of Ndc10 with Bir1 was dependent on phosphorylation. The loss of Ndc10 from the anaphase spindle prevented elongation of the spindle beyond 7 microm. We conclude that phosphorylation of the middle region of Bir1 is required to bring Ndc10 to the spindle at anaphase, which is required for full spindle elongation.
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PMID:Phosphorylation of the chromosomal passenger protein Bir1 is required for localization of Ndc10 to the spindle during anaphase and full spindle elongation. 1638 14


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