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1
Mossalayi, MD, JC Lecron, P. Goube de Laforest, G. Janossy, P. Debre, and J. Tanzer. "Characterization of prothymocytes with cloning capacity in human bone marrow." Blood 71, no. 5 (May 1, 1988): 1281–87. http://dx.doi.org/10.1182/blood.v71.5.1281.1281.
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Abstract The identity of human bone marrow (BM)-derived T cell precursors with colony forming capacity has led to controversy because of contamination with mature clonogenic T cells. We achieved 2 Log elimination of mature T cells from BM using a cocktail of monoclonal antibodies: CD2, CD3, CD4, CD6, and CD8 followed by two successive baby rabbit C' treatment. T cell depleted BM can generate colonies of CD2+, CD3+, Ti+, mostly CD4+, in the presence of PHA, rIL2, and a prothymocyte differentiating activity derived from phytohemagglutinin (PHA) induced mononuclear cells. These precursors could be enriched three- to sixfold by percoll gradient centrifugation and then significantly bypass the number of contaminant mature T cells as shown by limiting dilution analysis. Colony generation by marrow precursors was inhibited by the addition of autologous T cells. This inhibition was mostly caused by the T8+ subset. CFU-TL growth was dramatically inhibited by eliminating CD7+ cells suggesting their positivity for this surface marker. These precursors needed major histocompatibility complex (MHC) II-positive cells for optimal growth but lack DR themselves.
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Mossalayi, MD, JC Lecron, P. Goube de Laforest, G. Janossy, P. Debre, and J. Tanzer. "Characterization of prothymocytes with cloning capacity in human bone marrow." Blood 71, no. 5 (May 1, 1988): 1281–87. http://dx.doi.org/10.1182/blood.v71.5.1281.bloodjournal7151281.
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The identity of human bone marrow (BM)-derived T cell precursors with colony forming capacity has led to controversy because of contamination with mature clonogenic T cells. We achieved 2 Log elimination of mature T cells from BM using a cocktail of monoclonal antibodies: CD2, CD3, CD4, CD6, and CD8 followed by two successive baby rabbit C' treatment. T cell depleted BM can generate colonies of CD2+, CD3+, Ti+, mostly CD4+, in the presence of PHA, rIL2, and a prothymocyte differentiating activity derived from phytohemagglutinin (PHA) induced mononuclear cells. These precursors could be enriched three- to sixfold by percoll gradient centrifugation and then significantly bypass the number of contaminant mature T cells as shown by limiting dilution analysis. Colony generation by marrow precursors was inhibited by the addition of autologous T cells. This inhibition was mostly caused by the T8+ subset. CFU-TL growth was dramatically inhibited by eliminating CD7+ cells suggesting their positivity for this surface marker. These precursors needed major histocompatibility complex (MHC) II-positive cells for optimal growth but lack DR themselves.
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Brown, Julia, Nikolaos Patsoukis, and Vassiliki A. Boussiotis. "PD-1 Signals Inhibit Cell Cycle Progression by Mediating Upregulation of Both KIP and INK Family of Cdk Inhibitors." Blood 116, no. 21 (November 19, 2010): 585. http://dx.doi.org/10.1182/blood.v116.21.585.585.
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Abstract Abstract 585 The PD-1 pathway plays a critical role in the inhibition of T cell activation and the maintenance of T cell tolerance. PD-1 is expressed on activated T cells and limits T cell clonal expansion and effector function upon engagement with its ligands PD-L1 and PD-L2. PD-1 signals are vital for inhibition of autoimmunity whereas PD-1 ligation by PD-L1 and PD-L2 expressed on malignant cells has a detrimental effect on tumor-specific immunity. Furthermore, PD-1 signals result in T cell exhaustion in several chronic viral infections. The mechanism via which PD-1 signals mediate inhibition of T cell expansion is currently poorly understood. Here, we sought to determine the effects of PD-1 signals on mechanistic regulation of cell cycle progression mediated via TCR/CD3 and CD28 in primary human CD4+ T cells using anti-CD3/CD28 with or without agonist anti-PD-1 mAb conjugated to magnetic beads. Cell cycle analysis by ethynyl-deoxyuridine incorporation revealed that PD-1 induced blockade of cell cycle progression at the early G1 phase. To determine the molecular mechanisms underlying the blocked cell cycle progression we examined the expression and activation of cyclins and cdks and the regulation of cdk inhibitors that counterbalance the enzymatic activation of cyclin/cdk holoenzyme complexes. Our studies revealed that PD-1 mediated signals inhibited upregulation of Skp2, the SCF ubiquitin ligase that leads p27kip1 cdk inhibitor to ubiquitin-dependent degradation, and resulted in accumulation of p27kip1. Expression of cyclin E that is induced at the G1/S phase transition, and cyclin A that is synthesized during the S phase of the cell cycle, was dramatically reduced in the presence of PD-1 signaling. Strikingly, although expression of cdk4 and cdk2 was comparable between cells cultured in the presence or in the absence of PD-1, cdk2 enzymatic activation was significantly reduced in the presence of PD-1 signaling. Smad3 is a novel critical cdk substrate. Maximum cdk-mediated Smad3 phosphorylation occurs at the G1/S phase junction and requires activation of cdk2. Phosphorylation by cdk antagonizes TGF-β-induced transcriptional activity and antiproliferative function of Smad3 whereas impaired phosphorylation on the cdk-specific sites renders Smad3 more effective in executing its antiproliferative function. Based on those findings, we examined the effects of PD-1 signaling on Smad3 phosphorylation on cdk-specific and TGF-β-specific sites using site-specific phospho-Smad3 antibodies. Compared to anti-CD3/CD28 alone, culture in the presence of PD-1 induced impaired cdk2 activity, reduced levels of Smad3 phosphorylation on the cdk-specific sites and increased Smad3 phophorylation on the TGF-b-specific site. To determine whether the differential phosphorylation of Smad3 might differentially regulate Smad3 transcriptional activity in CD4+ T cells cultured in the presence versus the absence of PD-1, we examined expression of the INK family cdk4/6 inhibitor p15, a known downstream transcriptional target of Smad3. Expression of p15 was upregulated in CD4+ T cells cultured in the presence of PD-1 but not in cells cultured in the presence of CD3/CD28-coated beads alone. These results indicate that PD-1 signals inhibit cell cycle progression by mediating upregulation of both KIP and INK family of cdk inhibitors and Smad3 is a critical component of this mechanism, regulating blockade at the early G1 phase. Disclosures: No relevant conflicts of interest to declare.
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Harper, J. W., S. J. Elledge, K. Keyomarsi, B. Dynlacht, L. H. Tsai, P. Zhang, S. Dobrowolski, C. Bai, L. Connell-Crowley, and E. Swindell. "Inhibition of cyclin-dependent kinases by p21." Molecular Biology of the Cell 6, no. 4 (April 1995): 387–400. http://dx.doi.org/10.1091/mbc.6.4.387.
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p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.
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Swarbrick, Alexander, Christine S. L. Lee, Robert L. Sutherland, and Elizabeth A. Musgrove. "Cooperation of p27Kip1 and p18INK4c in Progestin-Mediated Cell Cycle Arrest in T-47D Breast Cancer Cells." Molecular and Cellular Biology 20, no. 7 (April 1, 2000): 2581–91. http://dx.doi.org/10.1128/mcb.20.7.2581-2591.2000.
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ABSTRACT The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G1 cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27Kip1among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27Kip1 and few were bound to p21Cip1. In vitro, recombinant His6-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His6-p27 in vitro or p27Kip1 in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18INK4c and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18INK4c led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27Kip1 and p18INK4c cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor β and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.
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Foster, James S., Donald C. Henley, Antonin Bukovsky, Prem Seth, and Jay Wimalasena. "Multifaceted Regulation of Cell Cycle Progression by Estrogen: Regulation of Cdk Inhibitors and Cdc25A Independent of Cyclin D1-Cdk4 Function." Molecular and Cellular Biology 21, no. 3 (February 1, 2001): 794–810. http://dx.doi.org/10.1128/mcb.21.3.794-810.2001.
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ABSTRACT Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G1/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16INK4a to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16INK4a inhibited G1/S transition induced in MCF-7 cells by 17-β-estradiol (E2) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G1 and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21Cip1 and p27Kip1 was decreased, however, in both control and p16INK4a-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E2 in control and p16INK4a-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16INK4a. Inhibition of Cdc25A activity in p16INK4a-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E2-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisenseCDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16INK4a-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21Cip1 and p27Kip1.
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Terada, Y., S. Inoshita, O. Nakashima, T. Yamada, M. Kuwahara, S. Sasaki, and F. Marumo. "Lovastatin inhibits mesangial cell proliferation via p27Kip1." Journal of the American Society of Nephrology 9, no. 12 (December 1998): 2235–43. http://dx.doi.org/10.1681/asn.v9122235.
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Mesangial cell proliferation is a key feature of glomerulonephritis. The hydroxymethylglutaryl-coenzyme A reductase inhibitor lovastatin is known to inhibit cell cycle progression. To determine the inhibitory mechanisms of mesangial cell proliferation by lovastatin, the cyclin-dependent kinase (CDK) activity, and expression of CDK inhibitor (p27Kip1, p21Cip1, and p16INK4) mRNA and protein were measured. Lovastatin inhibited phosphorylation of retinoblastoma protein and mesangial cell proliferation dose dependently. Lovastatin increased the p27Kip1 protein level but produced no changes in the abundance of the p27Kip1 mRNA level both in the presence and absence of mitogens. Treatment with lovastatin revealed the increment of both CDK2- and CDK4-bound-p27Kip1. The experiment using antisense oligonucleotide against p27Kip1 showed significant amelioration of lovastatin-induced cell cycle arrest. Lovastatin reduced both platelet-derived growth factor-stimulated CDK2 and CDK4 kinase activities. In conclusion, lovastatin inhibited mesangial proliferation via translational upregulation or impairment of p27Kip1 protein degradation. Lovastatin serves as a potential therapeutic approach to mesangial proliferative disease.
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Musgrove, Elizabeth A., Alexander Swarbrick, Christine S. L. Lee, Ann L. Cornish, and Robert L. Sutherland. "Mechanisms of Cyclin-Dependent Kinase Inactivation by Progestins." Molecular and Cellular Biology 18, no. 4 (April 1, 1998): 1812–25. http://dx.doi.org/10.1128/mcb.18.4.1812.
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ABSTRACT The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G1 phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of ∼120 and ∼200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.
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Salman Roghani, Roham, Ali Sanjari moghaddam, Gabrielle Rupprecht, Erdem Altunel, So Young Kim, Shannon McCall, Beatrice Thomas, Katie Ware, Jason Somarelli, and David S. Hsu. "A precision medicine drug discovery pipeline to identify dual CDK2/9 inhibition as a novel treatment for colorectal cancer." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e16056-e16056. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e16056.
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e16056 Background: Colorectal cancer (CRC) is the 3rdmost common form of cancer in the US, responsible for over 50,000 death each year. Therapeutic options for advanced colorectal cancer are limited, and there remains an unmet clinical need to identify new therapies to treat this deadly disease. To address this need, we have developed a precision medicine pipeline that integrates high throughput chemical screens with matched patient-derived cell lines and patient-derived xenografts (PDXs) to identify new treatments for CRC. Methods: We used high-throughput chemical screens of 2,100 compounds across five low-passage, patient-derived CRC cell lines. These results were validated using dose-response IC50curves for CDK1, CDK2, CDK9 or CDK1/2/9 inhibitors and by siRNA-mediated knockdown of CDK9 with or without CDK2 inhibition. Cell cycle arrest analysis was performed by flow cytometry and anaphase catastrophe was analyzed by immunofluorescence staining. For in vivo studies, matched PDXs were treated with either CDK2, CDK9 or dual CDK2/9 inhibitors. Results: We identified the CDK inhibitor drug class as among the most effective cytotoxic compounds across all five CRC lines. Further analysis of the CDK inhibitor class revealed that combined targeting of CDK1, 2, and 9 was the most effective, with IC50 in the range of 110 nM to 1.2 μM. We further validated the efficacy of combined CDK2/9 inhibition using siRNA-mediated knockdown of CDK9 in the presence of a CDK2 inhibitor(CVT-313), and showed that CDK9 knockdown acted synergistically with CDK2 inhibition. Dual CDK2/9 inhibition led to significant G2/M cell cycle arrest and anaphase catastrophe. Finally, combined CDK2/9 inhibition in vivo synergistically inhibited PDX tumor growth as compared to single-agent CDK inhibitors. Conclusions: Our precision medicine pipeline revealed CDK2/9 dual inhibition as a combinatorial therapy to treat CRC and can also be used to identify new and novel therapies
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Göke, R., P. Barth, A. Schmidt, B. Samans, and B. Lankat-Buttgereit. "Programmed cell death protein 4 suppresses CDK1/cdc2 via induction of p21Waf1/Cip1." American Journal of Physiology-Cell Physiology 287, no. 6 (December 2004): C1541—C1546. http://dx.doi.org/10.1152/ajpcell.00025.2004.
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We show that the recently discovered tumor suppressor pdcd4 represses the transcription of the mitosis-promoting factor cyclin-dependent kinase (CDK)1/cdc2 via upregulation of p21Waf1/Cip1. p21Waf1/Cip1 inhibits CDK4/6 and CDK2. Decrease of CDK4/6 and CDK2 enhances the binding of pRb to E2F/DP, which in turn together bind to and repress the cdc2 promoter. Upregulation of CDK1/cdc2 accompanied by a malignant change was previously reported in colon cancer. We show that expression of pdcd4 as an indirect suppressor of CDK1/cdc2 is lost in progressed carcinomas of lung, breast, colon, and prostate. Furthermore, it seems that localization and expression of pdcd4 directly correlate with tumor progression. Finally, the CDK1/cdc2 inhibitor roscovitine reduces the proliferation of several tumor cell lines, suggesting that inhibition of CDK1/cdc2 may be a useful strategy against malignant transformation. Therefore, pdcd4 might serve as a novel target for antineoplastic therapies.
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DINARINA, Ana, Laurent H. PEREZ, Amparo DAVILA, Markus SCHWAB, Tim HUNT, and Angel R. NEBREDA. "Characterization of a new family of cyclin-dependent kinase activators." Biochemical Journal 386, no. 2 (February 22, 2005): 349–55. http://dx.doi.org/10.1042/bj20041779.
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Progression through the cell cycle is regulated by CDKs (cyclin-dependent kinases), which associate with activating partners, named cyclins, to efficiently phosphorylate substrates. We previously reported the identification of RINGO, a Xenopus protein that can activate CDK1 and CDK2 despite lack of sequence similarity to cyclins, which plays a role in the regulation of the meiotic cell cycle in oocytes. In the present study we report the characterization of four mammalian RINGO proteins, which are 53–68% identical with Xenopus RINGO in a central core of about 75 residues. We show that all RINGO family members can bind to and activate CDK1 and CDK2, albeit with different efficiencies, but they do not bind to CDK4 or CDK6. The core RINGO sequences are critical for CDK activation. We also identified key residues in CDK2 that are required for RINGO binding. All RINGO proteins can also bind the CDK inhibitor p27Kip1, but with an inverse efficiency of their ability to bind to CDK1. Our results identify a new family of mammalian proteins that can activate CDKs and therefore potentially function as cell cycle regulators. The ability of RINGO proteins to activate CDK1 and CDK2 suggest also cyclin-independent roles for these kinases.
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Lu, Shuyan, Tae Sung, Marina Amaro, Brad Hirakawa, Bart Jessen, and Wenyue Hu. "Phenotypic Characterization of Targeted Knockdown of Cyclin-Dependent Kinases in the Intestinal Epithelial Cells." Toxicological Sciences 177, no. 1 (June 18, 2020): 226–34. http://dx.doi.org/10.1093/toxsci/kfaa092.
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Abstract Cyclin-dependent kinases (CDKs) are serine/threonine kinases that regulate cell cycle and have been vigorously pursued as druggable targets for cancer. There are over 20 members of the CDK family. Given their structural similarity, selective inhibition by small molecules has been elusive. In addition, collateral damage to highly proliferative normal cells by CDK inhibitors remains a safety concern. Intestinal epithelial cells are highly proliferative and the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. Using the rat intestinal epithelial (IEC6) cells as an in vitro model, we found that the selective CDK4/6 inhibitor palbociclib lacked potent anti-proliferative activity in IEC6 relative to the breast cancer cell line MCF7, indicating the absence of intestinal cell reliance on CDK4/6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, we chose common targets of CDK inhibitors (CDK 1, 2, 4, 6, and 9) for targeted gene knockdown to evaluate phenotypes. Surprisingly, only CDK1 and CDK9 knockdown demonstrated profound cell death or had moderate growth effects, respectively. CDK2, 4, or 6 knockdowns, whether single, double, or triple combinations, did not have substantial impact. Studies evaluating CDK1 knockdown under various cell seeding densities indicate direct effects on viability independent of proliferation state and imply a potential noncanonical role for CDK1 in intestinal epithelial biology. This research supports the concept that CDK1 and CDK9, but not CDKs 2, 4, or 6, are essential for intestinal cell cycle progression and provides safety confidence for interphase CDK inhibition.
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Niiya, Fumihiko, Xiaozhen Xie, Kyung S. Lee, Hiroki Inoue, and Toru Miki. "Inhibition of Cyclin-dependent Kinase 1 Induces Cytokinesis without Chromosome Segregation in an ECT2 and MgcRacGAP-dependent Manner." Journal of Biological Chemistry 280, no. 43 (August 23, 2005): 36502–9. http://dx.doi.org/10.1074/jbc.m508007200.
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Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.
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Schang, Luis M., Andrew Bantly, and Priscilla A. Schaffer. "Explant-Induced Reactivation of Herpes Simplex Virus Occurs in Neurons Expressing Nuclear cdk2 and cdk4." Journal of Virology 76, no. 15 (August 1, 2002): 7724–35. http://dx.doi.org/10.1128/jvi.76.15.7724-7735.2002.
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ABSTRACT Herpes simplex virus (HSV) establishes productive (lytic) infections in nonneuronal cells and nonproductive (latent) infections in neurons. It has been proposed that HSV establishes latency because quiescent neurons lack cellular factors required for productive infection. It has been further proposed that these putative factors are induced following neuronal stress, as a requirement for HSV reactivation. To date, the identity of these putative cellular factors remains unknown. We have demonstrated that cyclin-dependent kinase (cdk) 1, 2, or 7 is required for HSV replication in nonneuronal cells. Interestingly, cdks 1 and 2 are not expressed in quiescent neurons but can be induced in stressed neurons. Thus, cdks may be among the cellular proteins required for HSV reactivation whose neuronal expression is differentially regulated during stress. Herein, we determined that neuronal expression of nuclear cdk2, cdk4, and cyclins E and D2 (which activate cdks 2 and 4, respectively) was induced following explant cultivation, a stressful stimulus that induces HSV reactivation. In contrast, neuronal expression of cdk7 and cytoplasmic cdk4 decreased during explant cultivation, whereas cdk3 was detected in the same small percentage of neurons before and after explant cultivation and cdks 1, 5, and 6 were not detected in neuronal cell bodies. HSV-1 reactivated specifically in neurons expressing nuclear cdk2 and cdk4, and an inhibitor specific for cdk2 inhibited HSV-1 reactivation. We conclude that neuronal levels of cdk2 are among the factors that determine the outcome of HSV infections of neurons.
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Haller, Kerstin, Yalin Wu, Elisabeth Derow, Iris Schmitt, Kuan-Teh Jeang, and Ralph Grassmann. "Physical Interaction of Human T-Cell Leukemia Virus Type 1 Tax with Cyclin-Dependent Kinase 4 Stimulates the Phosphorylation of Retinoblastoma Protein." Molecular and Cellular Biology 22, no. 10 (May 15, 2002): 3327–38. http://dx.doi.org/10.1128/mcb.22.10.3327-3338.2002.
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ABSTRACT The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G1 phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21CIP. Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein.
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Shih, Li-Jane, Jia-Yu Wang, Jing-Yao Jheng, An-Ci Siao, Yen-Yue Lin, Yi-Wei Tsuei, Yow-Chii Kuo, Chih-Pin Chuu, and Yung-Hsi Kao. "Betel Nut Arecoline Induces Different Phases of Growth Arrest between Normal and Cancerous Prostate Cells through the Reactive Oxygen Species Pathway." International Journal of Molecular Sciences 21, no. 23 (December 3, 2020): 9219. http://dx.doi.org/10.3390/ijms21239219.
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Prostate cancer (PCa) is a reproductive system cancer in elderly men. We investigated the effects of betel nut arecoline on the growth of normal and cancerous prostate cells. Normal RWPE-1 prostate epithelial cells, androgen-independent PC-3 PCa cells, and androgen-dependent LNCaP PCa cells were used. Arecoline inhibited their growth in dose- and time-dependent manners. Arecoline caused RWPE-1 and PC-3 cell cycle arrest in the G2/M phase and LNCaP cell arrest in the G0/G1 phase. In RWPE-1 cells, arecoline increased the expression of cyclin-dependent kinase (CDK)-1, p21, and cyclins B1 and D3, decreased the expression of CDK2, and had no effects on CDK4 and cyclin D1 expression. In PC-3 cells, arecoline decreased CDK1, CDK2, CDK4, p21, p27, and cyclin D1 and D3 protein expression and increased cyclin B1 protein expression. In LNCaP cells, arecoline decreased CDK2, CDK4, and cyclin D1 expression; increased p21, p27, and cyclin D3 expression; had no effects on CDK1 and cyclin B1 expression. The antioxidant N-acetylcysteine blocked the arecoline-induced increase in reactive oxygen species production, decreased cell viability, altered the cell cycle, and changed the cell cycle regulatory protein levels. Thus, arecoline oxidant exerts differential effects on the cell cycle through modulations of regulatory proteins.
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Watad, Abdulla, Hannah Rowe, Tobias Russell, Qiao Zhou, Lisa K. Anderson, Almas Khan, Robert Dunsmuir, et al. "Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression." Annals of the Rheumatic Diseases 79, no. 8 (May 13, 2020): 1044–54. http://dx.doi.org/10.1136/annrheumdis-2020-217309.
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BackgroundThe human enthesis conventional T cells are poorly characterised.ObjectivesTo study the biology of the conventional T cells in human enthesis.MethodsCD4+ and CD8+ T cells were investigated in 25 enthesis samples using immunofluorescence, cytometrically, bulk RNAseq and quantitative real-time PCR following anti-CD3/CD28 bead stimulation to determine interleukin (IL)-17A and tumour necrosis factor (TNF) levels. T-cell receptor (TCR) repertoires were characterised and a search for putative T-cell reactivity was carried out using TCR3 database. The impact of pharmacological antagonism with retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti), methotrexate and phosphodiesterase type 4 inhibitor (PDE4i) was investigated.ResultsImmunofluorescence and cytometry suggested entheseal resident CD4+ and CD8+ T cells with a resident memory phenotype (CD69+/CD45RA-) and tissue residency gene transcripts (higher NR4A1/AhR and lower KLF2/T-bet transcripts). Both CD4+ and CD8+ T cells showed increased expression of immunomodulatory genes including IL-10 and TGF-β compared with peripheral blood T cells with entheseal CD8+ T cells having higher CD103, CD49a and lower SIPR1 transcript that matched CD4+ T cells. Following stimulation, CD4+ T cells produced more TNF than CD8+ T cells and IL-17A was produced exclusively by CD4+ T cells. RNAseq suggested both Cytomegalovirus and influenza A virus entheseal resident T-cell clonotype reactivity. TNF and IL-17A production from CD4+ T cells was effectively inhibited by PDE4i, while RORγti only reduced IL-17A secretion.ConclusionsHealthy human entheseal CD4+ and CD8+ T cells exhibit regulatory characteristics and are predicted to exhibit antiviral reactivity with CD8+ T cells expressing higher levels of transcripts suggestive of tissue residency. Inducible IL-17A and TNF production can be robustly inhibited in vitro.
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Kranenburg, O., V. Scharnhorst, A. J. Van der Eb, and A. Zantema. "Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells." Journal of Cell Biology 131, no. 1 (October 1, 1995): 227–34. http://dx.doi.org/10.1083/jcb.131.1.227.
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Studies on the molecular mechanisms underlying neuronal differentiation are frequently performed using cell lines established from neuroblastomas. In this study we have used mouse N1E-115 neuroblastoma cells that undergo neuronal differentiation in response to DMSO. During differentiation, cyclin-dependent kinase (cdk) activities decline and phosphorylation of the retinoblastoma gene product (pRb) is lost, leading to the appearance of a pRb-containing E2F DNA-binding complex. The loss of cdk2 activity is due to a decrease in cdk2 abundance whereas loss of cdk4 activity is caused by strong association with the cdk inhibitor (CKI) p27KIP1 and concurrent loss of cdk4 phosphorylation. Moreover, neuronal differentiation can be induced by overexpression of p27KIP1 or pRb, suggesting that inhibition of cdk activity leading to loss of pRb phosphorylation, is the major determinant for neuronal differentiation.
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Bae, Jooeun, Constantine Mitsiades, Rao Prabhala Prabhala, Tai Yu-Tzu, Jeff Martinson, Steven Hartson, Teru Hideshima, et al. "Hsp90 Is Critical for the Regulation of Human T Lymphocytes and NK Cell Phenotype and Function." Blood 106, no. 11 (November 16, 2005): 3464. http://dx.doi.org/10.1182/blood.v106.11.3464.3464.
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Abstract Hsp90 inhibitor has shown promising anti-tumor activity through the destabilization and eventual degradation of Hsp90 client proteins critical for cell survival. In this study, we examined the in vitro effects of Hsp90 inhibitor on the phenotype and function of human T lymphocytes and NK cells. We observed no significant effects of Hsp90 inhibitor treatment on cell survivals. However, Hsp90 inhibitor treatment for 24 hours led to irreversible down-regulation of expression of critical T-cell surface antigens including CD3, CD4, CD8, CD28, CD154 (CD40L) and TCRab. Among the antigens evaluated, expression of CD4 antigen was most significantly downregulated (untrt vs. trt = 326 vs. 88 in Mean Fluorescence Intensity) following Hsp90 inhibitor treatment. Decreased CD3+ T lymphocytes proliferation (untrt vs. trt = 222839 cpm vs. 111102 cpm, 3[H]-thymidine incorporation) and reduced IFN-g secretion (untrt vs. trt = 77 vs. 48 pg/ml) was observed upon stimulation with allogeneic dendritic cells following 24 hrs treatments of T cells with Hsp90 inhibitor. Furthermore, CD3+ T-cell proliferation in response to mitogen stimulation, as measured by flow cytometry using CFSE was decreased following Hsp90 inhibitor treatment (untrt vs. trt = 41% vs. 3%, CFSE). Specifically, the CD4+CD28+ (untrt vs. trt = 32% vs. 1%) and CD8+CD28+ (untrt vs. trt = 27% vs. 17%) activated T-cell subpopulations displayed a significant decrease in proliferation in response to mitogen. Similarly, NK cells displayed decreased activation receptor expression including CD2, CD11a, CD94, NKp30, NKp44, NKp46, and KARp50.3 and reduced cytotoxic activity against multiple myeloma cells (untrt vs. trt = 49% vs. 11% against MM1S cells, 65% vs. 8% against ARP cells) following Hsp90 inhibitor treatment. These studies demonstrate that Hsp90 inhibitor treatment significantly affects phenotype and function of human T-lymphocytes as well as NK cells, and suggest the need to monitor immune functions in patients being treated with Hsp90 inhibitor in our future studies.
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Li, Lequn, Nikolaos Patsoukis, Anoma Nellore, and Vassiliki A. Boussiotis. "Inhibition Of Cdk2 Promotes The Generation Of Inducible CD8+ T Regulatory Cells By Modulating The Epigenetic Regulator EZH2." Blood 122, no. 21 (November 15, 2013): 138. http://dx.doi.org/10.1182/blood.v122.21.138.138.
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Abstract Graft versus host disease (GvHD) remains the main cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation. In spite of the intense research efforts, control of GvHD remains incomplete and novel therapeutic approaches are required. Cdk2 has a central role in cell cycle re-entry of mature T lymphocytes and inhibition of Cdk2 is mandatory for induction of T cell anergy in vitro and tolerance in vivo. While Cdk2 is essential for expansion of activated T cells, it is not critical for survival of resting lymphocytes, hematopoiesis or thymocyte development. These properties make Cdk2 an attractive target for control of GvHD. To determine the effects of Cdk2 inhibition on T cell alloresponses in vivo, we used the B6D2F1 mouse model of allogeneic BMT and two different Cdk2 inhibitors, CYC202 (IC50=0.1 uM) and CYC205 (IC50=1 nM). Lethally irradiated B6D2F1(Kd) recipients were infused with bone marrow from C57BL/6(Kb) donors with (BMT) or without splenocytes (BM) and were subsequently treated with each Cdk2 inhibitor for three weeks. Treatment was administered daily during week 1, every other day on week 2, and twice a week on week 3. Effects of treatment on GvHD were assessed by body weight and survival during a 70-day period. Although BMT recipients treated with Cdk2 inhibitor displayed a transient initial weight loss, subsequently regained weight to levels comparable to control BM recipients. Furthermore, treated BMT recipient groups displayed significantly delayed GvHD mortality (p=0.0054). Recently, it was determined that inducible CD8+ Treg cells, have a central role in mediating protection from GvHD. Some immunosuppressive drugs have detrimental effects on Treg whereas others spare these cells or may even be beneficial to their proportional increase. To examine whether Cdk2 inhibitors induced Treg cells, we used GFP- T cells from Foxp3.GFP-KI mice (C57BL/6 background) as a source of T cells during BMT. Assessment of peripheral blood lymphocytes, splenocytes, peripheral lymph nodes and intestinal lymphoid cells (ILC) in BMT recipients revealed no differences in CD4+GFP+ Treg between treated and control groups. In contrast, the treated group displayed an increase of CD8+GFP+ Treg cells in these cell populations, predominantly ILC, which displayed a 5-fold increase of CD8+ Treg (p=0.05). To further investigate whether Cdk2 inhibitors had a selective effect on CD8+ Treg differentiation, we isolated CD4+GFP- and CD8+GFP- T cells from Foxp3.GFP-KI mice and subjected them to in vitro Treg polarizing with or without Cdk2 inhibitors. Inhibition of Cdk2 had almost no effect on CD4+GFP+ cells but induced a 2-4 fold increase of CD8+GFP+ cells. To determine whether Cdk2 inhibition induced its effect on CD8+ Treg differentiation by reducing the threshold of TGF-β-mediated signaling, we cultured CD8+GFP- cells with stable concentrations of Cdk2 inhibitors and decreasing concentrations of TGF-β. Cdk2 inhibition induced CD8+ Treg differentiation in the presence of TGF-β concentrations that failed to induce any significant numbers of CD8+ Treg cells when used alone. Expression of FOX family genes is regulated by transcriptional and epigenetic mechanisms. A critical epigenetic regulator of FOX transcription factors in cancer cells is the Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), which promotes histone H3 lysine 27 trimethylation (H3K27me3) and induces epigenetic gene silencing. Cdk1 and Cdk2 phosphorylate EZH2 at Thr350 in an evolutionarily conserved motif. Phosphorylation of Thr350 is important for EZH2 recruitment and maintenance of H3K27me3 levels at EZH2-target loci. We examined whether EZH2 becomes phosphorylated in CD8+ T cells and whether Cdk2 inhibition might affect this event. Upon polarizing CD8+ T cell culture, EZH2 displayed robust phosphorylation on Thr350, which was blocked by Cdk2 inhibition. This event temporally coincided with a 44-fold increase in Foxp3 mRNA expression compared to base line levels in control T cells. These results reveal an unexpected mechanism via which Cdk2 inhibitors mediate suppression of alloreactive T cells and protection from GvHD by inducing CD8+ Treg. Because Cdk-mediated EZH2 phosphorylation is a key mechanism governing EZH2 function to regulate epigenetic silencing, Cdk2 inhibition might have additional, yet unidentified implications on gene expression programs of alloreactive T cells. Disclosures: No relevant conflicts of interest to declare.
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NAKAMURA, K., N. YOKOYAMA, and I. IGARASHI. "Cyclin-dependent kinase inhibitors block erythrocyte invasion and intraerythrocytic development of Babesia bovis in vitro." Parasitology 134, no. 10 (July 18, 2007): 1347–53. http://dx.doi.org/10.1017/s0031182007002831.
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SUMMARYCyclin-dependent kinases (CDKs) are essential for the regulation of the eukaryotic cell cycle. A number of chemicals, which selectively inhibit the CDK activities, have been synthesized for the development of anti-cancer drugs. This report describes the inhibitory effect of purine derivatives known to be CDK inhibitors on the asexual growth of Babesia bovis. The 4 compounds, roscovitine, purvalanol A, CGP74514A, and CDK2 Inhibitor II, showed significantly suppressive effects on the in vitro growth of B. bovis. Three (roscovitine, purvalanol A, and CDK2 Inhibitor II) showed an inhibitory effect on the early stages of intraerythrocytic development of B. bovis. CGP74514A (CDK1-specific inhibitor) blocked the erythrocyte invasion by merozoites. Our data suggest the chemotherapeutic potential of the CDK inhibitors for babesiosis, and the target molecules of the compounds would participate in the process of successful erythrocyte invasion or intraerythrocytic development of B. bovis.
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Diehl, J. A., and C. J. Sherr. "A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase." Molecular and Cellular Biology 17, no. 12 (December 1997): 7362–74. http://dx.doi.org/10.1128/mcb.17.12.7362.
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Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.
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McConnell, Beth B., Fiona J. Gregory, Francesca J. Stott, Eiji Hara, and Gordon Peters. "Induced Expression of p16INK4a Inhibits Both CDK4- and CDK2-Associated Kinase Activity by Reassortment of Cyclin-CDK-Inhibitor Complexes." Molecular and Cellular Biology 19, no. 3 (March 1, 1999): 1981–89. http://dx.doi.org/10.1128/mcb.19.3.1981.
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ABSTRACT To investigate the mode of action of the p16 INK4a tumor suppressor protein, we have established U2-OS cells in which the expression of p16 INK4a can be regulated by addition or removal of isopropyl-β-d-thiogalactopyranoside. As expected, induction of p16 INK4a results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16 INK4a also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27 KIP1 . Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16 INK4a , this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16 INK4a . Sequestration of CDK4 by p16 INK4a allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16 INK4a , p27 KIP1 appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27 KIP1 and p21 CIP1 . Significantly, p16 INK4a itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.
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Ding, Qing-Ming, Tien C. Ko, and B. Mark Evers. "Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4." American Journal of Physiology-Cell Physiology 275, no. 5 (November 1, 1998): C1193—C1200. http://dx.doi.org/10.1152/ajpcell.1998.275.5.c1193.
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The cellular mechanisms regulating intestinal proliferation and differentiation remain largely undefined. Previously, we showed an early induction of the cyclin-dependent kinase (CDK) inhibitor p21Waf1/Cip1 in Caco-2 cells, a human colon cancer line that spontaneously differentiates into a small bowel phenotype. The purpose of our present study was to assess the timing of cell cycle arrest in relation to differentiation in Caco-2 cells and to examine the mechanisms responsible for CDK inactivation. Caco-2 cells undergo a relative G1/S block and cease to proliferate at day 3 postconfluency; an increase in the activity of terminally differentiated brush-border enzymes (sucrase and alkaline phosphatase) was noted at day 6 postconfluency. Cell cycle block was associated with suppression of both CDK2 and CDK4 activities, which are important for G1/S progression. Treatment of the CDK immune complexes with the detergent deoxycholate (DOC) resulted in restoration of CDK2, but not CDK4, activity at day 3postconfluency, suggesting the presence of inhibitory protein(s) binding to the cyclin/CDK2 complex at this time point. An increased binding of p21Waf1/Cip1 to CDK2 complexes at day 3 postconfluency was noted, suggesting a potential role for p21Waf1/Cip1in CDK2 inactivation; however, immunodepletion of p21Waf1/Cip1 from Caco-2 protein extracts demonstrated that p21Waf1/Cip1 is only partially responsible for CDK2 suppression at day 3postconfluency. A decrease in the cyclin E/CDK2 complex appears to contribute to the CDK2 inactivation noted at days 6 and 12 postconfluency. Taken together, our results suggest that multiple mechanisms contribute to CDK suppression during Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leads to G1 arrest and inhibition of proliferation that precede Caco-2 cell differentiation.
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Shapiro, Geoffrey I. "Cyclin-Dependent Kinase Pathways As Targets for Cancer Treatment." Journal of Clinical Oncology 24, no. 11 (April 20, 2006): 1770–83. http://dx.doi.org/10.1200/jco.2005.03.7689.
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Cyclin-dependent kinases (cdks) are critical regulators of cell cycle progression and RNA transcription. A variety of genetic and epigenetic events cause universal overactivity of the cell cycle cdks in human cancer, and their inhibition can lead to both cell cycle arrest and apoptosis. However, built-in redundancy may limit the effects of highly selective cdk inhibition. Cdk4/6 inhibition has been shown to induce potent G1 arrest in vitro and tumor regression in vivo; cdk2/1 inhibition has the most potent effects during the S and G2 phases and induces E2F transcription factor–dependent cell death. Modulation of cdk2 and cdk1 activities also affects survival checkpoint responses after exposure to DNA-damaging and microtubule-stabilizing agents. The transcriptional cdks phosphorylate the carboxy-terminal domain of RNA polymerase II, facilitating efficient transcriptional initiation and elongation. Inhibition of these cdks primarily affects the accumulation of transcripts with short half-lives, including those encoding antiapoptosis family members, cell cycle regulators, as well as p53 and nuclear factor-kappa B–responsive gene targets. These effects may account for apoptosis induced by cdk9 inhibitors, especially in malignant hematopoietic cells, and may also potentiate cytotoxicity mediated by disruption of a variety of pathways in many transformed cell types. Current work is focusing on overcoming pharmacokinetic barriers that hindered development of flavopiridol, a pan-cdk inhibitor, as well as assessing novel classes of compounds potently targeting groups of cell cycle cdks (cdk4/6 or cdk2/1) with variable effects on the transcriptional cdks 7 and 9. These efforts will establish whether the strategy of cdk inhibition is able to produce therapeutic benefit in the majority of human tumors.
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Wilkinson, M. F., J. Doskow, R. von Borstel, A. M. Fong, and C. L. MacLeod. "The expression of several T cell-specific and novel genes is repressed by trans-acting factors in immature T lymphoma clones." Journal of Experimental Medicine 174, no. 1 (July 1, 1991): 269–80. http://dx.doi.org/10.1084/jem.174.1.269.
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Cell surface proteins encoded by members of the immunoglobulin supergene family are sequentially expressed during T cell ontogeny. The molecular mechanisms responsible for the regulation of these surface molecules are not well understood. To investigate this issue, we used a series of well characterized T lymphoma cell clones with phenotypes characteristic of distinct stages of early thymocyte maturation. Somatic cell hybrids formed from these cell lines were employed to detect the presence of negative regulatory molecules. The expression of CD4 and CD8 was strongly repressed in hybrids formed between a CD4+ CD8+ lymphoma clone and "immature" CD4- CD8- lymphoma clones. Individual subunits of the T cell receptor (TCR)/CD3 complex displayed independent regulation in unique patterns in hybrid cells. Hybrids formed by fusing CD3+ and CD3- cells completely repressed CD3-delta mRNA expression while CD3-gamma, -epsilon, and -zeta transcripts were moderately inhibited or codominantly regulated. Similar to CD3-delta, interleukin 2R-alpha(IL-2R-alpha), and TCR-beta mRNA accumulation was trans-negatively regulated. Transcription rate measurements demonstrated that the inhibition of CD4, CD8, CD3-gamma, CD3-epsilon, TCR-beta, and IL-2R-alpha mRNA accumulation in hybrid cells was exerted, at least in part, at the transcriptional level. To test whether repressional regulation is a general feature of T cells, we examined the regulation of six novel genes which were selected solely on the basis of their differential expression between two of the cell lines used in this study. Five of the six novel gene transcripts were repressed in the somatic cell hybrids. Thus, inhibitor factors appear to play a general role in controlling T cell gene expression. The model system presented here may be useful for the identification and characterization of repressor molecules responsible for the regulation of genes expressed during T cell ontogeny.
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Major, Michael L., Rita Lepe, and Robert H. Costa. "Forkhead Box M1B Transcriptional Activity Requires Binding of Cdk-Cyclin Complexes for Phosphorylation-Dependent Recruitment of p300/CBP Coactivators." Molecular and Cellular Biology 24, no. 7 (April 1, 2004): 2649–61. http://dx.doi.org/10.1128/mcb.24.7.2649-2661.2004.
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ABSTRACT Previous liver regeneration studies demonstrated that the mouse forkhead box M1B (FoxM1B) transcription factor regulates hepatocyte proliferation through expression of cell cycle genes that stimulate cyclin-dependent kinase 2 (Cdk2) and Cdk1 activity. In this study, we demonstrated that disruption of the FoxM1B Cdk1/2 phosphorylation site at Thr residue 596 significantly reduced both FoxM1B transcriptional activity and Cdk phosphorylation of the FoxM1B T596A mutant protein in vivo. Retention of this FoxM1B 596 Cdk phosphorylation site was found to be essential for recruiting the histone acetyltransferase CREB binding protein (CBP) to the FoxM1B transcriptional activation domain. Consistent with these findings, dominant negative Cdk1 protein significantly reduced FoxM1B transcriptional activity and inhibited FoxM1B recruitment of the CBP coactivator protein. Likewise, Cdc25B-mediated stimulation of Cdk activity together with elevated levels of the CBP coactivator protein provided a 6.2-fold synergistic increase in FoxM1B transcriptional activity. Furthermore, mutation of the FoxM1B Leu 641 residue within an LXL motif (residues 639 to 641) inhibited recruitment of Cdk-cyclin complexes and caused significant reduction in both FoxM1B transcriptional activity and in vivo Cdk phosphorylation of the FoxM1B Thr 596 residue. We demonstrated that FoxM1B transcriptional activity requires binding of either S-phase or M-phase Cdk-cyclin complexes to mediate efficient Cdk phosphorylation of the FoxM1B Thr 596 residue, which is essential for recruitment of p300/CBP coactivator proteins.
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BARBERIS, Matteo, Luca DE GIOIA, Maria RUZZENE, Stefania SARNO, Paola COCCETTI, Piercarlo FANTUCCI, Marco VANONI, and Lilia ALBERGHINA. "The yeast cyclin-dependent kinase inhibitor Sic1 and mammalian p27Kip1 are functional homologues with a structurally conserved inhibitory domain." Biochemical Journal 387, no. 3 (April 26, 2005): 639–47. http://dx.doi.org/10.1042/bj20041299.
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In Saccharomyces cerevisiae, Sic1, an inhibitor of Cdk (cyclin-dependent kinase), blocks the activity of S-Cdk1 (Cdk1/Clb5,6) kinase that is required for DNA replication. Deletion of Sic1 causes premature DNA replication from fewer origins, extension of the S phase and inefficient separation of sister chromatids during anaphase. Despite the well-documented relevance of Sic1 inhibition of S-Cdk1 for cell cycle control and genome instability, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains obscure. In this paper, we show that Sic1 is functionally and structurally related to the mammalian Cki (Cdk inhibitor) p27Kip1 of the Kip/Cip family. A molecular model of the inhibitory domain of Sic1 bound to the Cdk2–cyclin A complex suggested that the yeast inhibitor might productively interface with the mammalian Cdk2–cyclin A complex. Consistent with this, Sic1 is able to bind to, and strongly inhibit the kinase activity of, the Cdk2–cyclin A complex. In addition, comparison of the different inhibitory patterns obtained using histone H1 or GST (glutathione S-transferase)–pRb (retinoblastoma protein) fusion protein as substrate (the latter of which recognizes both the docking site and the catalytic site of Cdk2–cyclin A) offers interesting suggestions for the inhibitory mechanism of Sic1. Finally, overexpression of the KIP1 gene in vivo in Saccharomyces cerevisiae, like overexpression of the related SIC1 gene, rescues the cell cycle-related phenotype of a sic1Δ strain. Taken together, these findings strongly indicate that budding yeast Sic1 and mammalian p27Kip1 are functional homologues with a structurally conserved inhibitory domain.
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Adams, P. D., W. R. Sellers, S. K. Sharma, A. D. Wu, C. M. Nalin, and W. G. Kaelin. "Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors." Molecular and Cellular Biology 16, no. 12 (December 1996): 6623–33. http://dx.doi.org/10.1128/mcb.16.12.6623.
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Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.
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Lim, Do Y., Yoonhwa Jeong, Angela L. Tyner, and Jung H. Y. Park. "Induction of cell cycle arrest and apoptosis in HT-29 human colon cancer cells by the dietary compound luteolin." American Journal of Physiology-Gastrointestinal and Liver Physiology 292, no. 1 (January 2007): G66—G75. http://dx.doi.org/10.1152/ajpgi.00248.2006.
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Luteolin is 3′,4′,5,7-tetrahydroxyflavone found in celery, green pepper, and perilla leaf that inhibits tumorigenesis in animal models. We examined luteolin-mediated regulation of cell cycle progression and apoptosis in the HT-29 human colon cancer cell line. Luteolin decreased DNA synthesis and viable HT-29 cell numbers in a concentration-dependent manner. It inhibited cyclin-dependent kinase (CDK)4 and CDK2 activity, resulting in G1 arrest with a concomitant decrease of phosphorylation of retinoblastoma protein. Activities of CDK4 and CDK2 decreased within 2 h after luteolin treatment, with a 38% decrease in CDK2 activity ( P < 0.05) observed in cells treated with 40 μmol/l luteolin. Luteolin inhibited CDK2 activity in a cell-free system, suggesting that it directly inhibits CDK2. Cyclin D1 levels decreased after luteolin treatment, although no changes in expression of cyclin A, cyclin E, CDK4, or CDK2 were detected. Luteolin also promoted G2/M arrest at 24 h posttreatment by downregulating cyclin B1 expression and inhibiting cell division cycle (CDC)2 activity. Luteolin promoted apoptosis with increased activation of caspases 3, 7, and 9 and enhanced poly(ADP-ribose) polymerase cleavage and decreased expression of p21CIP1/WAF1, survivin, Mcl-1, Bcl-xL, and Mdm-2. Decreased expression of these key antiapoptotic proteins could contribute to the increase in p53-independent apoptosis that was observed in HT-29 cells. We demonstrate that luteolin promotes both cell cycle arrest and apoptosis in the HT-29 colon cancer cell line, providing insight about the mechanisms underlying its antitumorigenic activities.
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Latham, K. M., S. W. Eastman, A. Wong, and P. W. Hinds. "Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases." Molecular and Cellular Biology 16, no. 8 (August 1996): 4445–55. http://dx.doi.org/10.1128/mcb.16.8.4445.
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Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.
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Alam, Samina, Brian S. Bowser, Michael J. Conway, Mohd Israr, Eric J. Ryndock, Long Fu Xi, and Craig Meyers. "Downregulation of Cdc2/CDK1 Kinase Activity Induces the Synthesis of Noninfectious Human Papillomavirus Type 31b Virions in Organotypic Tissues Exposed to Benzo[a]pyrene." Journal of Virology 84, no. 9 (February 24, 2010): 4630–45. http://dx.doi.org/10.1128/jvi.02431-09.
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ABSTRACT Epidemiological studies suggest that human papillomavirus (HPV)-infected women who smoke face an increased risk for developing cervical cancer. We have previously reported that exposure of HPV-positive organotypic cultures to benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, resulted in enhanced viral titers. Since BaP is known to deregulate multiple pathways of cellular proliferation, enhanced virion synthesis could result from carcinogen/host cell interaction. Here, we report that BaP-mediated upregulation of virus synthesis is correlated to an altered balance between cell cycle-specific cyclin-dependent kinase (CDK) activity profile compared with controls. Specifically, BaP treatment increased accumulation of hyperphosphorylated retinoblastoma protein (pRb) which coincided with increased cdc2/CDK1 kinase activity, but which further conflicted with the simultaneous upregulation of CDK inhibitors p16INK4 and p27KIP1, which normally mediate pRb hypophosphorylation. In contrast, p21WAF1 and p53 levels remained unchanged. Under these conditions, CDK6 and CDK2 kinase activities were decreased, whereas CDK4 kinase activity remained unchanged. The addition of purvalanol A, a specific inhibitor of CDK1 kinase, to BaP-treated cultures, resulted in the production of noninfectious HPV type 31b (HPV31b) particles. In contrast, infectivity of control virus was unaffected by purvalanol A treatment. BaP targeting of CDK1 occurred independently of HPV status, since BaP treatment also increased CDK1 activity in tissues derived from primary keratinocytes. Our data indicate that HPV31b virions synthesized in the presence of BaP were dependent on BaP-mediated alteration in CDK1 kinase activity for maintaining their infectivity.
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Katsikis, Peter D., Marcos E. Garcia-Ojeda, Javier F. Torres-Roca, Iwan M. Tijoe, Craig A. Smith, Leonore A. Herzenberg, and Leonard A. Herzenberg. "Interleukin-1β Converting Enzyme–like Protease Involvement in Fas-induced and Activation-induced Peripheral Blood T Cell Apoptosis in HIV Infection. TNF-related Apoptosis-inducing Ligand Can Mediate Activation-induced T Cell Death in HIV Infection." Journal of Experimental Medicine 186, no. 8 (October 20, 1997): 1365–72. http://dx.doi.org/10.1084/jem.186.8.1365.
Full textAbstract:
Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)–induced and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals. In this study, we extend and confirm these observations by using an inhibitor of interleukin-1β converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4+ and CD8+ T cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)– induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this AICD is mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different roles in the pathogenesis of HIV infection.
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Koga, Shinichiro, Nobuhiro Yamaguchi, Tomoko Abe, Masayoshi Minegishi, Shigeru Tsuchiya, Masayuki Yamamoto, and Naoko Minegishi. "Cell-Cycle-Dependent Oscillation of GATA2 Expression in Hematopoietic Cells." Blood 108, no. 11 (November 16, 2006): 4199. http://dx.doi.org/10.1182/blood.v108.11.4199.4199.
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Abstract Manipulation of hematopoietic stem cells (HSC) is a key issue in transplantation therapy and regenerative medicine, and methods enhancing proliferation of HSC with keeping pluripotency (self-renewal) are awaited. GATA2 is a transcription factor controlling proliferation with keeping pluripotency of HSC (self-renewal), although simple overexpression experiments of GATA2 do not induce proliferation of HSC. In this study, we showed the cell-cycle dependent oscillation of GATA2 expression, high in S phase but low in G1/S and M phase, in leukemic cells, and also in cord-blood CD34+ cells cultured under stimulation of Notch signals. Using green fluorescent protin-GATA2 fusion protein, we demonstrated the cell-cycle specificity of proteasome- dependent degradation of GATA2, which was inhibited by the treatment with Roscovitine, a selective inhibitor of cyclin- dependent kinase (CDK). Immunoprecipitation- immunoblotting analysis demonstrated phosphorylation of GATA2 at CDK-consensus motifs, S0P+1 and T0P+1, and the interaction of cyclin A, Cdk2, and Cdk4 with GATA2. Furthermore, mutants in CDK-consensus phosphorylation motifs exhibited altered expression profiles of GFP fusion proteins. These results indicate cell-cycle dependent control of GATA2 expression through the phosphorylation and interactions with cyclin-CDK systems, suggesting the possible effect of cell-cycle-specific induction of GATA2 expression on the self-renewal of HSC.
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Strausfeld, U. P., M. Howell, P. Descombes, S. Chevalier, R. E. Rempel, J. Adamczewski, J. L. Maller, T. Hunt, and J. J. Blow. "Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts." Journal of Cell Science 109, no. 6 (June 1, 1996): 1555–63. http://dx.doi.org/10.1242/jcs.109.6.1555.
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Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.
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Chan, F. K., J. Zhang, L. Cheng, D. N. Shapiro, and A. Winoto. "Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4." Molecular and Cellular Biology 15, no. 5 (May 1995): 2682–88. http://dx.doi.org/10.1128/mcb.15.5.2682.
Full textAbstract:
The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G1/S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.
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Greeve, MA, RK Allan, JM Harvey, and JM Bentel. "Inhibition of MCF-7 breast cancer cell proliferation by 5alpha-dihydrotestosterone; a role for p21(Cip1/Waf1)." Journal of Molecular Endocrinology 32, no. 3 (June 1, 2004): 793–810. http://dx.doi.org/10.1677/jme.0.0320793.
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Androgens inhibit the growth of breast cancer cells in vitro and in vivo by mechanisms that remain poorly defined. In this study, treatment of asynchronously growing MCF-7 breast cancer cells with the androgen, 5alpha-dihydrotestosterone (DHT), was shown to inhibit cell proliferation and induce moderate increases in the proportion of G1 phase cells. Consistent with targeting the G1-S phase transition, DHT pretreatment of MCF-7 cultures impeded the serum-induced progression of G1-arrested cells into S phase and reduced the kinase activities of cyclin-dependent kinase (Cdk)4 and Cdk2 to less than 50% of controls within 3 days. DHT treatment was associated with greater than twofold increases in the levels of the Cdk inhibitor, p27(Kip1), while p21(Cip1/Waf1) protein levels remained unchanged. During the first 24 h of DHT treatment, levels of Cdk4-associated p21(Cip1/Waf1) and p27(Kip1) were reduced coinciding with decreased levels of Cdk4-associated cyclin D3. In contrast, DHT treatment caused increased accumulation of Cdk2-associated p21(Cip1/Waf1), with no significant alterations in levels of p27(Kip1) bound to Cdk2 complexes. These findings suggest that DHT reverses the Cdk4-mediated titration of p21(Cip1/Waf1) and p27(Kip1) away from Cdk2 complexes, and that the increased association of p21(Cip1/Waf1) with Cdk2 complexes in part mediates the androgen-induced growth inhibition of breast cancer cells.
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Yang, Ying, Yujun Dai, Xuejiao Yang, Songfang Wu, and Yueying Wang. "DNMT3A Mutation-Induced CDK1 Overexpression Promotes Leukemogenesis by Modulating the Interaction between EZH2 and DNMT3A." Biomolecules 11, no. 6 (May 22, 2021): 781. http://dx.doi.org/10.3390/biom11060781.
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DNMT3A mutations are frequently identified in acute myeloid leukemia (AML) and indicate poor prognosis. Previously, we found that the hotspot mutation DNMT3A R882H could upregulate CDK1 and induce AML in conditional knock-in mice. However, the mechanism by which CDK1 is involved in leukemogenesis of DNMT3A mutation-related AML, and whether CDK1 could be a therapeutic target, remains unclear. In this study, using fluorescence resonance energy transfer and immunoprecipitation analysis, we discovered that increased CDK1 could compete with EZH2 to bind to the PHD-like motif of DNMT3A, which may disturb the protein interaction between EZH2 and DNMT3A. Knockdown of CDK1 in OCI-AML3 cells with DNMT3A mutation markedly inhibited proliferation and induced apoptosis. CDK1 selective inhibitor CGP74514A (CGP) and the pan-CDK inhibitor flavopiridol (FLA) arrested OCI-AML3 cells in the G2/M phase, and induced cell apoptosis. CGP significantly increased CD163-positive cells. Moreover, the combined application of CDK1 inhibitor and traditional chemotherapy drugs synergistically inhibited proliferation and induced apoptosis of OCI-AML3 cells. In conclusion, this study highlights CDK1 overexpression as a pathogenic factor and a potential therapeutic target for DNMT3A mutation-related AML.
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Chen, J., P. Saha, S. Kornbluth, B. D. Dynlacht, and A. Dutta. "Cyclin-binding motifs are essential for the function of p21CIP1." Molecular and Cellular Biology 16, no. 9 (September 1996): 4673–82. http://dx.doi.org/10.1128/mcb.16.9.4673.
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The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.
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Li, Lequn, Yoshiko Iwamoto, Alla Berezovskaya, and Vassiliki A. Boussiotis. "A Novel p27kip1-Smad3 Regulated Pathway Is Involved in Induction of T Cell Tolerance." Blood 108, no. 11 (November 16, 2006): 867. http://dx.doi.org/10.1182/blood.v108.11.867.867.
Full textAbstract:
Abstract Induction and maintenance of peripheral tolerance is essential for homeostasis of the immune system. In vivo studies demonstrate the significance of tolerance induction in preventing autoimmunity, graft rejection and GVHD. Upregulation of the cyclin-dependent kinase inhhibitor, p27, correlates with induction of T cell tolerance in vitro and in vivo. p27 interacts with cdk2, cdc2, grb2, and Rho family GTPases. Extensive studies support an essential role of cdks, particularly cdk2, in cell cycle re-entry. Cdk2 promotes cell cycle progression in part by phosphorylating Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors. Recent work indicates that cdk2 phosphorylates Smad2 and Smad3. Smad3 inhibits progression from G1 to S phase, and impaired phosphorylation on the cdk-specific sites renders it more effective in executing this function. In contrast, cdk-mediated phosphorylation of Smad3 reduces Smad3 transcriptional activity and antiproliferative function. In spite the strong correlation between p27 expression level and T cell tolerance, it remains unclear whether p27 has a causative role in induction of tolerance. Here, we examined the role of p27 during induction of tolerance of naïve T cells in vivo, using RAG2 deficient, DO11.10 TCR-transgenic T cells that lack the cyclin-cdk-binding domain of p27 (p27Δ) thereby disrupting only the interactions of p27 with cyclin-cdk complexes. We adoptively transferred CD4+ T cells from RAG2−/−DO11.10 TCR-transgenic mice (DO11.10) or RAG2−/−DO11.10 TCR-transgenic p27Δ mice (DO11.10/p27Δ) into syngeneic wild-type recipients and compared the development of immune responses to immunogenic or tolerizing stimulus in vivo. Following exposure to immunogenic or tolerizing stimulus, DO11.10 and DO11.10/p27Δ CD4+ T cells underwent equal numbers of divisions in vivo, and both cell types exhibited reduced number of divisions in response to tolerizing stimulus. Strikingly, only wild-type DO11.10 TCR-transgenic T cells were tolerized as determined by impaired cyclin E activation, proliferation, and IL-2 production upon antigen-specific rechallenge. Compared to primed wild-type DO11.10 cells, tolerized wild-type DO11.10 cells exhibited impaired cdk2 and cdc2 activity, reduced levels of Smad3 phosphorylation on cdk-specific sites, and increased Smad3-transactivation leading to upregulation of the cdk4/6-specific cdk inhibitor p15. In contrast, after either priming or tolerizing stimulus, DO11.10/p27Δ cells exhibited comparable cdk2 and cdc2 activity, cdk-mediated phosphorylation of Smad3, low-level Smad3 transactivation, and no upregulation of p15. Furthermore, knockdown of Smad3 by expression of Smad3 shRNA in wild-type DO11.10 T cells recapitulated the functional and molecular findings observed in DO11.10/p27Δ cells, preventing induction of tolerance and upregulation of p15, and resulting in production of IL-2 and cell cycle progression. In contrast, expression of Smad3 mutant resistant to cdk-mediated phosphorylation in DO11.10/p27Δ cells recapitulated the molecular and functional effects of tolerance and resulted in inhibition of IL-2 production, upregulation of p15 and blockade of cell cycle progression. These results show that p27 plays a causative role in the induction of tolerance of naïve T cells and Smad3 is a critical component of a pathway downstream of p27 regulating the induction of tolerance in vivo.
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Planas-Silva, M. D., and R. A. Weinberg. "Estrogen-dependent cyclin E-cdk2 activation through p21 redistribution." Molecular and Cellular Biology 17, no. 7 (July 1997): 4059–69. http://dx.doi.org/10.1128/mcb.17.7.4059.
Full textAbstract:
In order to elucidate the mechanisms by which estrogens and antiestrogens modulate the growth of breast cancer cells, we have characterized the changes induced by estradiol that occur during the G1 phase of the cell cycle of MCF-7 human mammary carcinoma cells. Addition of estradiol relieves the cell cycle block created by tamoxifen treatment, leading to marked activation of cyclin E-cdk2 complexes and phosphorylation of the retinoblastoma protein within 6 h. Cyclin D1 levels increase significantly while the levels of cyclin E, cdk2, and the p21 and p27 cdk inhibitors are relatively constant. However, the p21 cdk inhibitor shifts from its association with cyclin E-cdk2 to cyclin D1-cdk4, providing an explanation for the observed activation of the cyclin E-cdk2 complexes. These results support the notion that cyclin D1 has an important role in steroid-dependent cell proliferation and that estrogen, by regulating the activities of G1 cyclin-dependent kinases, can control the proliferation of breast cancer cells.
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Turco, MC, F. Alfinito, M. De Felice, A. Lamberti, S. Ferrone, and S. Venuta. "T-cell malignancies with mature phenotypes: altered cell cycle regulation by HLA class I molecules." Blood 78, no. 8 (October 15, 1991): 2045–52. http://dx.doi.org/10.1182/blood.v78.8.2045.2045.
Full textAbstract:
Abstract Soluble anti-HLA class I monoclonal antibodies (MoAbs) modulate normal T-lymphocyte proliferation induced via the CD3/Ti and the CD2 pathway, but do not induce proliferation of normal T lymphocytes in the absence of additional mitogenic stimuli. In this report, we show that anti-HLA class I MoAbs induce DNA synthesis in peripheral blood mononuclear cells from a patient with a CD4+CD8+T-prolymphocytic leukemia (T-PLL) and from a patient with a CD4-CD8+ T-chronic lymphocytic leukemia (T- CLL), in the absence of detectable additional mitogenic stimuli. Proliferation of leukemic T cells is induced by both whole Igs and Fab' fragments of anti-HLA class I MoAbs, arguing in favor of their direct interactions with the proliferating cells as the mechanism underlying the mitogenic effect. This interpretation is also supported by the ability of anti-HLA class I MoAbs to induce proliferation of leukemic T- cell preparations, depleted of accessory cells. DNA synthesis in T-CLL and T-PLL cells is preceded by expression of G1-specific messenger RNAs, ie. c-myc, 2F1, Tac, and interferon-gamma, in activated cells. Cell proliferation is inhibited by the protein kinase C inhibitor H7, indicating that activation of this enzyme is required for the mitogenic effect of anti-HLA class I MoAbs. The latter inhibit the proliferation of T-CLL cells as well as that of normal T cells stimulated with anti- CD3 MoAbs and enhance that of both types of cells stimulated with anti- CD2 MoAbs. In addition, anti-HLA class I MoAb Q6/64 in combination with anti-CD2 MoAb 9.6 or MoAb 9–1 induces proliferation of leukemic T cells to a greater extent than the individual MoAbs, but is not mitogenic for normal T cells. Anti-HLA class I MoAbs restore the cytolytic activity of T-CLL cells that is lost after 5 days of incubation of control medium, suggesting that HLA class I antigens may mediate a signal contributing to the activation state. The present results indicate that leukemic T-cell proliferation can be triggered via HLA class I molecules and suggest a potential role for these antigens in the in vivo growth of malignant clones.
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Turco, MC, F. Alfinito, M. De Felice, A. Lamberti, S. Ferrone, and S. Venuta. "T-cell malignancies with mature phenotypes: altered cell cycle regulation by HLA class I molecules." Blood 78, no. 8 (October 15, 1991): 2045–52. http://dx.doi.org/10.1182/blood.v78.8.2045.bloodjournal7882045.
Full textAbstract:
Soluble anti-HLA class I monoclonal antibodies (MoAbs) modulate normal T-lymphocyte proliferation induced via the CD3/Ti and the CD2 pathway, but do not induce proliferation of normal T lymphocytes in the absence of additional mitogenic stimuli. In this report, we show that anti-HLA class I MoAbs induce DNA synthesis in peripheral blood mononuclear cells from a patient with a CD4+CD8+T-prolymphocytic leukemia (T-PLL) and from a patient with a CD4-CD8+ T-chronic lymphocytic leukemia (T- CLL), in the absence of detectable additional mitogenic stimuli. Proliferation of leukemic T cells is induced by both whole Igs and Fab' fragments of anti-HLA class I MoAbs, arguing in favor of their direct interactions with the proliferating cells as the mechanism underlying the mitogenic effect. This interpretation is also supported by the ability of anti-HLA class I MoAbs to induce proliferation of leukemic T- cell preparations, depleted of accessory cells. DNA synthesis in T-CLL and T-PLL cells is preceded by expression of G1-specific messenger RNAs, ie. c-myc, 2F1, Tac, and interferon-gamma, in activated cells. Cell proliferation is inhibited by the protein kinase C inhibitor H7, indicating that activation of this enzyme is required for the mitogenic effect of anti-HLA class I MoAbs. The latter inhibit the proliferation of T-CLL cells as well as that of normal T cells stimulated with anti- CD3 MoAbs and enhance that of both types of cells stimulated with anti- CD2 MoAbs. In addition, anti-HLA class I MoAb Q6/64 in combination with anti-CD2 MoAb 9.6 or MoAb 9–1 induces proliferation of leukemic T cells to a greater extent than the individual MoAbs, but is not mitogenic for normal T cells. Anti-HLA class I MoAbs restore the cytolytic activity of T-CLL cells that is lost after 5 days of incubation of control medium, suggesting that HLA class I antigens may mediate a signal contributing to the activation state. The present results indicate that leukemic T-cell proliferation can be triggered via HLA class I molecules and suggest a potential role for these antigens in the in vivo growth of malignant clones.
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Albrecht, Jeffrey H., Brenda M. Rieland, Christopher J. Nelsen, and Cory L. Ahonen. "Regulation of G1 cyclin-dependent kinases in the liver: role of nuclear localization and p27 sequestration." American Journal of Physiology-Gastrointestinal and Liver Physiology 277, no. 6 (December 1, 1999): G1207—G1216. http://dx.doi.org/10.1152/ajpgi.1999.277.6.g1207.
Full textAbstract:
Recent studies suggest that cyclin D1 mediates progression of hepatocytes through G1 phase of the cell cycle. The present study further examines the regulation of cyclin D1-dependent kinase activity and the interplay between cyclin D1 and other G1phase regulatory proteins during liver regeneration. After 70% partial hepatectomy in rats, there was upregulation of kinase activity associated with cyclins (A, D1, D3, and E), cyclin-dependent kinases (Cdk2 and Cdk4), and Cdk-inhibitory proteins (p27, p107, and p130). Although cyclin D1/Cdk4 complexes were more abundant in the cytoplasmic fraction after partial hepatectomy, kinase activity was detected primarily in the nuclear fraction. Cytoplasmic cyclin D1/Cdk4 complexes were activated by recombinant cyclin H/Cdk7. Because endogenous Cdk7 activity was found in the nucleus, this suggests that activation of cyclin D1/Cdk4 requires nuclear importation and subsequent phosphorylation by cyclin H/Cdk7. Recombinant cyclin E/Cdk2 was inhibited by extracts from quiescent liver, and cyclin D1 could titrate out this inhibitory activity. Induction of cyclin D1 was accompanied by increased abundance of cyclin D1/p27 complexes, and most p27 was sequestered by cyclin D1 after partial hepatectomy. Thus cyclin D1 appears to play two roles during G1 phase progression in the regenerating liver: it forms a nuclear kinase complex, and it promotes activation of Cdk2 by sequestering inhibitory proteins such as p27. These experiments underscore the complexity of cyclin/Cdk regulatory networks in the regenerating liver.
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Shibuya, Kazuko, Jun Shirakawa, Tomie Kameyama, Shin-ichiro Honda, Satoko Tahara-Hanaoka, Akitomo Miyamoto, Masafumi Onodera, et al. "CD226 (DNAM-1) Is Involved in Lymphocyte Function–associated Antigen 1 Costimulatory Signal for Naive T Cell Differentiation and Proliferation." Journal of Experimental Medicine 198, no. 12 (December 15, 2003): 1829–39. http://dx.doi.org/10.1084/jem.20030958.
Full textAbstract:
Upon antigen recognition by the T cell receptor, lymphocyte function–associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12–independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1–mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.
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Davison, Elizabeth A., Christine S. L. Lee, Matthew J. Naylor, Samantha R. Oakes, Robert L. Sutherland, Lothar Hennighausen, Christopher J. Ormandy, and Elizabeth A. Musgrove. "The Cyclin-Dependent Kinase Inhibitor p27 (Kip1) Regulates Both DNA Synthesis and Apoptosis in Mammary Epithelium But Is Not Required for Its Functional Development during Pregnancy." Molecular Endocrinology 17, no. 12 (December 1, 2003): 2436–47. http://dx.doi.org/10.1210/me.2003-0199.
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Abstract Decreased expression of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is common in breast cancer and is associated with poor prognosis. p27 is also an important mediator of steroidal regulation of cell cycle progression. We have therefore investigated the role of p27 in mammary epithelial cell proliferation. Examination of the two major functions of p27, assembly of cyclin D1-Cdk4 complexes and inhibition of Cdk2 activity, revealed that cyclin D1-Cdk4 complex formation was not impaired in p27−/− mammary epithelial cells in primary culture. However, cyclin E-Cdk2 activity was increased approximately 3-fold, indicating that the CDK inhibitory function of p27 is important in mammary epithelial cells. Increased epithelial DNA synthesis was observed during pregnancy in p27−/− mammary gland transplants, but this was paralleled by increased apoptosis. During pregnancy and at parturition, development and differentiation of p27+/+ and p27−/− mammary tissue were indistinguishable. These results demonstrate a role for p27 in both the proliferation and survival of mammary epithelial cells. However, the absence of morphological and cellular defects in p27−/− mammary tissue during pregnancy raises the possibility that loss of p27 in breast cancer may not confer an overall growth advantage unless apoptosis is also impaired.
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Moulian, Nathalie, Jocelyne Bidault, Claude Planché, and Sonia Berrih-Aknin. "Two Signaling Pathways Can Increase Fas Expression in Human Thymocytes." Blood 92, no. 4 (August 15, 1998): 1297–307. http://dx.doi.org/10.1182/blood.v92.4.1297.
Full textAbstract:
Abstract Fas, a cell surface receptor, can induce apoptosis after cross-linking with its ligand. Fewer than 3% of human thymocytes strongly express Fas. We report that Fas antigen expression can be upregulated by two signaling pathways in vitro, one mediated by anti-CD3 and the other by interleukin-7 + interferon-γ. The two signaling pathways differed in several respects. (1) Fas expression increased in all thymic subsets after cytokine activation, but only in the CD4 lineage after anti-CD3 activation. (2) Fas upregulation was inhibited by cyclosporin A (a calcineurin inhibitor) in anti-CD3–activated but not in cytokine-activated thymocytes. (3) Cycloheximide (a metabolic inhibitor) inhibited Fas upregulation in cytokine-activated thymocytes but not in anti-CD3–activated thymocytes. (4) Cytokine-activated thymocytes were more susceptible than anti-CD3–activated thymocytes to Fas-induced apoptosis, a difference mainly accounted for by CD4+ cells. The nature of the stimulus might thus influence the susceptibility of human thymocytes to Fas-induced apoptosis. © 1998 by The American Society of Hematology.
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Moulian, Nathalie, Jocelyne Bidault, Claude Planché, and Sonia Berrih-Aknin. "Two Signaling Pathways Can Increase Fas Expression in Human Thymocytes." Blood 92, no. 4 (August 15, 1998): 1297–307. http://dx.doi.org/10.1182/blood.v92.4.1297.416k27_1297_1307.
Full textAbstract:
Fas, a cell surface receptor, can induce apoptosis after cross-linking with its ligand. Fewer than 3% of human thymocytes strongly express Fas. We report that Fas antigen expression can be upregulated by two signaling pathways in vitro, one mediated by anti-CD3 and the other by interleukin-7 + interferon-γ. The two signaling pathways differed in several respects. (1) Fas expression increased in all thymic subsets after cytokine activation, but only in the CD4 lineage after anti-CD3 activation. (2) Fas upregulation was inhibited by cyclosporin A (a calcineurin inhibitor) in anti-CD3–activated but not in cytokine-activated thymocytes. (3) Cycloheximide (a metabolic inhibitor) inhibited Fas upregulation in cytokine-activated thymocytes but not in anti-CD3–activated thymocytes. (4) Cytokine-activated thymocytes were more susceptible than anti-CD3–activated thymocytes to Fas-induced apoptosis, a difference mainly accounted for by CD4+ cells. The nature of the stimulus might thus influence the susceptibility of human thymocytes to Fas-induced apoptosis. © 1998 by The American Society of Hematology.
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Sandhu, C., J. Garbe, N. Bhattacharya, J. Daksis, C. H. Pan, P. Yaswen, J. Koh, J. M. Slingerland, and M. R. Stampfer. "Transforming growth factor beta stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells." Molecular and Cellular Biology 17, no. 5 (May 1997): 2458–67. http://dx.doi.org/10.1128/mcb.17.5.2458.
Full textAbstract:
The effects of transforming growth factor beta (TGF-beta) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1S, and 184A1L5R, which differ in their cell cycle responses to TGF-beta but express type I and type II TGF-beta receptors and retain TGF-beta induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-beta was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-beta-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p5INK4B protein stability. As cdk4- and cdk6-associated p15INK4B increased during TGF-beta arrest of sensitive cells, there was a loss of cyclin D1, p21Cip1, and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15INK4B complexes did not contain detectable cyclin. p15INK4B from both sensitive and resistant cells could displace in vitro cyclin D1, p21Cip1, and p27Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5R cell lysates. Thus, in TGF-beta arrest, p15INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-beta-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-beta is underlined by the observation that in TGF-beta-resistant 184A1L5R, although the p15 transcript increased, p15INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited.
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Franklin, David S., Virginia L. Godfrey, Deborah A. O'Brien, Chuxia Deng, and Yue Xiong. "Functional Collaboration between Different Cyclin-Dependent Kinase Inhibitors Suppresses Tumor Growth with Distinct Tissue Specificity." Molecular and Cellular Biology 20, no. 16 (August 15, 2000): 6147–58. http://dx.doi.org/10.1128/mcb.20.16.6147-6158.2000.
Full textAbstract:
ABSTRACT The presence of two families of seven distinct mammalian cyclin-dependent kinase (CDK) inhibitor genes is thought to mediate the complexity of connecting a variety of cellular processes to the cell cycle control pathway. The distinct pattern of tissue expression of CDK inhibitor genes suggests that they may function as tumor suppressors with different tissue specificities. To test this hypothesis, we have characterized two strains of double mutant mice lacking either p18INK4c and p27KIP1 or p18INK4cand p21CIP1/WAF1. Loss of both p18 and p27 function resulted in the spontaneous development by 3 months of age of at least eight different types of hyperplastic tissues and/or tumors in the pituitary, adrenals, thyroid, parathyroid, testes, pancreas, duodenum, and stomach. Six of these hyperplastic tissues and tumors were in endocrine organs, and several types of tumors routinely developed within the same animal, a phenotype reminiscent of that seen in combined human multiple endocrine neoplasia syndromes. The p18-p21 double null mice, on the other hand, developed pituitary adenomas, multifocal gastric neuroendocrine hyperplasia, and lung bronchioalveolar tumors later in life. G1 CDK2 and CDK4 kinase activities were increased in both normal and neoplastic tissues derived from mice lacking individual CDK inhibitors and were synergistically stimulated by the simultaneous loss of two CDK inhibitors. This indicates that an increase in G1 CDK kinase activity is a critical step during but is not sufficient for tumor growth. Our results suggest that functional collaborations between distinct CDK inhibitor genes are tissue specific and confer yet another level of regulation in cell growth control and tumor suppression.