Cell cycle arrest by the isoprenoids perillyl alcohol, geraniol, and farnesol is mediated by p21(Cip1) and p27(Kip1) in human pancreatic adenocarcinoma cells

Pancreatic cancer, the fourth leading cause of cancer-associated mortality in the United States, usually presents in an advanced stage and is generally refractory to chemotherapy. As such, there is a great need for novel therapies for this disease. The naturally derived isoprenoids perillyl alcohol, farnesol, and geraniol have chemotherapeutic potential in pancreatic and other tumor types. However, their mechanisms of action in these systems are not completely defined. In this study, we investigated isoprenoid effects on the cell cycle and observed a similar antiproliferative mechanism of action among the three compounds. First, when given in combination, the isoprenoids exhibited an additive antiproliferative effect against MIA PaCa-2 human pancreatic cancer cells. Furthermore, all three compounds induced a G(0)/G(1) cell cycle arrest that coincided with an increase in the expression of the cyclin kinase inhibitor proteins p21(Cip1) and p27(Kip1) and a reduction in cyclin A, cyclin B1, and cyclin-dependent kinase (Cdk) 2 protein levels. Immunoprecipitation studies demonstrated increased association of both p21(Cip1) and p27(Kip1) with Cdk2 as well as diminished Cdk2 kinase activity after isoprenoid exposure, indicating a cell cycle-inhibitory role for p21(Cip1) and p27(Kip1) in pancreatic adenocarcinoma cells. When siRNA was used to inhibit expression of p21(Cip1) and p27(Kip1) proteins in MIA PaCa-2 cells, conditional resistance to all three isoprenoid compounds was evident. Given similar findings in this cell line and in BxPC-3 human pancreatic adenocarcinoma cells, we conclude that the chemotherapeutic isoprenoid compounds perillyl alcohol, farnesol, and geraniol invoke a p21(Cip1)- and p27(Kip1)-dependent antiproliferative mechanism in human pancreatic adenocarcinoma cells.     

Primitive hematopoietic cells resist HIV-1 infection via p21

Hematopoietic stem cells are resistant to HIV-1 infection. Here, we report a novel mechanism by which the cyclin-dependent kinase inhibitor (CKI) p21(Waf1/Cip1/Sdi1) (p21), a known regulator of stem cell pool size, restricts HIV-1 infection of primitive hematopoietic cells. Modifying p21 expression altered HIV-1 infection prior to changes in cell cycling and was selective for p21 since silencing the related CKIs, p27(Kip1) and p18(INK4C), had no effect on HIV-1. We show that p21 blocked viral infection by complexing with HIV-1 integrase and aborting chromosomal integration. A closely related lentivirus with a distinct integrase, SIVmac-251, and the other cell-intrinsic inhibitors of HIV-1, Trim5alpha, PML, Murr1, and IFN-alpha, were unaffected by p21. Therefore, p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain how these cells remain an uninfected "sanctuary" in HIV disease.     

IL-7 promotes T cell proliferation through destabilization of p27Kip1

Interleukin (IL)-7 is required for survival and homeostatic proliferation of T lymphocytes. The survival effect of IL-7 is primarily through regulation of Bcl-2 family members; however, the proliferative mechanism is unclear. It has not been determined whether the IL-7 receptor actually delivers a proliferative signal or whether, by promoting survival, proliferation results from signals other than the IL-7 receptor. We show that in an IL-7-dependent T cell line, cells protected from apoptosis nevertheless underwent cell cycle arrest after IL-7 withdrawal. This arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 through a posttranslational mechanism. Overexpression of p27Kip1 induced G1 arrest in the presence of IL-7, whereas knockdown of p27Kip1 by small interfering RNA promoted S phase entry after IL-7 withdrawal. CD4 or CD8 T cells transferred into IL-7-deficient hosts underwent G1 arrest, whereas 27Kip1-deficient T cells underwent proliferation. We observed that IL-7 withdrawal activated protein kinase C (PKC)theta and that inhibition of PKCtheta with a pharmacological inhibitor completely blocked the rise of p27Kip1 and rescued cells from G1 arrest. The conventional pathway to breakdown of p27Kip1 is mediated by S phase kinase-associated protein 2; however, our evidence suggests that PKCtheta acts via a distinct, unknown pathway inducing G1 arrest after IL-7 withdrawal from T cells. Hence, IL-7 maintains T cell proliferation through a novel pathway of p27Kip1 regulation.     

Quantification of the cell-cycle inhibitors p27(Kip1) and p21(Cip1) in human atherectomy specimens: primary stenosis versus restenosis

Proliferation, a key determinator of vascular proliferative diseases, is dependent on cyclin/cyclin-dependent kinase (CDK) complexes, which are controlled by cyclin-dependent kinase inhibitors (CKIs) such as p27(Kip1) and p21(Cip1). Both have prognostic significance in various human malignancies. We have determined the levels of p27(Kip1) and p21(Cip1) in human directional coronary atherectomy specimens of primary lesions (n = 15) and lesions of in-stent restenosis (n = 18) in comparison to those of other vascular regions and have correlated CKI levels with clinical data. Quantitative immunoblotting demonstrated low expression of p27(Kip1) in primary lesions (5.9 +/- 0.5 ng/mg protein) compared with that in aorta (14.9 +/- 0.9 ng/mg), internal mammary artery (16.7 +/- 1.1 ng/mg), and carotid artery thrombendarterectomy specimens (16.5 +/- 1.7 ng/mg). Similarly, p27(Kip1) levels in lesions of in-stent restenosis were found to be significantly reduced (6.3 +/- 1.1 ng/mg; mean time of restenosis development 367 +/- 61 days). p27(Kip1) levels did, however, not have prognostic significance for the development of restenosis, and expression levels of proliferating cell nuclear antigen and CDK2 were similar in all groups examined, indicating low proliferative activity. Clinically, p27(Kip1) was not of value in predicting the development of restenosis. Furthermore, p27(Kip1) tissue levels were not increased in statin-treated patients, implying that the favorable effect of these drugs is not a result of p27(Kip1) stabilization. However, the relative content of p21(Cip1) was found to be significantly up-regulated in restenosis compared with that in primary lesions (225%) and the other vascular regions. Our data imply that negative-feedback mechanisms are still intact in coronary proliferative disease, thereby contrasting the finding of deregulated proliferation in malignancies.     

Bombyx mori cyclin‐dependent kinase inhibitor is involved in regulation of the silkworm cell cycle

Cyclin‐dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin‐dependent kinase (CDK)‐cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654‐bp‐long BmCKI‐L (the longer splice variant) encoding a protein with 217 amino acids and a 579‐bp‐long BmCKI‐S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI‐L and BmCKI‐S contain the Cip/Kip family conserved cyclin‐binding domain and the CDK‐binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181–210. Overexpression of BmCKI‐L or BmCKI‐S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI‐L or BmCKI‐S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI‐L and BmCKI‐S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI‐L overexpression (BmCKI‐L‐OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI‐L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm.     

The degradation of p27 and cancer

The cell cycle of eukaryotic cells is regulated by a series of protein complexes composed of cyclins and cyclin-dependent kinases (CDKs), the activity of which is suppressed by a group of CDK inhibitors (CKIs). Among the CKIs, p27 plays a pivotal role in the control of cell proliferation. Degradation of p27 is a critical event for reentry of cells into the cell cycle from G0 phase and occurs through ubiquitination by two ubiquitin ligase complexes (KPC and SCFSkP2) and subsequent degradation by the 26S-proteasome. A tumor suppressing function of p27 has been demonstrated in mouse models and studies of human tumors. This review will focus on the regulation of p27 proteolysis and its consequences for tumorigenesis.     

The synthetic ligand of peroxisome proliferator-activated receptor-gamma ciglitazone affects human glioblastoma cell lines

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that ligand activation of peroxisome proliferator-activated receptor (PPAR)-gamma can induce differentiation and inhibit proliferation of several cancer cells. In this study, we have investigated whether one PPARgamma ligand in particular, ciglitazone, inhibits cell viability and, additionally, whether it affects the cell cycle and apoptosis of human glioblastoma cell lines T98G, U-87 MG, A172, and U-118 MG. All glioblastoma cell lines were found to express PPARgamma protein, and following treatment with ciglitazone, localization was unchanged. Ciglitazone inhibited viability in a dose-dependent manner in all four tested glioblastoma cells after 24 h of treatment. Analysis of the cell cycle showed arrest in the G(1) phase and partial block in G(2)/M phase of the cell cycle. Cyclin D1 and cyclin B expression was decreased. Phosphorylation of Rb protein dropped as well. We found that ciglitazone was followed by increased expression of p27(Kip1) and p21(Waf1/Cip1). It also led to apoptosis induction: bax expression in T98G was elevated. Expression of the antiapoptotic protein bcl-2 was reduced in U-118 MG and U-87 MG and showed a slight decrease in A172 cells. Flow cytometry confirmed the induction of apoptosis. Moreover, PPARgamma ligand decreased telomerase activity in U-87 MG and U-118 MG cell lines. Our results demonstrate that ciglitazone inhibits the viability of human glioblastoma cell lines via induction of apoptosis; as a result, this ligand may offer potential new therapy for the treatment of central nervous system neoplasms.     

HMG-CoA reductase inhibitor simvastatin inhibits cell cycle progression at the G1/S checkpoint in immortalized lymphocytes from Alzheimer's disease patients independently of cholesterol-lowering effects

Recent work has suggested that statins may exert beneficial effects on patients suffering from Alzheimer's disease (AD). The pharmacological effects of statins extend beyond their cholesterol-lowering properties. Based on the antineoplastic and apoptotic effects of statins in several cell types, we hypothesized that statins may be able to protect neurons by controlling the regulation of cell cycle. A growing body of evidence indicates that neurodegeneration involves the activation of cell cycle machinery in postmitotic neurons. We and others have presented direct evidence to support the hypothesis that the failure of cell cycle control is not restricted to neurons in AD patients, but that it occurs in peripheral cells as well. For these reasons, we found it worthy to study the role of simvastatin on cell proliferation in immortalized lymphocytes from AD patients. We report here that simvastatin (SIM) inhibits the serum-mediated enhancement of cell proliferation in AD by blocking the events critical for G(1)/S transition. SIM induces a partial blockade of retinoblastoma protein phosphorylation and inhibition of cyclin E/cyclin-dependent kinase (CDK)2 activity associated with increased levels of the CDK inhibitors p21(Cip1) and p27(kip1). These effects of SIM on AD lymphoblasts are dependent on inhibition of the proteasome-mediated degradation of p21 and p27 proteins. The antiproliferative effect of this natural statin may provide a therapeutic approach for AD disease.     

Differential modulation of signaling pathways and apoptosis of ras-transformed 10T1/2 cells by the depsipeptide FR901228

(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone (FR901228), a natural anticancer depsipeptide, induces apoptosis of ras-transformed 10T1/2 cells whereas it induces growth arrest of nontransformed counterpart cells in G0/G1 phase of the cell cycle. Our study of the effect of FR901228 treatment on intracellular signaling pathways reveals a discriminating activity of FR901228 to regulate signaling cascades differently in ras-transformed 10T1/2 cells and nontransformed counterpart cells. Induction of apoptosis of ras-transformed cells by FR901228 correlates with suppression of the extracellular signal-regulated kinase (ERK) signaling pathway through reduction of Raf expression and deactivation of Mek and Erk, inhibition of the phosphoinositide-3 kinase (PI3-K) pathway indexed by suppression of Akt activity, suppression of p38 activity, and activation of caspase-3. Expression of p21(Cip1) is not induced in ras-transformed cultures undergoing apoptosis induced by FR901228. In contrast, FR901228 induces p21(Cip1) expression in nontransformed counterpart cultures growth-arrested in G0/G1 that is also accompanied by moderate induction of the kinase activities of Raf, Mek, Erk, and Akt, but not accompanied by activation of caspase-3 or changes in p38 activity. Our study indicates a potential value of FR901228 in the treatment of cancer cells involving aberrant regulation of Ras through preferential induction of the caspase cascade and suppression of the ERK, PI3-K, and p38 pathways.     

P27^Kip1和TGF-β1在原代APL细胞凋亡中作用机制的研究

本研究探讨三氧化二砷（arsenic trioxide,As203）对原代APL细胞凋亡及周期的影响,As2O3作用前后P27^Kip1和TGF-β1、cyclinE和bcl-2的变化以及P27^Kip1在As2O3诱导急性早幼粒细胞白血病细胞凋亡中的作用。用细胞形态学及流式细胞仪分别检测原代APL细胞凋亡和细胞周期改变,免疫组织化学法和RT-PCR技术检测药物处理前后细胞P27^Kip1、TGF-β1、cyclinE以及BCL-2蛋白和基因表达的变化。结果表明：As2O3与APL细胞共同培养24小时,浓度为1、5、10μmol／L时,凋亡细胞所占比例分别为1．42％、4．57％、10．67％;至48小时,凋亡细胞分别为8．92％、16．07％和18．90％,明显高于相同浓度的As2O3作用24小时（P〈0．05）;至72小时,细胞以碎片为主。同一时段随着As2O3浓度的增加,APL细胞凋亡不断增多;同一浓度As2O3作用于APL细胞,随着作用时间的延长,APL细胞凋亡也随之增多。5、10μmol／L的As2O3作用于APL细胞24、48、72小时可见细胞周期阻滞于G1期,G2期细胞比例均明显高于对照组（P〈0．05）,而1μmol／L As2O3作用后的细胞周期无明显变化。1、5和10μmol／L As2O3作用于APL细胞,P27^Kip1蛋白表达增高,P27Kip1 mRNA与β-actin灰度值之比（P27^Kip1/β-actin）分别为0．181、0．489和0．921,表明随着As2O3浓度的增高,P27Kipt mRNA表达水平显著上调（P〈0．05）,P27^Kip1表达与凋亡细胞数之间存在正相关（r=0．55,P〈0．05）;与对照组相比,TGF-β1蛋白和mRNA表达上调,伴有CyclinE、BCL-2蛋白和mRNA表达下调,TGF-β1表达与凋亡细胞数之间存在正相关（r=0．51,P〈0．05）,TGF-β1表达与P27^Kip1表达之间也存在正相关（r=0．31,P〈0．05）。结论：As2O3体外可诱导原代APL细胞凋亡,存在时间-剂量依赖关系,并使细胞周期阻滞于G1期。P27^Kip1表达的变化情况与As2O3诱导细胞凋亡的作用效果密切相关,As2O3诱导原代APL细胞凋亡的机制可能是通过上调TGF-β1,从而上调P27^Kip1拮抗CvclinE和BCL-2的作用,抑制细胞增殖,使细胞周期阻滞于G1期,从而诱导细胞发生凋亡。     

Curcumin induces G0/G1 arrest and apoptosis in hormone independent prostate cancer DU-145 cells by down regulating Notch signaling

ObjectiveCurcumin as an effective anticancer bioactive extract has been proved to induce apoptosis in many cancer cells. Notch signaling regulates prostate cancer apoptosis, but it is still unknown whether curcumin induces apoptosis in DU-145 cells by regulating Notch pathway. The aim of this study was to investigate the effect of curcumin on regulating Notch signaling and provide basic data for using curcumin in prostate cancer therapy.MethodsNotch pathway signal related proteins Notch 1, Jagged-1 and NICD were detected using Western blotting and RT-PCR. The proliferation and apoptosis were determined by MTT method and Elisa kits after curcumin treatment, respectively. Dual-Luciferase Reporter Assay was carried out to confirm that curcumin could target Notching signaling. In order to study whether Notch 1 expression could be downregulated by curcumin, Notch 1 siRNA and Notch 1 plasmid were used in Notch 1 down-regulation and over-expression. The effects of curcumin on cell cycle distribution and apoptosis related proteins expression were analyzed by flow cytometry and western blotting, separately.ResultsWe found that Notch 1 signaling was down regulated in Notch 1 siRNA or Notch 1 plasmid transfected 145 cells after curcumin treatment. Curcumin induced G0/G1 arrest in DU-145 cells, and G0/G1 phase related regulatory factors Cyclin D1 and CDK2 expressions were inhibited. Meanwhile, p21 and p27 were up regulated. The apoptosis related protein p53 expression was increased, and apoptosis suppressor Bcl-2 was inhibited in DU-145 after curcumin treatment. Additionally, Caspase-3 and Caspase-9 were activated by curcumin.ConclusionCurcumin induced apoptosis and G0/G1 arrest in DU-145 cells by down regulating Notch signaling.     

Induction of cell cycle regulatory proteins in anti-immunoglobulin- stimulated mature B lymphocytes

Progression through the cell cycle is a tightly controlled process that integrates signals generated at the plasma membrane with the proteins that form the cell cycle machinery. The current study chronicles the induction of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors in low density primary mouse B lymphocytes after anti- immunoglobulin plus interleukin 4 (IgM + IL-4) stimulation. In this system, > 85% of cells remain in the G0/G1 phase of cell cycle for an initial 24-h period, followed by entry of up to 50% of the cells into S phase, commencing around 30 h and peaking at 48 h. Extensive time course analyses of these anti-IgM + IL-4-stimulated B cells revealed that the G1-associated D-type cyclins D2 and D3 were induced by 3 h after stimulation, and that cyclins E, A, and B were subsequently induced sequentially, beginning at mid-G1, G1/S transition, and S phase, respectively. The G1-associated cyclin D1 was not expressed at any stage of the anti-Ig + IL-4-induced B cell cycle. cdk2, cdk4, and cdk6 were induced during G1, whereas cell division cycle-2 (cdc2) was induced concomitantly with S phase. Irrespective of their expression, the kinases cdk2 and cdc2 were only active from S phase onwards, suggesting that productive cyclin/kinase complex formation did not occur until that time. Cell cycle inhibitors p21 and p19 were induced by anti-Ig + IL-4, peaking in expression at mid-G1 and S phase, respectively. Stimulation of low density B cells with anti-Ig + IL-4 caused rapid down regulation of the p27 inhibitor, however this protein was reexpressed at 54-96 h after stimulation. In contrast, B cells stimulated with anti-CD40, a stimulus which induces long-term B cell proliferation, permanently down regulated p27. These findings are consistent with the concept that p27 reexpression contributes to the G1 arrest that follows antigen receptor crosslinking. Low density B cells cultured in the viability-enhancing cytokine IL-4 alone also showed induction of D2 and D3 cyclin expression. However, the D2 expression was transient, and the D3 expression was substantially lower than that observed in B cells induced to proliferate by anti-Ig + IL-4. This partial induction of D2 and D3 expression may explain IL-4's ability to promote B cell entry into G1 but not S phase of cell cycle, and furthermore, its ability to truncate G1 progression when B cells are subsequently stimulated with anti-Ig.     

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.     

FoxF1和FoxF2转录因子通过抑制p21Cip1 CDK调控早期胃癌能量代谢的机制

目的 探讨FoxFl和FoxF2转录因子通过抑制p21Cipl CDK调控早期胃癌能量代谢的机制.:方法 将对数生长期的HGC27细胞分为FoxFl组、FoxF2组和对照组,分别转染200 ng/mL pcDNA3.0 - FoxFl、200 ng/mL pcDNA3.0-FoxF2和等体积的磷酸盐缓冲液,采用CCK法...     

G(1) Phase growth arrest and induction of p21(Waf1/Cip1/Sdi1) in IB3-1 cells treated with 4-sodium phenylbutyrate

4-Sodium phenylbutyrate (4-PBA) has been used for many years in the treatment of urea cycle defects and has recently been studied as a chemotherapeutic agent for certain malignancies. 4-PBA has been shown to cause growth arrest, cellular differentiation, and apoptosis in certain malignant cells. Recently, it was shown that IB3-1 cells (a cystic fibrosis cell line, Delta508/W128X) treated with 4-PBA demonstrated a partial correction of the cystic fibrosis chloride channel defect. We were interested in evaluating the effect of 4-PBA on cell growth and cell cycle regulation in IB3-1 cells treated with 2 to 10 mM concentrations. We found that cells treated with 2 mM concentrations of 4-PBA for 96 h underwent a significant decrease in cell growth (P <.007). Using flow cytometry, we were able to demonstrate that growth arrest occurred at the G(1) phase of the cell cycle. This was detected as early as 24 h in IB3-1 cells treated with 5 mM 4-PBA (P <.03). Furthermore, the percentage of IB3-1 cells with less than a 2N DNA content increased with higher concentrations of 4-PBA, although this was not associated with an increase in apoptosis. Finally, p21(Waf1/Cip1/Sdi1) protein levels were induced in IB3-1 cells receiving 2 and 5 mM concentrations of 4-PBA as early as 24 h of exposure, suggesting that G(1) phase growth arrest in IB3-1 cells treated with 4-PBA is regulated through the p21(Waf1/Cip1/Sdi1) pathway.