Cyclin-dependent Kinase 2 Protein Levels Research Articles

Maintenance of cellular annexin A1 level is essential for PI3K/AKT/mTOR-mediated proliferation of pancreatic beta cells.

Annexin A1 (AnxA1, also known as lipocortin-1), is a calcium-dependent phospholipid binding protein with diverse functions. Previous studies have indicated that AnxA1 is associated with age-related β-cell dysfunction and aging, which lead to decreased β-cell proliferation capacity. However, it has been uncertain whether AnxA1 affects the proliferation of pancreatic beta (β) cells. In the present study, we reduced AnxA1 expression in the MIN6 islet β-cell line using small interfering RNA (AnxA1-siRNA), then measured cell cycle distribution and cellular proliferation. We also measured the expression levels of cell cycle-related proteins such as cyclin D1, cyclin E, and cyclin-dependent kinase 2 (CDK2) by Western blot analysis. We investigated the phosphatidylinositol 3-kinase (PI3K)/ serine/threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway to explore the potential mechanism underlying the observed effects. Knockdown of AnxA1 expression using siRNA reduced the rates of MIN6 cell proliferation. The proportions of cells in S and G2/M phases also decreased upon inhibition of AnxA1. Moreover, AnxA1 protein expression in MIN6 cells was positively related to the protein levels of cyclin D1, cyclin E, and CDK2. Activation of the PI3K/Akt/mTOR signaling pathway by AnxA1 may be involved in the signaling cascade to regulate cell proliferation. This study identified a positive correlation between AnxA1 protein and pancreatic β-cell proliferation. AnxA1 protein expression might affect the proliferation of MIN6 cells via regulation of cyclin D1, cyclin E, and CDK2 proteins, as well as the PI3K/Akt/mTOR signaling pathway.

Expression of Long Noncoding RNA LIPCAR Promotes Cell Proliferation, Cell Migration, and Change in Phenotype of Vascular Smooth Muscle Cells.

BackgroundThe long noncoding RNA LIPCAR is a type of transcription product (>200 nucleotides long). Recent studies demonstrated that LIPCAR is a potential biomarker in cardiovascular disease and can predict survival in patients with cardiovascular disease. Therefore, the present study explored the role of LIPCAR in the regulation of proliferation, migration, and change in phenotype of vascular smooth muscle cells.Material/MethodsHuman vascular smooth muscle cells (VSMCs) were treated with 20 g/mL oxidatively modified low-density lipoprotein (ox-LDL) or 20 ng/ml platelet-derived growth factor BB (PDGF-BB) for 24 h, then the expression levels of LIPCAR were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. LIPCAR-overexpressing plasmids were transfected into VSMCs. After transfection, cell proliferation and migration were measured using the Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The levels of α-smooth muscle actin (α-SMA) a molecular marker of the contractile VSMC phenotype, were measured using Western blot and immunofluorescence assays. Protein levels of cyclin-dependent kinase-2 (CDK2), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor A (VEGF-A), and angiopoietin-2 (Ang-2) were assessed by Western blot. The level of tissue factor (TF) was measured by enzyme-linked immunosorbent assay (ELISA).ResultsTreatment with PDGF-BB or ox-LDL significantly increased levels of LIPCAR in VSMCs. Overexpression of LIPCAR markedly promoted cell proliferation and migration. Further, upregulation of LIPCAR increased CDK2, p21, PCNA, MMP2, MMP9, VEGF-A, Ang-2, and TF expression and decreased p21 expression. In addition, LIPCAR significantly decreased α-SAM expression.ConclusionsTogether, our data suggest that overexpression of LIPCAR promotes cell proliferation, migration, and phenotypic switch of vascular smooth muscle cells.

Rhomboid domain-containing protein 1 promotes breast cancer progression by regulating the p-Akt and CDK2 levels

BackgroundOur previous work revealed that rhomboid domain-containing protein 1 (RHBDD1) participates in the modulation of cell growth and apoptosis in colorectal cancer cells. This study aimed to investigate the function of RHBDD1 in regulating breast cancer progression and its underlying molecular basis.MethodsImmunohistochemistry was performed to evaluate RHBDD1 expression in 116 breast cancer tissue and 39 adjacent normal tissue and expression of RHBDD1, phospho-Akt (p-Akt) and cyclin-dependent kinase 2 (CDK2) in the same 84 breast cancer specimens. RHBDD1-knock-out cells were established using breast cancer cell lines. In vitro studies were carried out to estimate the function of RHBDD1 in cell proliferation, migration and invasion. Fluorescence microscopy assay and flow cytometric analysis were used to measure apoptosis and cell cycle regulation. RNA sequencing and western blot analysis were used to investigate the molecular mechanisms of RHBDD1.ResultsRHBDD1 was highly up-regulated in breast cancer tissue compared with that in normal tissue and associated with pathological tumor (pT) stage, pathological tumor-node-metastasis (pTNM) stage and estrogen receptor (ER) expression. RHBDD1 up-regulation was associated with poor prognosis in several subtypes of breast cancer. Deletion of RHBDD1 promoted apoptosis and suppressed proliferation, migration and invasion in breast cancer cells. RHBDD1 deletion suppressed Akt activation and decreased CDK2 protein level via proteasome pathway, thus inhibited cell cycle progression and G1/S phase transition. Moreover, the protein level of RHBDD1, p-Akt and CDK2 was significantly positively correlated in breast cancer tissue.ConclusionsOur study reveals that RHBDD1 promotes breast cancer progression by regulating p-Akt and CDK2 protein levels, and might be a potential biomarker and prognostic indicator for breast cancer patients.

Epigallocatechin gallate targets FTO and inhibits adipogenesis in an mRNA m6A-YTHDF2-dependent manner.

N6-methyladenosine (m6A) modification of mRNA plays a role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, plays a critical role in anti-obesity and anti-adipogenesis. High-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HPLC-QqQ-MS/MS) was performed to determine the m6A levels in 3T3-L1 preadipocytes. The effects of EGCG on the m6A levels in specific genes were determined by methylated RNA immunoprecipitation coupled with quantitative real-time PCR (meRIP-qPCR). Several adipogenesis makers and cell cycle genes were analyzed by quantitative real-time PCR (qPCR) and western blotting. Lipid accumulation was evaluated by oil red O staining. All measurements were performed at least for three times. Here we showed that EGCG inhibited adipogenesis by blocking the mitotic clonal expansion (MCE) at the early stage of adipocyte differentiation. Exposing 3T3-L1 cells to EGCG reduced the expression of fat mass and obesity-associated (FTO) protein, an m6A demethylase, which led to increased overall levels of RNA m6A methylation. Cyclin A2 (CCNA2) and cyclin dependent kinase 2 (CDK2) play vital roles in MCE. The m6A levels of CCNA2 and CDK2 mRNA were dramatically enhanced by EGCG. Interestingly, EGCG increased the expression of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), which recognized and decayed methylated mRNAs, resulting in decreased protein levels of CCNA2 and CDK2. As a result, MCE was blocked and adipogenesis was inhibited. FTO overexpression and YTHDF2 knockdown in 3T3-L1 cells significantly increased CCNA2 and CDK2 protein levels and ameliorated the EGCG-induced adipogenesis inhibition. Thus, m6A-dependent CCNA2 and CDK2 expressions mediated by FTO and YTHDF2 contributed to EGCG-induced adipogenesis inhibition. Our findings provide mechanistic insights into how m6A is involved in the EGCG regulation of adipogenesis and shed light on its anti-obesity effect.

MiR-302b regulates cell cycles by targeting CDK2 via ERK signaling pathway in gastric cancer.

To investigate the molecular mechanism of miR‐302b in the regulation of cell proliferation and cell cycle regulation in gastric cancer. Samples of tumor and adjacent normal tissues were collected from 30 gastric cancer patients. Bioinformatics and the dual luciferase report were used for verification of the relationship between miR‐302b expression and cyclin‐dependent kinase 2 (CDK2). RT‐PCR and western blot were used to examine CDK2 mRNA and protein levels. The impacts of miR‐302b on CDK2 expression and extracellular signal‐regulated kinase (ERK) signaling pathway were assessed in cells transfected with miR‐302b analogs and CDK2 overexpression carrier, respectively. We used 3‐(4,5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) to assay gastric cancer cell growth after transfection, flow cytometry to analyze cell cycle. Compared with normal tissues, miR‐302b expression was significantly lower in gastric cancer tissues, which was significantly related to lymph node metastasis, metastasis distance, and TNM staging. miR‐302b expression was increased in miR‐302b mimics transfected cells and was significantly decreased in miR‐302b inhibitors transfected cells. CDK2 is a target gene of miR‐302b. Decreased miR‐302b and increased CDK2 expressions can significantly promote proliferation and G1/S phase transformation in gastric cancer. miR‐302b promoted the proliferation of gastric cancer cells through upregulation of CDK2, thereby inhibiting ERK pathway, which can in turn inhibit the promoting ability of miR‐302b on proliferation. The upregulation of miR‐302b reduced the expression of CDK2, and inhibited ERK signaling pathway, thereby inhibiting cell proliferation and G1/S phase conversion rate. Therefore, miR‐302b provides new perspectives for research of cell regulation and proliferation in gastric cancer, and new targets for gastric cancer diagnosis and treatment.

Tissue Inhibitor of Matrix Metalloproteinases-1 Knockdown Suppresses the Proliferation of Human Adipose-Derived Stem Cells.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional matrix metalloproteinase, and it is involved in the regulation of cell proliferation and apoptosis in various cell types. However, little is known about the effect of TIMP-1 expression on the proliferation of adipose-derived stem cells (ADSCs). Therefore, TIMP-1 expression in the ADSCs was firstly detected by western blotting, and TIMP-1 gene was knocked down by lentivirus-mediated shRNA. Cell proliferation was then evaluated by MTT assay and Ki67 staining, respectively. Cell cycle progression was determined by flow cytometry. The changes of p51, p21, cyclin E, cyclin-dependent kinase 2 (CDK2), and P-CDK2 caused by TIMP-1 knockdown were detected by western blotting. The results indicated that ADSCs highly expressed TIMP-1 protein, and the knockdown of TIMP-1 inhibited cell proliferation and arrested cell cycle progression at G1 phase in the ADSCs possibly through the upregulation of p53, p21, and P-CDK2 protein levels and concurrent downregulation of cyclin E and CDK2 protein levels. These findings suggest that TIMP-1 works as a positive regulator of cell proliferation in ADSCs.

Qufeng Tongluo Prescription (祛风通络方) inhibits mesangial cell proliferation and promotes apoptosis through regulating cell cycle progression

To study the effects and possible underlying mechanism of Qufeng Tongluo Prescription (, QFTL) on the regulation of mesangial cells (MCs) proliferation and apoptosis. The MCs used in this experiment have undergone five passages induced by lipopolysaccharide (LPS). Changes in the proliferation, apoptosis, cell cycle regulatory proteins and mRNA expression levels of the MCs after administration of Benazepril or QFTL were measured by methyl thiazolyl tetrazolium (MTT) reduction assay, flow cytometry, Western blot and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The addition of Benazepril or QFTL serum inhibited LPS-induced MC proliferation after treatment for 24, 48 and 72 h, respectively (P<0.05 or P<0.01). Moreover, the inhibitory effect is more significant in the QFTL group at 48 h (P<0.05). Compared with the control group, LPS-induced cell proliferation decreased the number of cells in G1 phase versus cells in S and G2/M phases, while the addition of QFTL and Benazepril serum increased the ratio of cells at G1 phase (P<0.05 or P<0.01) to cells at S phase (P<0.01), implicating the cell cycle inhibition effect exerted by QFTL. LPS decreased the level of MC apoptosis, compared with the control group (P<0.05), while QFTL and Benazepril serum increased the level of MC apoptosis (P<0.01). Moreover, the difference between the QFTL group and the Benazepril group was statistically significant (P<0.01). Compared with the control group, the protein and mRNA expression levels of cylinD1, cyclin dependent kinase 2 (CDK2) and p21 were significantly increased (P<0.05 or P<0.01), p27 was decreased but with no statistical significance (P>0.05); After being treated with QFTL and Benazepril serum, the protein and mRNA expression levels of cylinD1, CDK2, p21 were decreased and p27 increased significantly (P<0.05 or P<0.01); Compared with the Benazepril group, QFTL show better effects on protein and mRNA expression levels of cylinD1, CDK2 (P<0.05 or P<0.01) and p21 protein expression (P<0.05). QFTL inhibits MCs proliferation, promotes MCs apoptosis through an underlying mechanism of down-regulating the protein and mRNA expression levels of cylinD1, CDK2, p21 and up-regulation of the expression level of p27.

Water extract of Hedyotis Diffusa Willd suppresses proliferation of human HepG2 cells and potentiates the anticancer efficacy of low-dose 5-fluorouracil by inhibiting the CDK2-E2F1 pathway

Hedyotis Diffusa Willd (HDW), a Chinese herbal medicine, has been widely used as an adjuvant therapy against various cancers, including hepatocellular carcinoma (HCC). However, the underlying anticancer mechanisms are yet to be elucidated. In the present study, the anticancer effects of HDW were evaluated and the efficacy and safety of HDW combined with low-dose 5-fluorouracil (5-FU) were investigated. HepG2 cells were cultured invitro and nude mouse xenografts were established invivo. The proliferation of HepG2 cells was measured using the MTT method and flow cytometry. The mRNA and protein expression levels of cyclin-dependent kinase2 (CDK2), cyclinE and E2F1 were examined using relative quantitative real-time PCR and western blot analysis, respectively. The results showed that water extract of HDW remarkably inhibited HepG2 cell proliferation in a dose-dependent manner via arrest of HepG2 cells at the G0/G1phase and induction of Sphase delay. This suppression was accompanied by a great decrease of E2F1 and CDK2 mRNA expression. In addition, HDW remarkably potentiated the anticancer effect of low-dose 5-FU in the absence of overt toxicity by downregulating the mRNA and protein levels of CDK2, cyclinE and E2F1. Our findings support the use of HDW as adjuvant therapy of chemotherapy and suggest that HDW may potentiate the efficiency of low-dose 5-FU in treating HCC.

NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer

Centrosome amplification can drive chromosomal instability (CIN) which is a major source of tumor initiation. The present study aimed to investigate the impact of nuclear factor kappa B (NF-κB) on centrosome amplification of Hep-2 cells. Immunofluorescence was performed to display centrosomes. BAY11-7082 was used as an inhibitor of NF-κB to assess the inhibition of centrosome amplification, and cyclin-dependent kinase 2 (CDK2), ensuring cell cycle cycle coordination with centrosome cycle was detected by Western blotting. Furthermore, a 1556-bp fragment of the CDK2 promoter was analyzed using the TRANSFAC-TESS software. Luciferase assay, including a series of truncated CDK2 promoters and site mutations, was carried out to determine NF-κB binding sites in the CDK2 promoter. Electrophoresis mobility shift and chromatin immunoprecipitation assays were applied to confirm whether NF-κB indeed binds to the 5'-promoter region of the CDK2 gene. To reveal the clinical significance of CDK2 expression in laryngeal squamous cell cancer, mRNA and protein levels were assessed by RT-PCR and Western blotting, respectively. We found that the transcription factor NF-κB plays a role in centrosome amplification in Hep-2 cells. Centrosome amplification is reduced by inhibition of the NF-κB pathway. Moreover, expression of the p65 subunit of NF-κB is sufficient to promote centrosome amplification and increase in CDK2 protein levels. We further identified a functional NF-κB binding site located in the CDK2 promoter. Single mutation of the NF-κB site III (construct mutIII) however resulted in 76±5% (p<0.01) luciferase activity reduction. Electromobility shift assays and chromatin immunoprecipitaton results suggest that NF-κB indeed binds to this responsive element associating with CDK2 expression and centrosome amplification. RT-PCR and Western blotting results revealed that both mRNA and protein levels of CDK2 were significantly higher in tumor tissues than those in paired adjacent normal laryngeal tissues.

Millimeter wave treatment promotes chondrocyte proliferation by upregulating the expression of cyclin-dependent kinase 2 and cyclin A

We investigated the effects of millimeter wave treatment on the expression of the cell cycle regulating proteins cyclin-dependent kinase 2 (CDK2) and cyclin A in chondrocytes. Knee articular cartilage from SD rats was used to establish cultured primary chondrocytes. After identification using toluidine blue staining, passage 2 chondrocytes were randomly divided into different groups and treated with nocodazole or millimeter wave. The RNA expression of CDK2 and cyclin A was measured using RT-PCR, and their protein levels were detected by Western blotting. Cell cycle analysis showed that nocodazole treatment significantly increased the number of G0/G1 and G2/M stage chondrocytes and decreased the amount of S phase cells. In contrast, millimeter wave treatment significantly decreased the number of G0/G1 and G2/M chondrocytes and increased the number of S phase cells. The mRNA and protein levels of CDK2 and cyclin A consistently demonstrated a reverse trend, with the lowest levels in the chondrocytes treated with nocodazole. The expression of CDK2 and cyclin A was higher in chondrocytes receiving millimeter wave treatment than in untreated cells. In conclusion, millimeter wave treatment induces CDK2 and cyclin A expression, accelerates S-phase entry and G2/M transition and promotes chondrocyte cell cycle progression.

Serum starvation induces G1 arrest through suppression of skp2-CDK2 and CDK4 in SK-OV-3 cells

Recent studies have suggested that Skp2, an SCF-type ubiquitin ligase, positively regulates cell cycle through degradation of p27, which is an inhibitor of cyclin-dependent kinase 2 (CDK2), which drives cells from the G1 to S phase of cell cycles. In the present study, we examined key regulatory proteins involved in serum starvation-induced cell cycle arrest in human ovarian cancer cells, SK-OV-3. Cell cycle analysis showed that cells were arrested at the G1 phase after serum starvation. Western blot analysis showed that the protein levels of CDK4 and CDK2 were significantly decreased in SK-OV-3 cells. Consistently, Roscovitine, an inhibitor of CDK2, induced cell cycle arrest in normally proliferating cells and a chemical inhibitor of CDK4, 3-ATA [3-Amino-9-thio(10H)-acridone], was found to induce growth arrest. We also found that the protein level of Skp2 was dramatically decreased in response to serum starvation. Moreover, CDK2 protein, which allows cell cycle transit from the G1 to the S phase, was decreased when the Skp2 expression was inhibited by specific siRNA of Skp2, but CDK4 was not decreased. Therefore, these results suggest that serum starvation induces G1 arrest through suppression of Skp2-dependent CDK2 activity and Skp2-independent CDK4 activity in human SK-OV-3 ovarian cancer cells.

IKKα controls p52/RelB at the skp2 gene promoter to regulate G1- to S-phase progression

The IkappaB-inducing kinase (IKK) is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma. IKK-regulated signaling pathways are believed to promote the proliferation of normal cells as well as the aberrant proliferation of cancer cells. The molecular mechanisms linking the IKK signaling pathway components to the cell cycle machinery are not entirely understood. To study the function(s) of the catalytic subunits of the IKK complex, we used pancreatic cancer cells, with constitutive IKK activity. We show that the G1 phase of the cell cycle is specifically regulated by the IKKalpha subunit, which regulates the stability of the cyclin-dependent kinase inhibitor p27(Kip1). Increased p27(Kip1) protein levels following the transfection of IKKalpha-specific siRNAs are a result of the downregulation of the F-box protein S-phase kinase-associated protein 2 (skp2). Additionally, we demonstrate that IKKalpha signaling regulates the transcription of the skp2 gene by controlling the composition of a RelB-containing NF-kappaB complex. Together, this work defines a novel IKKalpha-regulated growth pathway involving the p52/RelB-dependent transcriptional regulation of the skp2 gene.

Methylglyoxal stimulated proliferation of vascular smooth muscle cell through enhanced cytoplasmic CDK2 activity

Elevated MG levels and over-proliferation of vascular smooth muscle cells (VSMCs) in spontaneously hypertensive rats has been reported. Present study investigated the effect of MG on VSMCs proliferation. Application of MG at the range of 0.01 to 10 μM significantly increased DNA synthesis and proliferation of cultured VSMCs, which was abolished by co-application of N-acetylcysteine (NAC) or superoxide dismutase (SOD). Cyclin-dependent kinase 2 (CDK2) activity in cytoplasmic fraction, but not nuclear fraction, of MG-treated cells was increased, as compared with that of control cells. Activity of cyclin A-CDK2 complexes, but not cyclin E-CDK2, was responsible for the increased cytoplasmic CDK2 activity. Protein levels of CDK2, cyclin A, cyclin E and phosphorylation at residue Thr160 of CDK2 were not altered by MG treatment. However, phosphorylation at residue Tyr15 of CDK2 was significantly decreased in MG-treated cells compared to control cells, which may account the increased cytoplasmic CDK2 activity. Increased cytoplasmic CDK2 activity and decreased phosphorylation on Tyr15 of CDK2 in MG-treated cells were prevented by co-application of NAC or SOD. In conclusion, MG at physiologically relevant concentrations stimulated VSMCs proliferation through an enhanced cytoplasmic CDK2 activity. Increased MG level, therefore, may underscore increased proliferation of VSMC in some cardiovascular disorders. (Grants from Canadian Institutes of Health Research (CIHR, MOP-68938) and Heart and Stroke Foundation of Canada to L. Wu.)

Role of cyclins in cAMP inhibition of glomerular mesangial cell proliferation.

MC (mesangial cell) proliferation is closely linked to the progression of glomerular disease. It has been reported that cAMP effectors suppress MC proliferation, inhibiting activation of MAPK (mitogen-activated protein kinase). In fibroblasts, activation of MAPK induces the expression of type D cyclin, whereas, in MCs, this induction has not been shown. In the present study, we explored the effects of cAMP on MAPK and expression of cell-cycle-regulated proteins. PDGF (platelet-derived growth factor) stimulated MAPK activity, up-regulated protein levels of cyclin D1, CDK2 (cyclin-dependent kinase 2) and PCNA (proliferating cell nuclear antigen), decreased the protein level of p27 and increased DNA synthesis. Fsk (forskolin) or PD98059 suppressed PDGF-induced DNA synthesis. Both agents inhibited PDGF-stimulated mRNA and protein expression of cyclin D1 and CDK2. Fsk or PD98059 also inhibited protein expression of PCNA and blocked a decrease in p27 protein. Fsk induced the phosphorylation of Raf-1 at Ser259, which was inhibited by KT5720. These data suggest that cAMP inhibits MC proliferation through inhibition of MAPK activity, and this mechanism partly involves alteration in the levels of cell-cycle-regulated proteins.

The Tumor Suppressor Interferon Regulatory Factor 1 Interferes with SP1 Activation to Repress the Human CDK2 Promoter

Cell growth control by interferons (IFNs) involves up-regulation of the tumor suppressor interferon regulatory factor 1 (IRF1). To exert its anti-proliferative effects, this factor must ultimately control transcription of several key genes that regulate cell cycle progression. Here we show that the G1/S phase-related cyclin-dependent kinase 2 (CDK2) gene is a novel proliferation-related downstream target of IRF1. We find that IRF1, but not IRF2, IRF3, or IRF7, selectively represses CDK2 gene transcription in a dose- and time-dependent manner. We delineate the IRF1-responsive repressor element between nt -68 to -31 of the CDK2 promoter. For comparison, the tumor suppressor p53 represses CDK2 promoter activity independently of IRF1 through sequences upstream of nt -68, and the CDP/cut/Cux1 homeodomain protein represses transcription down-stream of -31. Thus, IRF1 repression represents one of three distinct mechanisms to attenuate CDK2 levels. The -68/-31 segment lacks a canonical IRF responsive element but contains a single SP1 binding site. Mutation of this element abrogates SP1-dependent enhancement of CDK2 promoter activity as expected but also abolishes IRF1-mediated repression. Forced elevation of SP1 levels increases endogenous CDK2 levels, whereas IRF1 reduces both endogenous SP1 and CDK2 protein levels. Hence, IRF1 represses CDK2 gene expression by interfering with SP1-dependent transcriptional activation. Our findings establish a causal series of events that functionally connect the anti-proliferative effects of interferons with the IRF1-dependent suppression of the CDK2 gene, which encodes a key regulator of the G1/S phase transition.

Overexpression of CDC25A associated with poor prognosis in breast cancer

Cell-cycle regulation is often altered in cancer; mutations cause the abolition of cell-cycle checkpoints resulting in uncontrolled cell proliferation. The cell-cycle gene, CDC25A, activates cyclin-dependent kinase 2 (CDK2), promoting entry into S (DNA synthesis) phase of the cell cycle. Overexpression of CDC25A, which has been shown previously to have oncogenic potential, eliminates this checkpoint. Cangi and colleagues 1. Cangi M.G. et al. Role of the Cdc25A phosphatase in human breast cancer. J. Clin. Invest. 2000; 106: 753-761 Crossref PubMed Scopus (169) Google Scholar examined primary breast tumours samples and found that CDC25A was overexpressed in,50% of those tested. This overexpression was associated with abnormally increased CDK2 activity, in the absence of any increase in CDK2 protein production. CDC25A was then inactivated in a breast cancer cell line by transfection with CDC25A antisense oligos. This treatment blocked CDK2 activation, without affecting CDK2 protein levels, showing that CDC25A was responsible for the increase in CDK2 activity. Cangi et al. further investigated whether CDC25A overexpression is associated with outcome in 144 patients with breast carcinomas of,1 cm diameter, who tend to have a good prognosis. Patients with tumours that overexpressed CDC25A had an increased risk of dying of the disease (24% mortality vs 5% mortality in patients with tumours that did not express CDC25A). The results of this study suggest that it might be useful to measure CDC25A and CDK2 levels before designing treatment regimes, and that CDC25A and CDK2 represent promising therapeutic targets for breast cancer treatment.

Interleukin-6 Induces G1Arrest through Induction of p27Kip1, a Cyclin-Dependent Kinase Inhibitor, and Neuron-like Morphology in LNCaP Prostate Tumor Cells

Prostate carcinoma cells express high levels of interleukin-6 (IL-6) and IL-6 receptor. In this study, we examined the effect of IL-6 on LNCaP human prostate carcinoma cells. IL-6 induces G1growth arrest of LNCaP. Following IL-6 treatment of LNCaP, Western blot analysis showed that the protein levels of cyclin-dependent kinase-2 (CDK2), CDK4, and CDK6 were decreased, while accumulation of CDK inhibitor p27Kip1was rapidly and markedly induced.In vitrokinase assays revealed that the CDK-associated histone H1 and CDK4- and CDK6-associated pRb kinase activities were significantly inhibited in IL-6-treated LNCaP. Further, a significant amount of p27Kip1was co-precipitated with CDK2, CDK4 and CDK6, as detected in immunoprecipitation experiments. Thus, IL-6-induced G1arrest appears to be due to the accumulation of p27Kip1. In addition, IL-6-treated LNCaP cells induced neuron-like morphological changes. Since neuroendocrine differentiation is observed in most prostate carcinomas, these findings raise the possibility that IL-6 may be involved in neuroendocrine differentiationin vivo.