Cyclin-dependent Kinase Inhibitor Protein Research Articles

Insulin promotes vascular smooth muscle cell proliferation via microRNA-208-mediated downregulation of p21

Abnormal vascular smooth muscle cell (VSMC) proliferation is involved in the development of vascular diseases. However, the mechanisms by which insulin exerts this effect are not completely known. We hypothesize that microRNAs might be involved in insulin-induced VSMC proliferation. VSMC proliferation was determined by [H]-thymidine incorporation; microRNAs were determined by microRNA chips and real-time PCR; and p21expression was determined by immunoblotting. In this study, we found that insulin increased VSMC proliferation and miR-208 expression. Overexpression of miR-208 increased basal and insulin-mediated VSMC proliferation. Although a miR-208 inhibitor, by itself, had no effect on VSMC proliferation, it reduced the insulin-mediated cell proliferation. Moreover, miR-208 increased the transformation of cell cycle from G0/G1 phase to the S phase. Bioinformatics analysis found that p21, a member of the cyclin-dependent kinase (CDK)-inhibitory protein family, may be the target of miR-208. Insulin decreased p21 expression in VSMCs; transfection of miR-208 also decreased p21 protein expression. In the presence of miR-208 inhibitor, the inhibitory effect of insulin on p21 expression in VSMCs was partially blocked. The interaction between miR-208 and p21 was direct. Using a luciferase reporter with entire wild-type p21 3'UTR or a mutant p21 3'UTR in HEK293 cells, we found that miR-208 decreased but neither miR-208 mimic nor the mutant p21 3'UTR had any significant effect on the luciferase activity. This study indicates that miRNAs, miR-208, in particular, are involved in the insulin-induced VSMC proliferation via downregulation of its potential target, p21, a key member of CDK-inhibitory protein family.

Current understanding and management of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a kind of rare head and neck cancer in Japan. However, NPC has some unique features. It is one of the most popular cancers in southern China, Southeast Asia, the Arctic, and the middle East/north Africa. This distinctive racial, ethnical, and geographic predisposition to NPC implies that both genetic susceptibility and environmental factors contribute to the development of this tumor. NPC is an Epstein-Barr virus - associated tumor. Consistent elevation of EBV antibody titers is a well-established risk factor of development of NPC. Not only pathophysiological relationship, but also molecular mechanism of EBV-mediated carcinogenesis has been enthusiastically investigated. LMP1, an EBV primary oncogene, upregulates each step of metastasis, and contribute to highly metastatic feature of NPC. A tumor suppressor gene p53 is mostly intact and overexpressed in NPC whereas expression of p16, a cyclin-dependent kinase inhibitory protein, is downregulated in 2/3 of NPC. Intention modulated radiotherapy (IMRT) is now getting prevalent for the treatment of NPC because of complicated structure and location of nasopharynx. A good therapeutic result can be achieved by distributing a high dose to the tumor while keeping down normal tissue complications by reducing radiation dose to normal tissues. Chemotherapy is important to control distant metastasis of chemoradiosensitive NPC, and thus, should play an important role. However, most effective combination of anti-tumor drugs, protocol of chemoradiotherapy has not well-established. Finally, molecular targeting therapy, including targeting EBV gene product, has been developing and on the way to the clinical use.

Galbanic acid decreases androgen receptor abundance and signaling and induces G1 arrest in prostate cancer cells

Androgen receptor (AR) signaling is crucial for the genesis and progression of prostate cancer (PCa). We compared the growth responses of AR(+) LNCaP and LNCaP C4-2 vs. AR(-) DU145 and PC-3 PCa cell lines to galbanic acid (GBA) isolated from the resin of medicinal herb Ferula assafoetida and assessed their connection to AR signaling and cell cycle regulatory pathways. Our results showed that GBA preferentially suppressed AR(+) PCa cell growth than AR(-) PCa cells. GBA induced a caspase-mediated apoptosis that was attenuated by a general caspase inhibitor. Subapoptotic GBA downregulated AR protein in LNCaP cells primarily through promoting its proteasomal degradation, and inhibited AR-dependent transcription without affecting AR nuclear translocation. Whereas docking simulations predicted binding of GBA to the AR ligand binding domain with similarities and differences with the AR antagonist drug bicalutamide (Bic), LNCaP cell culture assays did not detect agonist activity of GBA. GBA and Bic exerted greater than additive inhibitory effect on cell growth when used together. Subapoptotic GBA induced G(1) arrest associated with an inhibition of cyclin/CDK4/6 pathway, especially cyclin D(1) without the causal involvement of cyclin-dependent kinase (CDK) inhibitory proteins P21(Cip1) and P27(Kip1) . In summary, the novelty of GBA as an anti-AR compound resides in the distinction between GBA and Bic with respect to AR protein turnover and a lack of agonist effect. Our observations of anti-AR and cell cycle arrest actions plus the anti-angiogenesis effect reported elsewhere suggest GBA as a multitargeting drug candidate for the prevention and therapy of PCa.

P27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27Kip1 Cdk inhibitor, Ink4 proteins, or Rb. Here, we use tamoxifen-inducible mouse models to delete p27Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.

Genetic Evidence That Intestinal Notch Functions Vary Regionally and Operate through a Common Mechanism of Math1 Repression

Notch signaling is active in many sites, and its diverse activities must require tissue-specific intermediaries, which are largely unknown. In the intestinal epithelium, Notch promotes crypt cell proliferation and inhibits goblet cell differentiation. Pharmacologic studies suggest that the latter effect occurs through the transcription factor Math1/Atoh1, which specifies all intestinal secretory cells. We tested this hypothesis using mouse mutants. Genetic loss of the Notch effector RBP-Jκ alone increases all intestinal secretory lineages, with variation between proximal and distal gut segments. This secretory cell excess observed with RBP-Jκ loss was blocked in the absence of Math1 in RBP-Jκ(Fl/Fl);Math1(Fl/Fl);Villin-Cre((ER-T2)) mice. Loss of both factors also restored progenitor replication, proving that Math1 is epistatic to Notch signaling in both secretory cell differentiation and crypt cell proliferation. Investigating mechanisms downstream of Math1, we found that expression of the known Notch effector protein Hes1 was predictably lost in RBP-Jκ(-/-) mice but surprisingly recovered in RBP-Jκ;Math1 compound conditional mutants. Furthermore, the cell cycle inhibitors p27(Kip1) and p57(Kip2) were selectively overexpressed in duodenal and ileal crypts, respectively, in RBP-Jκ-deficient mice. Regional activation of these products was completely abrogated in the absence of Math1. Thus, all intestinal Notch effects channel through the tissue-restricted factor Math1, which promotes secretory differentiation and cell cycle exit by regionally distinct mechanisms. Our data further suggest that, besides transmitting Notch signals, the transcription factor Hes1 acts downstream of Math1 to regulate expression of cell cycle inhibitors and intestinal crypt cell replication.

Regulation of Migration and Proliferation of Glioblastoma Cells by Modulating the Acid Sensitive Ion Channel‐1 (ASIC‐1) Activity

Inhibition of glioma cell specific cation channels is a promising opportunity for the therapy of glioblastoma multiforme. One of the major features of high grade glioma cells is their extreme ability to migrate and proliferate through brain parenchyma. The movement of cations through ion channels is an important requirement for the metastasis of glioma cells. We have previously reported that high grade malignant glioma cells express an amiloride-sensitive cation conductance which is not present in normal astrocytes or low grade glioma cells. In the present study, we have investigated the potential role of a component of this channel, Acid Sensitive Ion Channel-1 (ASIC-1) to regulate migration and the cell cycle process in glioma cells. We found that the blocking of ASIC-1 by Psalmotoxin-1 (PcTX-1) (100 nM), a tarantula spider toxin, significantly inhibits the migration and proliferation of D54MG and U251 glioma cells both in transwell migration and in-vitro scratch wound assays. Both PcTx-1 and benzamil (100mM), an amiloride analog, caused a significant cell cycle arrest of D54MG cells in G0/G1 phases (by 30% and 40%, respectively) and reduced accumulation in the S phase (by 50% and 86%, respectively), and G2/M phase (by 78% and 80%, respectively) (n=5, P<0.01) at 24h of incubation. An unfolded control peptide (100nM) had no significant effect compared to PcTX-1 and benzamil. PcTX-1 also induced an upregulation of cyclin dependent kinase inhibitor (CDKI) proteins p21Cip1/Waf1 and p27Kip1 in D54MG cells. We also found similar effects in D54MG cells by reducing the [Na+]O in the incubation medium. Knockdown of ASIC-1 expression had similar effects on p21Cip1/Waf1 and p27Kip1 expression and inhibited the cell cycle. Our data implicate the potentialrole of glioma specific cation conductance in migration and in progressionof the cell cycle in glioma cells. This study was supported by NIH grants CA101952 and DK037206.

Recruitment of cyclin-dependent kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1

Cyclin-dependent protein kinases (CDKs) are important regulators of cellular processes and are functionally integrated into the replication of human cytomegalovirus (HCMV). Recently, a regulatory impact of CDK activity on the viral mRNA export factor pUL69 was shown. Here, specific aspects of the mode of interaction between CDK9/cyclin T1 and pUL69 are described. Intracellular localization was studied in the presence of a novel selective CDK9 inhibitor, R22, which exerts anti-cytomegaloviral activity in vitro. A pronounced R22-induced formation of nuclear speckled aggregation of pUL69 was demonstrated. Multi-labelling confocal laser-scanning microscopy revealed that CDK9 and cyclin T1 co-localized perfectly with pUL69 in individual speckles. The effects were similar to those described recently for the broad CDK inhibitor roscovitine. Co-immunoprecipitation and yeast two-hybrid analyses showed that cyclin T1 interacted with both CDK9 and pUL69. The interaction region of pUL69 for cyclin T1 could be attributed to aa 269-487. Moreover, another component of CDK inhibitor-induced speckled aggregates was identified with RNA polymerase II, supporting earlier reports that strongly suggested an association of pUL69 with transcription complexes. Interestingly, when using a UL69-deleted recombinant HCMV, no speckled aggregates were formed by CDK inhibitor treatment. This indicated that pUL69 is the defining component of aggregates and generally may represent a crucial viral interactor of cyclin T1. In conclusion, these data emphasize that HCMV inter-regulation with CDK9/cyclin T1 is at least partly based on a pUL69-cylin T1 interaction, thus contributing to the importance of CDK9 for HCMV replication.

Cell-cycle control as a target for calcium, hormonal and developmental signals: the role of phosphorylation in the retinoblastoma-centred pathway

During the life cycle of plants, both embryogenic and post-embryogenic growth are essentially based on cell division and cell expansion that are under the control of inherited developmental programmes modified by hormonal and environmental stimuli. Considering either stimulation or inhibition of plant growth, the key role of plant hormones in the modification of cell division activities or in the initiation of differentiation is well supported by experimental data. At the same time there is only limited insight into the molecular events that provide linkage between the regulation of cell-cycle progression and hormonal and developmental control. Studies indicate that there are several alternative ways by which hormonal signalling networks can influence cell division parameters and establish functional links between regulatory pathways of cell-cycle progression and genes and protein complexes involved in organ development. An overview is given here of key components in plant cell division control as acceptors of hormonal and developmental signals during organ formation and growth. Selected examples are presented to highlight the potential role of Ca(2+)-signalling, the complex actions of auxin and cytokinins, regulation by transcription factors and alteration of retinoblastoma-related proteins by phosphorylation. Auxins and abscisic acid can directly influence expression of cyclin, cyclin-dependent kinase (CDK) genes and activities of CDK complexes. D-type cyclins are primary targets for cytokinins and over-expression of CyclinD3;1 can enhance auxin responses in roots. A set of auxin-activated genes (AXR1-ARGOS-ANT) controls cell number and organ size through modification of CyclinD3;1 gene expression. The SHORT ROOT (SHR) and SCARECROW (SCR) transcriptional factors determine root patterning by activation of the CYCD6;1 gene. Over-expression of the EBP1 gene (plant homologue of the ErbB-3 epidermal growth factor receptor-binding protein) increased biomass by auxin-dependent activation of both D- and B-type cyclins. The direct involvement of auxin-binding protein (ABP1) in the entry into the cell cycle and the regulation of leaf size and morphology is based on the transcriptional control of D-cyclins and retinoblastoma-related protein (RBR) interacting with inhibitory E2FC transcriptional factor. The central role of RBRs in cell-cycle progression is well documented by a variety of experimental approaches. Their function is phosphorylation-dependent and both RBR and phospho-RBR proteins are present in interphase and mitotic phase cells. Immunolocalization studies showed the presence of phospho-RBR protein in spots of interphase nuclei or granules in mitotic prophase cells. The Ca(2+)-dependent phosphorylation events can be accomplished by the calcium-dependent, calmodulin-independent or calmodulin-like domain protein kinases (CDPKs/CPKs) phosphorylating the CDK inhibitor protein (KRP). Dephosphorylation of the phospho-RBR protein by PP2A phosphatase is regulated by a Ca(2+)-binding subunit.

H 2O 2 scavenging inhibits G1/S transition by increasing nuclear levels of p27 KIP1

The aim of the present study was to evaluate cell cycle regulation by scavenging H 2O 2 in tumor cells. A significant arrest in the G1 phase of the cell cycle was demonstrated in CH72-T4 carcinoma cells exposed to catalase, associated with a decrease in cyclin D1 and an increase in the CDK inhibitory protein p27 KIP1. Moreover, we found a differential intracellular distribution of p27 KIP1, which remained in the nucleus after catalase treatment. In vivo experiments showed an increase in nuclear levels of p27 KIP1 associated with the inhibition of tumor growth by H 2O 2 scavenging, confirming in vitro results. To conclude, H 2O 2 scavenging may induce cell cycle arrest through the modulation of cyclin D1 and p27 KIP1 levels and nuclear localization of p27 KIP1. To our knowledge, this is the first report that demonstrates that the modulation of ROS alters the intracellular localization of a key regulatory protein of G1/S transition.

New Insight Into the Role of the Cdc34 Ubiquitin-Conjugating Enzyme in Cell Cycle Regulation via Ace2 and Sic1

The Cdc34 ubiquitin-conjugating enzyme plays a central role in progression of the cell cycle. Through analysis of the phenotype of a mutant missing a highly conserved sequence motif within the catalytic domain of Cdc34, we discovered previously unrecognized levels of regulation of the Ace2 transcription factor and the cyclin-dependent protein kinase inhibitor Sic1. In cells carrying the Cdc34(tm) mutation, which alters the conserved sequence, the cyclin-dependent protein kinase inhibitor Sic1, an SCF(Cdc4) substrate, has a shorter half-life, while the cyclin Cln1, an SCF(Grr1) substrate, has a longer half-life than in wild-type cells. Expression of the SIC1 gene cluster, which is regulated by Swi5 and Ace2 transcription factors, is induced in CDC34(tm) cells. Levels of Swi5, Ace2, and the SCF(Grr1) targets Cln1 and Cln2 are elevated in Cdc34(tm) cells, and loss of Grr1 causes an increase in Ace2 levels. Sic1 levels are similar in CDC34(tm) ace2Δ and wild-type cells, explaining a paradoxical increase in the steady-state level of Sic1 protein despite its reduced half-life. A screen for mutations that interact with CDC34(tm) uncovered novel regulators of Sic1, including genes encoding the polyubiquitin chain receptors Rad23 and Rpn10.

Prolonged Mechanical Ventilation Induces Cell Cycle Arrest in Newborn Rat Lung

RationaleThe molecular mechanism(s) by which mechanical ventilation disrupts alveolar development, a hallmark of bronchopulmonary dysplasia, is unknown.ObjectiveTo determine the effect of 24 h of mechanical ventilation on lung cell cycle regulators, cell proliferation and alveolar formation in newborn rats.MethodsSeven-day old rats were ventilated with room air for 8, 12 and 24 h using relatively moderate tidal volumes (8.5 mL.kg−1).Measurement and Main ResultsVentilation for 24 h (h) decreased the number of elastin-positive secondary crests and increased the mean linear intercept, indicating arrest of alveolar development. Proliferation (assessed by BrdU incorporation) was halved after 12 h of ventilation and completely arrested after 24 h. Cyclin D1 and E1 mRNA and protein levels were decreased after 8–24 h of ventilation, while that of p27Kip1 was significantly increased. Mechanical ventilation for 24 h also increased levels of p57Kip2, decreased that of p16INK4a, while the levels of p21Waf/Cip1 and p15INK4b were unchanged. Increased p27Kip1 expression coincided with reduced phosphorylation of p27Kip1 at Thr157, Thr187 and Thr198 (p<0.05), thereby promoting its nuclear localization. Similar -but more rapid- changes in cell cycle regulators were noted when 7-day rats were ventilated with high tidal volume (40 mL.kg−1) and when fetal lung epithelial cells were subjected to a continuous (17% elongation) cyclic stretch.ConclusionThis is the first demonstration that prolonged (24 h) of mechanical ventilation causes cell cycle arrest in newborn rat lungs; the arrest occurs in G1 and is caused by increased expression and nuclear localization of Cdk inhibitor proteins (p27Kip1, p57Kip2) from the Kip family.

Expression of p27kip1 and skp2 protein in lung carcinoma tissue

Objective To explore the relationship between the expression of S-phase kinase-associated protein 2 ( skp2),cyclin dependent kinase inhibitor protein 27 ( p27kip1) and lung carcinogenesis.Methods The protein expression of p27kip1 and skp2 was detected by immunohistochemistry ( SP method)in 56 cases of lung carcinoma and 19 cases of paraneoplastic normal tissues as control group.Results The positive expression rate of p27kip1 protein was 46.42% in lung carcinoma,while that was 89.47% in paraneoplastic normal tissue ( P ＜0.01 ).The positive expression rate of skp2 protein was 32.14% in lung carcinoma,while that was 10.52% in paraneoplastic normal tissue (P ＜ 0.05 ).There was a negative correlation between the expression of p27kip1 and skp2 in lung corcinoma ( P ＜ 0.01 ).Conclusion The detection of p27kip1 and skp2 proteins is helpful to clinical diagnosis and prognostic evaluation of lung corcinoma. Key words: Lung carcinoma; Cyclin; skp2

Epigenetic Dysregulation in Lymphoid Malignancies

AbstractAbstract SCI-29Greater understanding of the role of the epigenetic mechanisms and cancer cell epigenome in the pathogenesis of cancer in general and lymphoid malignancies in particular has emerged, which is yielding insights into how to therapeutically target these novel mechanisms. Epigenetic mechanisms include histone modifications, DNA methylation, nucleosome remodeling and small non-coding RNAs. In transformed cells, the methylome involves global DNA hypomethylation mostly targeting DNA repeats, and hypermethylation of CpG islands in the promoter regions of TSGs. For example, methylation and silencing of cell cycle dependent kinase inhibitors p16 and p15, and of DNA repair protein MGMT, is observed in lymphoma. A disequilibrium in the lysine (K) acetylation/deacetylation of the N-terminal tail of the core histone (H) proteins due to increased histone deacetylase (HDAC) activity leads to aberrant transcription repression of genes involved in regulation of proliferation and differentiation of lymphoid progenitors in lymphoid malignancies. Additionally, increased HDAC levels and activity may directly deacetylate and modify the transcriptional activity of transcription factors, or HDACs may be recruited along with co-repressors by transcriptional repressors such as Bcl-6 to the promoters of the repressed genes in lymphoma. Histone lysine methylation, regulated by the reciprocal activity of a specific histone methyl transferase (HMTs) and histone de-methylase, also modifies gene expression based on the lysine residue that is affected. For example, increased levels and activity of the polycomb repressive complex (PRC) 2 protein EZH (enhancer of zeste) 2, a histone methyl transferase induces the repressive chromatin mark H3K27 trimethylation (me3), which is erased by the demethylase, UTX. Mutations in EZH2 and UTX have been observed in transformed cells. Conversely, the methylation status of the permissive chromatin mark H3K4me3 and gene expression is reciprocally controlled by the HMTase, MLL, and the demethylases JARID1A and LSD1. PRC1 protein, BMI-1, is over-expressed and implicated in lymphoid malignancies. Genetic abnormalities of cell fate transcription factors (CFTFs), e.g., MYB, PAX5 and IKAROS, which recruit PRC1 and PRC2 complexes and their member proteins, e.g., BMI and EZH2, also deregulate the expression of developmentally regulated genes involved in stem cell behavior, related to self-renewal, proliferation and differentiation of transformed cells. PRC proteins EZH2 and BMI, in turn, can recruit DNMTs and promote de novo DNA methylation of TSGs. Levels and activity of the non-coding small (MiRs 101 and 26a for EZH2; MiR 29 for DNMT3a and DNMT3b; and MiR15a and 16 for BMI) may also be deregulated in transformed cells. Interestingly, the levels of MiRs are epigenetically regulated. Collectively, the complex interplay of the epigenetic mechanisms in the pathogenesis of lymphoid malignancies underscores the importance of targeting the deregulated mechanisms in a combinatorial fashion. Inhibitors of chromatin modifying enzymes including HDACs and DNMTs are approved for therapy by the FDA and, given alone or in combination, have shown efficacy in clinical trials in hematologic malignancies. Combined inhibition of EZH2 and HDACs also displays anti-lymphoma and anti-leukemia synergy. In the presentation, the emerging knowledge related to the role of epigenetic mechanisms in the pathogenesis of lymphoid malignancies, as well as the potential for combined therapeutic targeting of the deregulated mechanisms will be highlighted. Disclosures:Bhalla:Novartis: Honoraria, Research Funding; Merck: Honoraria.

A Novel Pyrazolo[1,5-a]pyrimidine Is a Potent Inhibitor of Cyclin-Dependent Protein Kinases 1, 2, and 9, Which Demonstrates Antitumor Effects in Human Tumor Xenografts Following Oral Administration

Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Here, we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC₅₀= 3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A, E, and D1, and cell cycle block in the S and G₂/M phases. Consistent with these findings, 4k demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI₅₀= 280 nmol/L). Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.

Electrospray ionization‐mass spectrometry conformational analysis of isolated domains of an intrinsically disordered protein

The highly dynamic and heterogeneous molecular ensembles characterizing intrinsically disordered proteins (IDP) in solution pose major challenges to the conventional methods for structural analysis. Electrospray ionization-mass spectrometry (ESI-MS) allows direct detection of distinct conformational components, effectively capturing also partially folded and short-lived states. We report the description of two complementary fragments (1-186 and 187-284) of the IDP Sic1, a cyclin-dependent protein kinase inhibitor of yeast Saccharomyces cerevisiae. Structural heterogeneity is noted in both cases, but the two fragments reveal slightly different conformational properties. The results are consistent with previously reported differences between the two protein moieties and corroborate the feasibility of IDP conformational analysis by ESI-MS.

MS4a4B, a CD20 Homologue in T Cells, Inhibits T Cell Propagation by Modulation of Cell Cycle

MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells

Penta-O-galloyl-β-D-glucose induces G1arrest and DNA replicative S-phase arrest independently of P21 cyclin-dependent kinase inhibitor 1A, P27 cyclin-dependent kinase inhibitor 1B and P53 in human breast cancer cells and is orally active against triple-negative xenograft growth

IntroductionNatural herbal compounds with novel actions different from existing breast cancer (BCa) treatment modalities are attractive for improving therapeutic efficacy and safety. We have recently shown that penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG) induced S-phase arrest in prostate cancer (PCa) cells through inhibiting DNA replicative synthesis and G1 arrest, in addition to inducing cell death at higher levels of exposure. We and others have shown that PGG through intraperitoneal (i.p.) injection exerts a strong in vivo growth suppression of human PCa xenograft models in athymic nude mice. This study aims to test the hypothesis that the novel targeting actions of PGG are applicable to BCa cells, especially those lacking proven drugable targets.MethodsMono-layer cell culture models of p53-wild type estrogen receptor (ER)-dependent MCF-7 BCa cells and p53-mutant ER-/progesterone receptor (PR)- and Her2-regular (triple-negative) MDA-MB-231 BCa were exposed to PGG for a comprehensive investigation of cellular consequences and molecular targets/mediators. To test the in vivo efficacy, female athymic mice inoculated with MDA-MB-231 xenograft were treated with 20 mg PGG/kg body weight by daily gavage starting 4 days after cancer cell inoculation.ResultsExposure to PGG induced S-phase arrest in both cell lines as indicated by the lack of 5-bromo2'-deoxy-uridine (BrdU) incorporation into S-phase cells as well as G1 arrest. Higher levels of PGG induced more caspase-mediated apoptosis in MCF-7, in strong association with induction of P53 Ser15 phosphorylation, than in MDA-MB-231 cells. The cell cycle arrests were achieved without an induction of cyclin dependent kinase (CDK) inhibitory proteins P21Cip1 and P27Kip1. PGG treatment led to decreased cyclin D1 in both cell lines and over-expressing cyclin D1 attenuated G1 arrest and hastened S arrest. In serum-starvation synchronized MCF-7 cells, down-regulation of cyclin D1 was associated with de-phosphorylation of retinoblastoma (Rb) protein by PGG shortly before G1-S transition. In vivo, oral administration of PGG led to a greater than 60% inhibition of MDA-MB231 xenograft growth without adverse effect on host body weight.ConclusionsOur in vitro and in vivo data support PGG as a potential drug candidate for breast cancer with novel targeting actions, especially for a triple negative BCa xenograft model.

Persistent P21Cip1 Induction Mediates G1 Cell Cycle Arrest by Methylseleninic Acid in DU145 Prostate Cancer Cells

The induction of G(1) cell cycle arrest and apoptosis by second-generation selenium compounds (e.g., methylselenol precursors such as methylseleninic acid, MSeA) may contribute to their anti-cancer activities. We have documented previously induction of G(1) arrest and apoptosis by MSeA in association with upregulation of cyclin-dependent kinase inhibitor (CDKI) proteins p21Cip1 and/or p27Kip1 in DU145 prostate cancer cells. However, whether these CDKIs play a critical mediator role in G(1) arrest and apoptosis by MSeA has not been addressed. In the present work, we show exposure of p53-mutant DU145 cells to sub-apoptotic concentrations of MSeA induced p21cip1 mRNA (3 h) and protein (6 h) much faster than p27kip1 mRNA (12 h) and protein (12 h). Knocking down of p21 by siRNA completely abolished G(1) arrest induction by MSeA in DU145 cells, yet si-p27 RNA had no attenuation effect on the G(1) arrest. Depletion of p21Cip1 alone or both p21Cip1 and p27Kip1 increased MSeA-induced caspase-mediated apoptosis. Immunoprecipitation detected increased binding of p21Cip1 to CDK2 and CDK6 in MSeA-exposed DU145 cells. In DU145 xenografts from mice acutely treated with MSeA p.o., the induction of p21Cip1 was observed at 72 h of daily exposure. In p53-wild type LNCaP PCa cells and p53-null PC-3 PCa cells, MSeA modestly and transiently upregulated p21Cip1 protein level, subsiding to basal level by 24 h, without affecting P27Kip1 abundance in the same duration. Si-p21 RNA knockdown in these cells have only a partial effect to reverse G(1) arrest induction by MSeA. Together, our data support persistent, p53-independent, p21Cip1 induction as a critical mediator of MSeA-induced G(1) arrest in DU145 PCa cells, however, p21Cip1 induction and G(1) arrest were not necessary for, and may antagonize, caspase-mediated apoptosis.

Abstract 2882: Penta-O-galloyl-beta-D-glucose induces DNA replicative S arrest and G1 arrest independent of p21Cip1, p27Kip1 and p53 in human breast cancer cells and is orally active against breast cancer xenograft

Abstract Natural herbal compounds with novel actions different from existing breast cancer targeting modalities are attractive for improving treatment and prevention efficacy and safety. We have recently shown that penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG), a naturally occurring gallotannin polyphenol compound, induced S-phase arrest in prostate cancer cells through inhibiting DNA replicative synthesis, in addition to the induction of G1 arrest and cell deaths at higher levels of exposure. We and others have shown that PGG through i.p. injection exerts a strong in vivo growth suppression of human prostate cancer xenograft models in athymic nude mice. We hypothesize that the novel DNA replication targeting action and the cellular arrest and death effects of PGG are applicable to breast cancer cells and PGG could be a novel agent for treatment or prevention of breast cancer, especially those without proven drugable targets such as triple-negative breast cancer. In cell culture studies, exposure to serum achievable level of PGG induced rapid S arrest in p53-wild type ER-dependent MCF-7 breast cancer cells and in p53-mutant ER-/PR- and Her2-negative (triple-negative) MDA-MB-231 breast cancer cells as indicated by the lack of BrdU incorporation into S-phase cells. Higher levels of PGG induced more caspase-mediated apoptosis in MCF-7 than in MDA-MB-231 cells. In addition to S-arrest, PGG induced G1 arrest in both cell lines. Contrary to a study by others with questionable design, we have found no induction of CDK inhibitory proteins p21Cip1 and p27Kip1 with G1 arrest. PGG treatment led to decreased cyclin D1 in both cell lines and over-expressing cyclin D1 abolished G1 arrest and hastened S arrest. In serum-starvation synchronized MCF-7 cells, we observed a close association of down-regulation of cyclin D1 and de-phosphorylation of Rb by PGG shortly before G1-S transition stimulated by serum. Though PGG inhibited estrogen-stimulated cell growth in MCF-7 cells, it had little effect on estrogen receptor-alpha level. To test the in vivo efficacy of PGG, we treated female athymic mice inoculated with MDA-MB-231 xenograft with 10 mg/kg body weight by daily gavage and compared with weekly paclitaxel treatment at the same dosage. Oral administration of PGG led to a better growth inhibitory efficacy than the taxane drug. Together, our in vitro and in vivo data support PGG as a potential chemopreventve or treatment agent for breast cancer with novel targeting actions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2882.

Regulation Of Migration and Proliferation of Glioma Cells by Modulating the Ion Channel Activity.

The movement of cations through ion channels is essential for the migration of glioma cells. We have previously reported that malignant glioma cells express an amiloride sensitive cation conductance that is not detected in normal astrocytes. In the present study, we investigate the potential role of this ion channel to regulate the migration and cell cycle processes of glioma cells. We found that the blocking of Acid Sensitive Ion Channel 1 by Psalmotoxin‐1 (40 amino acid synthetic peptide) significantly inhibited both migration and proliferation of D54MG and U251 glioma cells, using in vitro transwell migration and in‐vitro scratch wound assays. We also found that both PcTx‐1 and benzamil caused a significant cell cycle arrest of D54MG cells in G0/G1 (by 18% and 19%, respectively), and reduced accumulation in the S phase (by 61% and 49%, respectively), and G2/M phase (by 51% and 32%, respectively) (n=3–4; P&lt;0.01) at 24h of incubation. PcTX‐1 and benzamil showed the same effect after 48h of incubation time. An unfolded control peptide had no significant effect compared to PcTX‐1. PcTX‐1 also induced upregulation of cyclin dependent kinase inhibitor (CDKI) proteins (p21Cip1 and p27Kip1) in D54MG cells. Thus, our data implicate the potential role of this cation conductance in migration and in progression of the cell cycle in glioma cells. This study was supported by NIH grants CA101952 and DK037206.