Cell Cycle Regulatory Mechanisms Research Articles

Protein kinase D1 induces G1-phase cell-cycle arrest independent of Checkpoint kinases by phosphorylating Cell Division Cycle Phosphatase 25.

Protein Kinase D1 (PrKD1) functions as a tumor and metastasis suppressor in several human cancers by influencing cell-cycle progression. However, the exact mechanism of cell-cycle regulation by PrKD1 is unclear. Overexpression and ectopic expression of PrKD1 induces G1 arrest in cancer cell lines. Because checkpoint kinases (CHEKs) are known to play a role in progression through the G1 phase, we downregulated CHEK1, which did not overcome the G1 arrest induced by PrKD1. Using in vitro phosphorylation and Western blot assays, we showed that PrKD1 phosphorylates all CDC25 isoforms (known substrates of CHEK kinases), independent from CHEK kinases, suggesting that direct phosphorylation of CDC25 by PrKD1 may be an alternate mechanism of G1 arrest. The study has identified a molecular mechanism for the influence of PrKD1 in cell-cycle progression.

Inhibitors of cyclin-dependent kinases as new class of drugs in oncology. Achievements and development prospects in treatment of ER+ /HER 2- breast cancer

The review is devoted to a new class of drugs - inhibitors of cy-clin-dependent kinases. The discovery of the genetic and molecular mechanisms of cell cycle regulation, and as a result, the emergence of CDK4 / 6 inhibitors was a breakthrough in the treatment of ER+ HER2- metastatic breast cancer and changed the paradigm of hormone therapy in this group of patients. We consider the current role of CDK4 / 6 inhibitors in treating patients with hormone-sensitive breast cancer, as well as the prospects for the future use of this class of drugs. The article presents the results of the main registration studies of the FDA-approved inhibitors of CDK4 / 6: palbocyclyb, ribocyclyb, abemocyclib; a comparative analysis of their efficacy and toxicity profile was carried out.

Cypermethrin Induced Liver Toxicity: Altered Gene Expression and DNA Methylation

Cypermethrin, a type II pyrethroid, is a widely used insecticide worldwide. It is considered a safe insecticide to be used in pet shampoos and household cleaning agents thus ignoring the possible health hazards caused by it. Cypermethrin may contaminate food, water, fruits or get deposited on furniture, mats or floors leading to acute or chronic disorders in non‐target organisms. Very limited studies with low and non‐frequent dose mimicking the unintentional cypermethrin intake in non‐target organisms including human beings have been carried out. The present study was aimed at evaluating the toxic effect of cypermethrin in the liver of Balb/c mice exposed to a low and once in a week oral dose (1/10 LD50) for 28 days. Serum was extracted from blood after the dosing period and utilized for Liver Function Tests. The gene regulatory studies were performed by amplifying the cDNA, employing specific primers for p21 and p53 and other cell cycle regulatory genes through PCR (Polymerase Chain Reaction). DNA methylation studies were carried out by bisulfite treatment of DNA followed by Bisulphite specific PCR. The methylation status was found out by analyzing the gene promoter regions through sequencing.The changes in liver enzyme activities and histo‐architecture of liver cells show that sub‐chronic exposure to cypermethrin causes liver damage. It also induces a significant increase in hepatic markers enzymes (ALT, AST, ALP) along with necrosis, vacuolar degeneration, increased kupffer cells and a decrease in the number of hepatocytes. A significant change in the gene expression of p21 and p53 is seen in the test animals pointing towards the relation of liver injury by cypermethrin with cell cycle regulatory mechanisms. The gene promoter region of p53 gene show alteration in methylation status in the test animal sample as compared to the control.Sub‐chronic exposure to cypermethrin causes significant changes in liver enzyme activities, cell architecture, cell cycle regulatory gene expression and methylation status of the concerned genes. These genes along with the other cell cycle regulatory genes can be used as biomarkers for liver injury caused by low doses of cypermethrin.Support or Funding Information1. University Grant Commission, New Delhi, India.2. DST‐PURSE grant, New Delhi, India.3. Institute of Forensic Science and Criminology, Panjab University, India.4. Centre for Stem cell and Tissue Engineering, Panjab University, India.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative analysis of anatomy, gene expression of Vaccinium corymbosum cyclins and cyclin dependent kinases during the flower bud and fruit ontogeny

Cell-cycle system controls flower and fruit growth, size and mass precisely, accompanied by cumulative cell proliferation and cell expansion. Vaccinium corymbosum shows significant variation in berry size, but its underlying anatomical and molecular mechanisms are poorly known. In this study, single fruit mass and cell layers of Southern highbush blueberry cultivars 'O'Neal' and 'Bluerain' were analyzed, and 12 cell-cycle related genes were isolated and detected its expression tendency. The results showed that blueberry flower buds had a lower contribution to mature fruit mass, and fruit growth exhibited a double-sigmoid pattern. Ovary wall (or pericarp) was the vital component for final fruit size, and approximately 70% cell layers of pericarp were produced before anthesis. Cell division in the blueberry fruit was arrested at pre- and post-anthesis. The sequences of Vccyclins and VcCDKs were relatively conserved, and its expression tendency followed flower bud and fruit developmental pattern, while VcCycA2L, VcCycB2L, VcCycD3;1L, VcCycT1;4L, VcCDKB2;2, VcCDKC1L and VcCDKE1 might be directly involved in cell proliferation and cell expansion. However, the difference of expression pattern between two cultivars was not quite significant, indicated that isolated Vccyclins and VcCDKs were not the key factors for blueberry fruit size. Collectively, this study expands our understanding of the complex cell division and cell expansion mechanisms, and cell-cycle regulatory mechanisms were involved in V. corymbosum flower and fruit during ontogeny.

Supercritical CO2 fluid extraction of croton crassifolius Geisel root: Chemical composition and anti-proliferative, autophagic, apoptosis-inducing, and related molecular effects on A549 tumour cells

BackgroundThe use of plant essential oils as pharmaceuticals is a fast-growing market especially in China. Throughout the 20th century, a rapid increase took place in the use of many essential oil-derived products in the medicinal industry as nutraceuticals, medicinal supplements, and pharmaceuticals. PurposeThe objective of this study was to explore the chemical composition of Croton crassifolius essential oil as well as its potential anti-tumour properties and related anti-proliferative, autophagic, and apoptosis-inducing effects. MethodsSupercritical CO2 fluid extraction technology was used to extract CCEO and the chemical constituents of the essential oil were identified by comparing the retention indices and mass spectra data taken from the NIST library with those calculated based on the C7-C40 n-alkanes standard. The cytotoxic activity and anti-proliferative effects of CCEO were evaluated against five cancer cell lines and one normal human cell line via CCK-8 assays. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of CCEO treatments in controlling cancer cell proliferation was assessed by cell cycle analysis, clonal formation assays, RT-qPCR, and western blot analysis. Autophagic and apoptosis-inducing effects of oils and the associated molecular mechanisms were assessed by flow cytometry, cell staining, reactive oxygen species assays, RT-qPCR, and western blot analysis. ConclusionForty compounds representing 92.90% of the total oil were identified in CCEO. The results showed that CCEO exerted a measurable selectivity for cancer cell lines, especially for A549 with the lowest IC50 value of 25.00 ± 1.62 μg/mL. Assessment of the anti-proliferative effects of CCEO on A549 cells showed that the oil inhibited cell proliferation and colony formation in a dose- and time-dependent manner. Investigation of the molecular mechanisms of cell cycle regulation confirmed that the oil arrested A549 cells in G2/M phase by decreasing the expression of cyclin B1-CDK1 and cyclin A-CDK1 and increasing the expression of cyclin-dependent kinase inhibitor (CKI) P21 at both the transcriptional and translational levels. Autophagy staining assays and western blot analysis revealed that CCEO promoted the formation of autophagic vacuoles in A549 cells and increased the expression of autophagy-related proteins beclin-1 and LC3-II in a dose-dependent manner. A series of apoptosis analyses indicated that CCEO induces apoptosis through a mitochondria-mediated intrinsic pathway. This study revealed that CCEO is a promising candidate for development into an anti-tumour drug of the future.

Targeting the Cardiomyocyte Cell Cycle for Heart Regeneration.

Adult mammalian cardiomyocytes (CMs) exhibit limited proliferative capacity, as cell cycle activity leads to an increase in DNA content, but mitosis and cytokinesis are infrequent. This makes the heart highly inefficient in replacing with neoformed cardiomyocytes lost contractile cells as occurs in diseases such as myocardial infarction and dilated cardiomyopathy. Regenerative therapies based on the implant of stem cells of diverse origin do not warrant engraftment and electromechanical connection of the new cells with the resident ones, a fundamental condition to restore the physiology of the cardiac syncytium. Consequently, there is a growing interest in identifying factors playing relevant roles in the regulation of the CM cell cycle to be targeted in order to induce the resident cardiomyocytes to divide into daughter cells and thus achieve myocardial regeneration with preservation of physiologic syncytial performance. Despite the scientific progress achieved over the last decades, many questions remain unanswered, including how cardiomyocyte proliferation is regulated during heart development in gestation and neonatal life. This can reveal unknown cell cycle regulation mechanisms and molecules that may be manipulated to achieve cardiac self-regeneration. We hereby revise updated data on CM cell cycle regulation, participating molecules and pathways recently linked with the cell cycle, as well as experimental therapies involving them.

Drosophila Caliban mediates G1-S transition and ionizing radiation induced S phase checkpoint

ABSTRACTCell cycle progression is precisely regulated by diverse extrinsic and intrinsic cellular factors. Understanding the underlying mechanisms of cell cycle regulation is essential to address how normal development and tissue homeostasis are achieved. Here, we present a novel cell cycle regulator Caliban (Clbn), the Drosophila ortholog of human Serologically defined colon cancer antigen 1 (SDCCAG1) gene. We show that ionizing radiation induces expression of clbn, and over-expression of clbn blocks G1-to-S cell cycle transition in Drosophila, while flies loss of clbn have defective S phase checkpoint in response to irradiation. Mechanistically, induced expression of clbn suppressed E2F1 activity and down-regulates the DNA replication and expression of its downstream target cyclin E, a key regulator of G1-to-S transition. Meanwhile, clbn over-expression leads to upregulation of the CDK inhibitor Dacapo (Dap), and upregulated Dap is decreased when e2f1 is over-expressed. Furthermore, expression of clbn is down-regulated in cells with e2f1 over-expression or rbf1 knockdown, indicating that Clbn and E2F1 act antagonistically in mediating G1-to-S transition. Thus we provide genetic evidence that Clbn works together with E2F1 in regulating cell cycle progression, and Clbn is required for S phase cell cycle checkpoint in response to DNA damage.

The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans.

In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival.

Effect of pivaloyl-substituted pyrrole containing heterocyclic compounds on DNA repair pathways in Ewing sarcoma cells

Aim. To examine deoxyribonucleic acid (DNA) damage repair and cell cycle regulatory mechanisms of Ewing sarcoma cells exposed to pivaloyl-substituted pyrrole containing heterocyclic compounds.
 Methods. The study was performed on A673 Ewing sarcoma cell line. The tumor cells were incubated for 48 h in the presence of pivaloyl-substituted pyrrole containing heterocyclic compounds (compounds №20 and №24). Western blot analysis was utilized to examine expression of the markers of DNA single-strand (phosphorylated forms of ATR and Chk1) and double-strand breaks (phosphorylated forms of H2AX, АТМ, DNA-PK, BRCA-1, Chk-2). Analysis of the cell cycle phases was performed by flow cytometry (BD FacsCanto, USA).
 Results. Pivaloyl-substituted pyrrole containing heterocyclic compounds substantially increased the expression of histone 2A phosphorylated on serine 138 (γ-H2AX) that indicates DNA damage (double-strand breaks). Under exposure to pivaloyl-substituted pyrrole containing heterocyclic compounds the studied cells increased expression of phosphorylated forms of ATM-kinase and BRCA-1. Also cell cycle disorders leading to substantial G2/M arrest and enhanced apoptosis of tumor cells were observed.
 Conclusion. Pivaloyl-substituted pyrrole containing heterocyclic compounds induced DNA double-strand breaks in A673 Ewing sarcoma cell line; in response to DNA damage in tumor cells, the mechanisms of DNA double-strand breaks repair were activated; despite activation of DNA repair mechanisms, A673 cells underwent cell cycle arrest in the G2/M-phase and apoptosis.

Chemical Waves in Embryonic Cell Cycles

AbstractIn most metazoans, early embryonic development is characterized by a rapid series of cleavage divisions. At the core of the coordination of these divisions is the oscillatory dynamic of Cyclin‐dependent kinase 1 (Cdk1), which arises through the integration of a negative feedback with delay and positive feedback. The regulation of this oscillator in large embryos is emerging as an ideal model for quantitative studies of how spatiotemporal coordination is achieved in large complex tissues. Recent work has demonstrated that the reaction‐diffusion dynamics regulating the cell cycle can generate traveling waves of enzymatic activity, which ensure the synchronization of cell division processes. Here, we will review how the mechanisms of cell cycle regulation can give rise to chemical waves, and highlight recent experiments on the coordination of cell division through traveling waves.

Reduced Expression of Mismatch Repair Genes MSH6/MSH2 Directly Promotes Pituitary Tumor Growth via the ATR-Chk1 Pathway.

The mechanisms of pituitary adenoma (PA) pathogenesis and proliferation remain largely unknown. To clarify the role of mismatch repair (MMR) genes in the molecular mechanism of PA proliferation. We performed quantitative analyses by real-time polymerase chain reaction and immunohistochemistry to detect MMR gene and protein expression in human PAs (n = 47). We also performed correlation analyses of expression levels and tumor volume doubling time (TVDT; n = 31). Specifically, correlation analyses were performed between genes with significant correlation and ataxiatelangiectasia and Rad3-related (ATR) expression in cell-cycle regulatory mechanism ATR-checkpoint kinase 1 (Chk1) pathway (n = 93). We investigated the effect of reduced gene expression on cell proliferation and ATR gene expression in AtT-20ins cells and primary cultures of human PAs. Expression of mutS homologs 6 and 2 (MSH6 and MSH2) was positively associated with TVDT (R = 0.52, P = 0.003, and R = 0.44, P = 0.01), as were the corresponding protein levels. Gene expression was positively associated with ATR expression (R = 0.47, P < 0.00001, and R = 0.49, P < 0.00001). In AtT-20ins, the reduction of MSH6 and/or MSH2 expression by small interfering RNA significantly promoted cell proliferation by decreasing ATR expression. This effect was also observed in primary culture. Reduction of MSH6 and MSH2 expression at the messenger RNA and protein levels could be involved in direct PA proliferation by promoting cell-cycle progression or decreasing the rate of apoptosis through interference with the function of the ATR-Chk1 pathway.

Linking biological and physical aging: Dynamical scaling of multicellular regeneration.

The fight against biological aging (bio-aging) is long-standing, with the focus of intense research aimed at maintaining high rates of tissue regeneration to promote health and longevity. Nevertheless, there are overwhelming complexities associated with the quantitative analysis of aging. In this study, we sought to quantify bio-aging based on physical aging, by mapping instances of multicellular regeneration to the relaxation of physical systems. An experiment of delayed wound healing assays was devised to obtain delay-dependent healing data. The experiment confirmed the slowdown of healing events, which fitted dynamical scaling just as relaxation events do in physical aging. The scaling exponent, which describes the aging rate in physics, is here similarly proposed as an indicator of the deterioration rate of tissue-regenerative power. Parallel equation-based and cell-based simulations also revealed that asymmetric cell cycle-regulatory mechanisms under strong growth-inhibitory conditions predominantly control the critical slowdown of healing analogous to physical criticality. By establishing a direct link between physical aging and biological aging, we are able to estimate the aging rate of tissues and to achieve an integrated understanding of bio-aging mechanism which may improve the modulation of regeneration for clinical use.

The Toxoplasma Centrocone Houses Cell Cycle Regulatory Factors.

ABSTRACTOur knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures, and we named this factor ECR1 for essential for chromosome replication 1. ECR1 was discovered by complementation of a temperature-sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40°C similar to a ts mutant carrying a defect in topoisomerase. ECR1 is a 52-kDa protein containing divergent RING and TRAF-Sina-like zinc binding domains that are dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the unique spindle compartment of the Apicomplexa (centrocone) of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughter parasites separate from the mother parasite, ECR1 is downregulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex, and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the cyclin-dependent kinase (CDK)-related kinase, T. gondii Crk5 (TgCrk5), which displays a similar cell cycle expression and localization during tachyzoite replication. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates that this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.

Abstract 114: TG02 induces cell cycle arrest in glioblastoma

Abstract Dysregulated cell cycle contributes to the limitless replicative potential of cancer cells. The cyclin-dependent kinases (CDKs) and cyclins, the CDK-regulatory proteins are the key cell cycle machinery and can be targets for cancer therapy. TG02, a pyrimidine-based multi-kinase inhibitor has an anti-glioma effect which was demonstrated by our previous studies. An in vitro kinase spectrum assay of TG02 demonstrated inhibitory effects in several CDKs at the nanomolar level. To further investigate the molecular mechanisms of TG02-induced cell cycle regulation in glioma, a cell cycle analysis was performed by flow cytometry using the Click-It Edu Flow Cytometric Assay Kit in GSC923 and U251 cell lines, a human stem-like cell and patient derived cell line, respectively. Protein expression of CDKs and cyclins was tested by Western blotting. RNAseq analysis using Next Generation Sequencing was performed (only GSC923) and followed by a supervised hierarchical clustering analysis of cell cycle pathways that were derived from Qiangen’s Ingenuity Pathway Analysis (IPA). Our results showed that TG02 treatment results in G2/M-phase arrest in glioma cell lines. The protein expression of CDK1, CDK2, cyclin A2 and cyclinB1 are downregulated in both TG02 treated cell lines. Additionally, RNAseq analysis revealed downregulation of cyclins A2, B1 and B2 and significant irregulation in mRNA expression of G2/M checkpoint and spindle formation proteins, indicating G2 checkpoint and mitosis progression are also affected by TG02. Taken together, we demonstrated that TG02 induced cell cycle G2/M arrest through downregulating CDK and cyclin expression signaling in the preclinical models of glioblastoma. These findings strongly support the further investigation of TG02 as a potential therapy for malignant gliomas. &amp;lt;!–EndFragment–&amp;gt; Citation Format: Yu-Ting Su, Robert Chen, Hallie Lappin, Herui Wang, Dragan Maric, Orieta Celiku, Aiguo Li, Mark R. Gilbert, Jing Wu. TG02 induces cell cycle arrest in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 114. doi:10.1158/1538-7445.AM2017-114

Abstract 2358: Impact of MDM2 inhibition on cell cycle regulation through Aurora Kinase B - CDK1 axis in prostate cancer cells

Abstract Cyclin-dependent kinases (CDKs) are critical regulators of cell cycle progression and therefore overexpression of CDKs contributes to the proliferation of cancer cells. A variety of genetic and epigenetic events cause over activity of the CDKs in human cancers, and their inhibition can lead to cell cycle arrest and apoptosis. In malignant cells, both elevated expression of CDKs and their modulators including cyclins, and loss of expression of CDK inhibitors result in deregulated CDK activity, providing a selective growth advantage for cancer progression. In this respect, the multifunctional mouse double minute 2 homolog (MDM2) oncoprotein has been gaining a significant amount of attention towards better understanding the cell cycle regulatory mechanisms. It is well established that MDM2 gene amplification can occur in diverse human malignancies including prostate cancer. Also, MDM2 oncoprotein has been shown to exert both p53-dependent and p53-independent roles in oncogenesis. It has been well established in several laboratories, including ours, that overexpression of MDM2, can eventually lead to the inactivation of cell cycle control and loss of apoptotic ability in many tumors. However, the mechanisms underlying the regulation of CDK1 by MDM2 in cancer cells have not been fully identified. Therefore, the main objective of our study was to understand the impact of MDM2 overexpression on CDK1 that regulates cell cycle progression and apoptosis. Our preliminary data from human cell cycle PCR array experiments revealed the expression profile of genes that are involved in different phases of cell cycle regulation in LNCaP-MST cells with and without nutlin-3 treatment. Our study clearly demonstrated a significant increase in the expression level of Aurora Kinase B (AURKB), CDC25C and CDK1 in MDM2 transfected LNCaP-MST cells as compared with non-transfected LNCaP cells. However, after treating the cells with 20 µM of MDM2 specific inhibitor nutlin-3, for 24 h, the expression levels of the above mentioned proteins were significantly altered when compared to untreated controls. In addition, inhibition of MDM2 with nutlin-3 leads to increased expression of pro-apoptotic proteins p53, p21, and Bax. Our results offer significant evidence towards the effectiveness of MDM2 inhibition in causing cell cycle arrest via blocking the transmission of signals through AURKB-CDK1 axis and inducing apoptosis in cancer cells. It is clearly evident from our data that MDM2 overexpression probably is the primary cause for CDK1 up-regulation in the LNCaP-MST cells, which might have occurred possibly through activation of AURKB. However, further studies in this direction should shed more light on the intracellular mechanisms involved in the regulation of CDK1 in MDM2 positive cancers. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Thanigaivelan Kanagasabai, Khalid Alhazzani, Thiagarajan Venkatesan, Sivanesan Dhandayuthapani, Ali Alaseem, Appu Rathinavelu. Impact of MDM2 inhibition on cell cycle regulation through Aurora Kinase B - CDK1 axis in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2358. doi:10.1158/1538-7445.AM2017-2358

Metformin suppressed human cytomegalovirus (hCMV) replication and its potential molecular mechanisms in human fibroblasts

Abstract Background Chronic hCMV infection may contribute to T-cell immunosenenscence and chronic inflammation, and is shown to be associated with frailty and mortality in old adults. Metformin is a first-line treatment for type 2 diabetes mellitus. Emerging data have shown its additional beneficial effects including cardiovascular protection as well as anti-inflammatory, anti-cancer, and anti-aging activities. The objective of this study was to investigate the effect of metformin on hCMV infection in human fibroblasts and its underlying mechanisms. Methods Metformin was added (at concentration of 0, 1mM, 3mM, or 5mM) 2 h before inoculation of hCMV (Towne strain) at multiplicity of infection (MOI) of 0.01 or 0.1 to 80% confluent MRC-5 culture. Cells were cultured for additional 24h to 96 h. Viral plaques were observed under microscope, replication measured by qPCR (primers targeted to hCMV UL123 gene), cell cycle effect determined using flow cytometer, and hCMV protein expression (IE1/IE2, pp28, ICP36) and signaling pathway molecules analyzed by Western blot. Results and discussion Metformin at 3mM potently suppressed hCMV viral plaque formation, replication, and protein production. While hCMV infection promoted MRC-5 cells from G2 to M and S phase, metformin treatment led to G0/G1 arrest. Metformin suppressed expression of cyclin D, CDK4, and p21. It had no effect on CK2 or CDk6 expression. While AMPK pathway antagonist compound C suppressed AMPKa phosphorylation from both hCMV and metformin, it did not antagonize suppressive effect of metformin on hCMV protein expression. Taken together, metformin may suppress hCMV infection in MRC-5 cells via cell cycle regulation and molecular mechanisms independent of AMPK pathway.

Expression of FOXO3a and Correlation With Histopathologic Features in Retinoblastoma.

Forkhead box (FOX) transcription factors are a class of highly conserved proteins, which serve critical cellular functions including cell cycle regulation. The downstream mechanisms of cell cycle regulation involve preservation of retinoblastoma protein function. Its deactivation by phosphorylation and translocation from nucleus to cytoplasm leads to cell proliferation. FOXO3a has been found to be dysregulated in few cancers. However, no study has been reported on role of FOXO3a in retinoblastoma. We assessed the expression of FOXO3a in sections of archived tissue blocks of enucleated/exenterated specimens of retinoblastoma by immunohistochemistry. The histopathologic features were reviewed and correlated with its expression. Effect of FOXO3a expression on survival was assessed. FOXO3a expression was assessed in 100 sections. Six samples did not contain any viable tissue. Retrospective data of 94 patients revealed that median age at presentation was 36 months with male:female ratio of 1.9:1. Fifty-one percent of patients were International Retinoblastoma Staging System stage 1. Of the 94 sections, 68 (72%) showed cytoplasmic expression. Choroidal invasion was associated with cytoplasmic FOXO3a (P=0.04). A trend was also noted in optic nerve cut end involvement (P=0.07). No other histopathologic features were found to be associated with FOXO3a expression. The overall survival and progression-free survival were not found to be affected by FOXO3a expression. Cytoplasmic expression of FOXO3a is frequently found in retinoblastoma and may be involved in pathogenesis. Activation by relocation of FOXO3a to nucleus may activate nonmutated retinoblastoma and may be a potential target of treatment in retinoblastoma.

Cell Cycle Regulation in Treatment of Breast Cancer.

Cell cycle progression and cell proliferation are under precise and orchestrated control in normal cells. However, uncontrolled cell proliferation caused by aberrant cell cycle progression is a crucial characteristic of cancer. Understanding cell cycle progression and its regulation sheds light on cancer treatment. Agents targeting cell cycle regulators (such as CDKs) have been considered as promising candidates in cancer treatment. Although the first-generation pan-CDK inhibitors failed in clinical trials because of their adverse events and low efficacy, new selective CDK 4/6 inhibitors showed potent efficacy with tolerable safety in preclinical and clinical studies. Here we will review the mechanisms of cell cycle regulation and targeting key cell cycle regulators (such as CDKs) in breast cancer treatment. Particularly, we will discuss the mechanism of CDK inhibitors in disrupting cell cycle progression, the use of selective CDK4/6 inhibitors in treatment of advanced, hormone receptor (HR)-positive postmenopausal breast cancer patients, and other clinical trials that aim to extend the utilization of these agents.

Replication fork passage drives asymmetric dynamics of a critical nucleoid-associated protein in Caulobacter.

In bacteria, chromosome dynamics and gene expression are modulated by nucleoid-associated proteins (NAPs), but little is known about how NAP activity is coupled to cell cycle progression. Using genomic techniques, quantitative cell imaging, and mathematical modeling, our study in Caulobacter crescentus identifies a novel NAP (GapR) whose activity over the cell cycle is shaped by DNA replication. GapR activity is critical for cellular function, as loss of GapR causes severe, pleiotropic defects in growth, cell division, DNA replication, and chromosome segregation. GapR also affects global gene expression with a chromosomal bias from origin to terminus, which is associated with a similar general bias in GapR binding activity along the chromosome. Strikingly, this asymmetric localization cannot be explained by the distribution of GapR binding sites on the chromosome. Instead, we present a mechanistic model in which the spatiotemporal dynamics of GapR are primarily driven by the progression of the replication forks. This model represents a simple mechanism of cell cycle regulation, in which DNA-binding activity is intimately linked to the action of DNA replication.

Defective T Memory Cell Differentiation after Varicella Zoster Vaccination in Older Individuals.

Vaccination with attenuated live varicella zoster virus (VZV) can prevent zoster reactivation, but protection is incomplete especially in an older population. To decipher the molecular mechanisms underlying variable vaccine responses, T- and B-cell responses to VZV vaccination were examined in individuals of different ages including identical twin pairs. Contrary to the induction of VZV-specific antibodies, antigen-specific T cell responses were significantly influenced by inherited factors. Diminished generation of long-lived memory T cells in older individuals was mainly caused by increased T cell loss after the peak response while the expansion of antigen-specific T cells was not affected by age. Gene expression in activated CD4 T cells at the time of the peak response identified gene modules related to cell cycle regulation and DNA repair that correlated with the contraction phase of the T cell response and consequently the generation of long-lived memory cells. These data identify cell cycle regulatory mechanisms as targets to reduce T cell attrition in a vaccine response and to improve the generation of antigen-specific T cell memory, in particular in an older population.