A novel mechanism for regulation of calmodulin signaling by RC3

RC3, also known as neurogranin, is a small neuronal IQ domain protein whose only known function is to bind calmodulin (CaM). The hypothesis tested in this work was that RC3 alters the dynamics of the interaction of Ca 2+-CaM with CaM-kinase II, so that there is less CaM-kinase II activation for a given Ca2+ stimulus. To evaluate this hypothesis, we investigated the affinity and kinetics of the interactions of CaM with Ca 2+, RC3 and CaM-kinase II. We quantitated the interaction of the four CaM-kinase II isoforms with CaM and found that the KD for binding of CaM to CaM-kinase II ranged from 7 nM to 60 nM. Using stopped-flow fluorimetry, we determined the kinetics of the interaction of Ca2+-CaM with αCaM-kinase II, and found that the association rate constant is 2.1 × 10 M −1s−1 and the dissociation rate constant is 1.6 s−1. We investigated the effects of RC3 and αCaM-kinase II on the affinity of CaM for Ca2+ and found that both proteins alter the rate of dissociation of Ca2+ from CaM. RC3 increases the rate of dissociation of Ca2+ from the C-terminal binding sites of CaM from 9 s−1 to ∼500 s−1 , while αCaM-kinase II causes a decrease in the rate of dissociation from all four Ca2+ binding sites. Measurement of the rate of dissociation of Ca2+ from CaM in the presence of both RC3 and αCaM-kinase II revealed a role for RC3 in accelerating the dissociation of the Ca 2+-CaM-αCaM-kinase II complex at the end of a Ca2+ signal. We characterized the interaction of RC3 with apo-CaM and Ca 2+-CaM and found that the KD for both of these interactions is about 1 μM. We also directly tested whether RC3 slowed the dynamics of the binding of CaM to αCaM-kinase II and found that RC3 had no effect for large changes in Ca2+, and a modest effect for small changes in Ca2+ levels. Our overall conclusion is that the ability of RC3 to alter the interaction of Ca2+ with CaM allows RC3 to alter the dynamics of interaction of CaM with Ca2+-dependent targets such as CaM-kinase II. ^

Mechanism of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in head and neck squamous cell carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer and neuroblastoma in clinical trials. 4HPR induces growth inhibition and apoptosis in various cancer cells including head and neck squamous cell carcinoma (HNSCC) cells. 4HPR induces apoptosis by several mechanisms including increasing reactive oxygen species (ROS), or inducing mitochondrial permeability transition (MPT). 4HPR has also been shown to modulate the level of different proteins by transcriptional activation or posttranslational modification in various cellular contexts. However, the mechanism of its action is not fully elucidated. In this study, we explored the mechanism of 4HPR-induced apoptosis in HNSCC cells. ^ First, we identified proteins modulated by 4HPR by using proteomics approaches including: Powerblot western array and 2-dimensional polyacrylamide gel electrophoresis. We found that 4HPR modulated the levels of several proteins including c-Jun. Further analysis has shown that 4HPR induced activation of Activator Protein 1 (AP-1) components, c-Jun and ATF-2. We also found that 4HPR increased the level of Heat shock protein (Hsp) 70 and phosphorylation of Hsp27. ^ Second, we found that 4HPR induced prolonged activation of JNK, p38/MAPK and extracellular signal-regulated kinase (ERK). We also demonstrated that the activation of these kinases is required for 4HPR-induced apoptosis. JNK inhibitor SP600125 and siRNA against JNK1 and JNK2 suppressed, while overexpression of JNK1 enhanced 4HPR-induced apoptosis. p38/MAPK inhibitor PD169316 and MEK1/2 inhibitor PD98059 also suppressed 4HPR-induced apoptosis. We also demonstrated that activation of JNK, p38/MAPK and ERK is triggered by ROS generation induced by 4HPR. We also found that translation inhibitor, cycloheximide, suppressed 4HPR-induced apoptosis through inhibition of 4HPR-induced events (e.g. ROS generation, cytochrome c release, JNK activation and suppression of Akt). We also demonstrated that MPT is involved in 4HPR-induced apoptosis. ^ Third, we demonstrated the presence of NADPH oxidase in HNSCC 2B cells. We also found that 4HPR increased the level of the p67phox, a subunit of NADPH oxidase which participates in ROS production and apoptosis induced by 4HPR. ^ The novel insight into the mechanism by which 4HPR induces apoptosis can be used to improve design of future clinical studies with this synthetic retinoid in combination with specific MAPK modulators. ^

Can a Newly Proposed Mechanism for Allocating Contracts in U.S. Electricity Wholesale Markets Lead to Lower Prices? A Game Theoretic Analysis

This study of the wholesale electricity market compares the cost-minimizing performance of the auction mechanism currently in place in U.S. markets with the performance of a proposed replacement. The current mechanism chooses an allocation of contracts that minimizes a fictional cost calculated using pay-as-offer pricing. Then suppliers are paid the market clearing price. The proposed mechanism uses the market clearing price in the allocation phase as well as in the payment phase. In concentrated markets, the proposed mechanism outperforms the current mechanism even when strategic behavior by suppliers is taken into account. The advantage of the proposed mechanism increases with increased price competition.

Targeting dysregulated nuclear proteins androgen receptor and enhancer of zeste homolog 2: Clinical implication and mechanism underline

Cancer is the most devastating disease that has tremendous impacts on public health. Many efforts have been devoted to fighting cancer through either translational or basic researches for years. Nowadays, it emerges the importance to converge these two research directions and complement to each other for battling with cancer. Thus, our study aims at both translational and basic research directions. The first goal of our study is focus on translational research to search for new agents targeting prevention and therapy of advanced prostate cancer. Hormone refractory prostate cancer is incurable and lethal. Androgen receptor (AR) mediates androgen's effect not only on the tumor initiation but also plays the major role in the relapse transition of prostate cancer. Here we demonstrate that emodin, a natural compound, can directly target AR to suppress prostate cancer cell growth in vitro and prolong the survival of C3(1)/SV40 transgenic mice in vivo. Emodin treatment resulted in repressing androgen-dependent transactivation of AR by inhibiting AR nuclear translocation. Emodin decreased the association of AR and heat shock protein 90 and increased the association of AR and MDM2, which in turn, induces AR degradation through a proteasome-mediated pathway in a ligand independent manner. Our work indicates a new mechanism for the emodin-mediated anticancer effect and justifies further investigation of emodin as a therapeutic and preventive agent for prostate cancer. The second goal of our study is try to elucidate the fundamental tumor biology of cancer progression then provide the rationale to develop more efficient therapeutic strategy. Enhancer of zeste homologue 2 (EZH2) plays an important role in many biological processes through its intrinsic methyltransferase activity to trimethylate lysine 27 in histone H3. Although overexpression of EZH2 has been shown to be involved in cancer progression, the detailed mechanisms are elusive. Here, we show that Akt phosphorylates EZH2 at serine 21 and suppresses its methyltransferase activity by impeding the binding to its substrate histone H3, resulting in a decrease of lysine 27 trimethylation and derepression of silenced genes, thus promotes cell proliferation and tumorigenicity. Our results also show that histone methylation is not permanent but regulated in a dynamic manner and that the Akt signaling pathway is involved in the regulation of this epigenetic modification through phosphorylation of EZH2, thus contributing to oncogenic processes. ^

Mechanism of IFN-induced apoptosis and sensitization by the proteasome inhibitor bortezomib in bladder cancer cells

Bladder cancer is the fifth most common cancer with more than 50,000 cases diagnosed each year. Interferon-α (IFNα) is mostly used in combination with BCG for the treatment of transitional cell carcinoma (TCC). To examine the effects of IFNα on bladder cancer cells, I analyzed a panel of 20 bladder cancer cell lines in terms of their sensitivity to IFNα-induced apoptosis and the underlying mechanisms. I identified three categories: cells that die after 48hr, after 72h, and cells resistant even after 72hr of IFNα treatment. Examination of the IFN-signal transduction pathway revealed that the defect was not due to abrogation of IFN signaling. Further analysis demonstrated dependency of IFN-induced apoptosis on caspase-8, implicating the role of death receptors in IFN-induced cell death. Of the six most-IFN-sensitive cell lines, the majority upregulated Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at the mRNA and protein level and IFN-induced cell death was mediated through TRAIL, while a minority of the most IFN-sensitive cells undergo apoptosis through a TNFα-dependent mechanism. IFNα resistance was due to either absence of TRAIL upregulation at the mRNA or protein level, resistance to exogenous rhTRAIL itself or lack of sensitization to IFN-induced cell death. Downregulation of XIAP, or XIAP inactivation through its regulator NFκB has been reported to sensitize tumor cells to death receptor-induced cell death. Baseline and IFN-inducible XIAP levels were examined in the most and least IFN-sensitive cells, knocking down XIAP and the p65 subunit of NFκB enhanced IFN-induced cell death, implicating XIAP downregulation as a mechanism through which bladder cancer cells are sensitized to IFN-induced apoptosis. To determine whether or not the proteasome inhibitor Bortezomib (BZ) sensitizes bladder cancer cells to IFN-induced cell death, the combined effects of IFN+BZ and the underlying molecular mechanisms were examined both in vitro and in vivo using two bladder xenograft models. In both models, tumor growth inhibition was the result of either increased cell death of tumor cells exerted by the two agents and/or inhibition of angiogenesis. In vitro, MAP downregulation in response to the combined treatment of IFN+BZ accounts for one of the mechanisms mediating IFN+BZ cell death in bladder cancer cells. ^

A mechanism of insulin-like growth factor binding protein 2-induced cell mobility in glioma

Insulin-like growth factor binding protein 2 (IGFBP2) is a protein known to be overexpressed in a majority of glioblastoma multiforme (GBM) tumors. While it is known the IGFBP2 is involved in promoting GBM tumor cell invasion, no mechanism exists for how the protein is involved in signal transduction pathways leading to enhanced cell invasion. ^ We follow up on preliminary microarray data on IGFBP2-overexpressing GBM cells and protein sequence analysis of IGFBP2 in generating the hypothesis that IGFBP2 interacts with integnn α5 in regulating cell mobility. Microarray data showing upregulation of integrin α5 by IGFBP2 is validated and evidence of protein-protein interaction between IGFBP2 and integrin α5 is found. The exact binding domain on IGFBP2 responsible for its interaction with integrin α5 is also determined, confirming our initial findings and reaffirming that the IGFBP2/integrin α5 interaction is specific. Disruption of this interaction resulted in attenuation of IGFBP2-enhanced cell mobility. Further, we found that cell mobility is only enhanced when IGFBP2 and integrin α5 are both overexpressed and able to interact with each other. ^ We also determined fibronectin to be a critical player in the activation of the IGFBP2/integrin α5 pathway. The activation of this pathway appears to be progressive and initiates once GBM cells have sufficiently established anchorage. ^

The low molecular weight (LMW) isoforms of cyclin E provide a novel mechanism of cell cycle deregulation

Cyclin E, in complex with cyclin dependent kinase 2 (CDK2), is a positive regulator of G1 to S phase progression of the cell cycle. Deregulation of G1/S phase transition occurs in the majority of tumors. Cyclin E is overexpressed and post-translationally generates low molecular weight (LMW) isoforms in breast cancer, but not normal cells. Such alteration of cyclin E is linked to poor prognosis. Therefore, we hypothesized that the LMW isoforms of cyclin E provide a novel mechanism of cell cycle de-regulation in cancer cells. Insect cell expression system was used to explore the biochemical properties of the cyclin E isoforms. Non-tumorigenic (76NE6) and tumorigenic (T47D) mammary epithelial cells transfected with the cyclin E isoforms and breast tumor tissue endogenously expressing the LMW isoforms were used to study the biologic consequences of the LMW isoforms of cyclin E. All model systems studied show that the LMW forms (compared to full-length cyclin E) have increased kinase activity when partnered with CDK2. Increases in the percentage of cells in S phase and colony formation were also observed after overexpression of LMW compared to full-length cyclin E. The LMW isoforms of cyclin E utilize several mechanisms to attain their hyper-activity. They bind CDK2 more efficiently, and are resistant to inhibition by cyclin dependent kinase inhibitors (CKIs) as compared to full-length cyclin E. In addition, the LMW isoforms sequester the CKIs from full-length cyclin E abrogating the overall negative regulation of cyclin E. Despite their correlation with adverse biological consequences, the direct role of the LMW isoforms of cyclin E in mediating tumorigenesis remained unanswered. Subsequent to LMW cyclin E expression in 76NE6 cells, they lose their ability to enter quiescence and exhibit genomic instability, both characteristic of a tumor cell phenotype. Furthermore, injection of 76NE6 cells overexpressing each of the cyclin E isoforms into the mammary fat pad of nude mice revealed that the LMW isoforms of cyclin E yield tumors, whereas the full-length cyclin E does not. In conclusion, the LMW isoforms of cyclin E utilize several mechanisms to acquire a hyperactive phenotype that results in deregulation of the cell cycle and initiates the tumorigenic process in otherwise non-transformed mammary epithelial cells. ^

The role and mechanism of supressor of cytokine signaling 1 in brain metastasis

Brain metastasis, which occurs in 40%-60% of patients with advanced melanoma, has led directly to death in the majority of cases. Unfortunately, little is known about the biological and molecular basis of melanoma brain metastases. In our previous study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The increased activation of Stat3 is also responsible for affecting melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affecting the expression of bFGF, VEGF, and MMP-2 in vivo and in vitro. Interestingly, a member of a new family of cytokine-inducible inhibitors of signal transduction, termed suppressors of cytokine signaling 1 (SOCS1) was found to negatively regulate the Janus kinase signal transducer and activator of transcription (Jak/STAT) signaling cascade. Here we report that restoration of SOCS1 expression by transfecting of SOCS1-expressing vector effectively inhibited melanoma brain metastasis through inhibiting Stat3 activation and further affecting melanoma angiogenesis and melanoma cell invasion in vitro, and significantly affected the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in vitro and in vivo. In addition, we used cDNA array to compare mRNA expression in the SOCS1-transfected and vector-transfected cell lines and found some genes are tightly correlated to the restoration of SOCS1. One of them is Caveolin-1 (Cav-1). Cav-1 was reported to function as a tumor suppressor gene by several groups. Finally, the Cav-1 expression is up-regulated in SOCS1-overexpressing cell line. Further study found the regulation of Cav-1 by SOCS1 occurs through inhibiting Stat3 activation. Activated Stat3 binds directly to Cav-1 promoter and the Cav-1 promoter within -575bp is essential for active Stat3 binding. My studies reveal that Stat3 activation and SOCS1 expression play important roles in melanoma metastases. Moreover, the expression between SOCS1, Stat3 and Cav-1 forms a feedback regulation loop. ^

A novel mechanism of 3-bromopyruvate-induced cell death through targeting hexokinase and ubiquitination

Increased dependence on aerobic glycolysis for energy (ATP) supply has been observed in various human cancer cells. It is plausible to exploit this metabolic alteration for therapeutic benefits by inhibiting glycolysis to preferentially abolish cancer energy metabolism and kill the malignant cells. 3-Bromopyruvate has been shown to be a potent inhibitor of glycolysis capable of inducing severe ATP reduction and cell death in various cancer cell lines, especially cancer cells with mitochondrial defects or under hypoxic conditions. However, the detailed mechanisms of this novel anticancer agent still remain unclear. My study demonstrated that 3-Bromopyruvate caused a covalent modification of hexokinase II, a key glycolytic enzyme, and disrupted its association with mitochondria. This led to mitochondrial permeability transition and a substantial release of apoptosis-inducing faction (AIF) prior to cytochrome c release. Dissociation of HK II from mitochondria using a cell permeable specific peptide also induced the release of AIF and cytochrome c, and caused substantial cell death. HK II-targeted peptide did not cause significant change in mitochondria respiration and glycolysis activity, suggesting that dissociation of this molecule from mitochondria alone can also cause cell death, and that this may be a novel mechanism by which 3-Bromopyruvate exerts its potent cytotoxic action, in addition to its inhibition of the enzyme activity. Another significant new discovery was that 3-Bromopyruvate induced rapid reduction of protein ubiquitination in vivo, which occurred within several hours of drug incubation and before ATP reduction and cell death. Further mechanistic studies showed that this was due to the inhibition the ubiquitin activating enzyme E1 and the conjugating enzyme E2. Knocking down ubiquitin protein expression by siRNA did not suppress mitochondria respiration and glycolysis, but caused significant cell death. Taken together, this study demonstrated that induction of HK II dissociation from mitochondria and inhibition of glycolysis are two newly discovered mechanisms that contribute to the potent anticancer activity of 3-Bromopyruvate, and identified this compound as a valuable chemical tool for research in protein ubiquitination. ^

Mitochondrial dysfunction leads to NOX activation: A novel mechanism to maintain high glycolysis in cancer cells

Increased glycolysis and oxidative stress are common features of cancer cells. These metabolic alterations are associated with mitochondrial dysfunction and can be caused by mitochondrial DNA (mtDNA) mutations, oncogenic signals, loss of tumor suppressor, and tumor tissue hypoxia. It is well established that mitochondria play central roles in energy metabolism, maintenance of redox balance, and regulation of apoptosis. However, the biochemical and molecular mechanisms that maintain high glycolysis in cancer cells (the Warburg effect) with mitochondrial dysfunction and oxidative stress remain to be determined. The major goals of this study were to establish a unique experimental system in which the mitochondrial respiratory function can be regulated as desired, and to use this system to investigate the mechanistic link between mitochondrial dysfunction and the Warburg effect along with oxidative stress in cancer cells. To achieve these goals, I have established a tetracycline-inducible system in which a dominant negative form of mitochondrial DNA polymerase y (POLGdn) expression could be regulated by tetracycline; thus controlling mitochondrial respiratory function. Using this cell system, I demonstrated that POLGdn expression resulted in mitochondrial dysfunction through decreasing mtDNA content, depletion of mtDNA encoded mRNA and protein expression. This process was mediated by TFAM proteasome degradation. Mitochondrial dysfunction mediated by POLGdn expression led to a significant increase in cellular glycolysis and oxidative stress. Surprisingly, mitochondrial dysfunction also resulted in increased NAD(P)H oxidase (NOX) enzyme activity, which was shown to be essential for maintaining high glycolysis. Chemical Inhibition of NOX activity by diphenyliodonium (DPI) preferentially impacted the survival of mitochondrial defective cells. The colon cancer HCT116-/- cells that have lost transcriptional regulation of the mitochondrial assembling enzyme SCO2, leading to compromised mitochondrial respiratory function, were found to have increased NOX activity and were highly sensitive to DPI treatment. Ovarian epithelial cells with Ras transformation also exhibited an increase in NOX gene expression and NOX enzyme activity, rendering the cells sensitive to DPI inhibition especially under hypoxic condition. These data together suggest that NOX plays a novel role in maintaining high glycolysis in cancer cells with mitochondrial defects, and that NOX may be a potential target for cancer therapy. ^

The mechanism of action of a novel benzo[c]phenanthridine alkaloid, NK314 and the cellular responses

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that is currently in clinical trials as an antitumor compound, based on impressive activities in preclinical models. However, its mechanism of action is unknown. The present investigations were directed at determining the mechanism of action of this agent and cellular responses to NK314. My studies demonstrated that NK314 intercalated into DNA, trapped topoisomerase IIα in its cleavage complex intermediate, and inhibited the ability of topoisomerase IIα to relax super-coiled DNA. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIα, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. This indicates topoisomerase IIα is the target of NK314 in the cells. NK314 caused phosphorylation of the histone variant, H2AX, which is considered a marker of DNA double-strand breaks. DNA double-strand breaks were also evidenced by pulsed-field gel electrophoresis and visualized as chromosomal aberrations after cells were treated with NK314 and arrested in mitosis. Cell cycle checkpoints are activated following DNA damage. NK314 induced significant G2 cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G2 checkpoint pathway was activated in response to NK314, which can be abrogated by the Chk1 inhibitor UCN-01. Cell cycle checkpoint activation may be a defensive mechanism that provides time for DNA repair. DNA double-strand breaks are repaired either through ATM-mediated homologous recombination or DNA-PK-mediated non-homologous end-joining repair pathways. Clonogenic assays demonstrated a significant decrease of colony formation in both ATM deficient and DNA-PK deficient cells compared to ATM repleted and DNA-PK wild type cells respectively, indicating that both ATM and DNA-PK play important roles in the survival of the cells in response to NK314. The DNA-PK specific inhibitor NU7441 also significantly sensitized cells to NK314. In conclusion, the major mechanism of NK314 is to intercalate into DNA, trap and inhibit topoisomerase IIα, an action that leads to the generation of double-strand DNA breaks, which activate ATM and DNA-PK mediated DNA repair pathways and Chk1 mediated G2 checkpoint pathway. ^

Molecular mechanism of jet fuel induced immune suppression

Dermal exposure to jet fuel suppresses the immune response. Immune regulatory cytokines, and biological modifiers, including platelet activating factor, prostaglandin E2, and interleukin-10 have all been implicated in the pathway leading to immunosuppression. It is estimated that approximately 260 different hydrocarbons are found in JP-8 (jet propulsion-8) jet fuel, and the identity of the immunotoxic compound is not known. The recent availability of synthetic jet fuel (S-8), which is devoid of aromatic hydrocarbons, made it feasible to design experiments to test the hypothesis that the aromatic hydrocarbons are responsible for jet fuel induced immune suppression. Applying S-8 to the skin of mice does not up-regulate the expression of epidermal cyclooxygenase-2 nor does it induce immune suppression. Adding back a cocktail of 7 of the most prevalent aromatic hydrocarbons found in jet fuel to S-8 up-regulated cyclooxygenase-2 expression and induced immune suppression. Cyclooxygenase-2 induction can be initiated by reactive oxygen species (ROS). JP-8 treated keratinocytes increased ROS production, S-8 did not. Antioxidant pre-treatment blocked jet fuel induced immune suppression and cyclooxygenase-2 up-regulation. Accumulation of reactive oxygen species induces oxidant stress and affects activity of ROS sensitive transcription factors. JP-8 induced activation of NFκB while S-8 did not. Pre-treatment with antioxidants blocked activation of NFκB and parthenolide, an NFκB inhibitor, blocked jet fuel induced immune suppression and cyclooxygenase-2 expression in skin of treated mice. p65 siRNA transfected keratinocytes demonstrated NFκB is critically involved in jet fuel induced COX-2 expression. These findings clearly implicate the aromatic hydrocarbons found in jet fuel as the agents responsible for inducing immune suppression, in part by the production of reaction oxygen species, NFκB dependent up-regulation of cyclooxygenase-2, and the production of immune regulatory factors and cytokines. ^

Anticancer activity of OSW-1: Induction of apoptotic pathway in leukemia and autophagic death in pancreatic cancer through a calcium mediated mechanism

OSW-1 is a natural compound found in the bulbs of Orninithogalum saudersiae, a member of the lily family. This compound exhibits potent antitumor activity in vitro with the IC50 values in the low nanomolar concentration range and demonstrating its ability to kill drug resistant cancer cells. In an effort to discover the unknown mechanism of action of this novel compound as a potential anticancer agent, the main objective of this research project was to test the cytotoxicity of OSW-1 against various cancer lines, and to elucidate the biochemical and molecular mechanism(s) responsible for the anticancer activity of OSW-1. My initial investigation revealed that OSW-1 is effective in killing various cancer cells including pancreatic cancer cells and primary leukemia cells resistant to standard chemotherapeutic agents, and that non-malignant cells were less sensitive to this compound. Further studies revealed that in leukemia cells, OSW-1 causes a significant increase in cytosolic calcium and activates rapid calcium-dependent apoptosis by the intrinsic pathway. Additionally, OSW-1 treatment leads to the degradation of the ER chaperone GRP78/BiP implicated in the survival of cancer cells. Meanwhile, it shows a reduced sensitivity in respiration-deficient sub-clones of leukemia cells which had higher basal levels of Ca2+. Mechanistically, it was further demonstrated that cytosolic Ca2+ elevations were observed together with Na+ decreases in the cytosol, suggesting OSW-1 caused the calcium overload through inhibition of the Na+/Ca 2+exchanger (NCX). Although similar calcium disturbances were observed in pancreatic cancer cells, mechanistic studies revealed that autophagy served as an initial pro-survival mechanism subsequent to OSW-1 treatment but extended autophagy caused inevitable cell death. Furthermore, combination of OSW-1 with autophagy inhibitors significantly enhances the cytotoxicity against pancreatic cancer cells. Taken together, this study revealed the novel mechanism of OSW-1 which is through inhibition of the Na+/Ca2+ exchanger and provides a basis for using this compound in combination with other agents for the treatment of pancreatic cancer which is resistant to available anticancer drugs. ^

G870A polymorphism of cyclin D1 gene influences cancer risk - From epidemiological study to mechanism analysis

Alternate splicing of the cyclin D1 gene gives rise to transcript a and b which encode two protein isoforms cyclin D1a and cyclin D1b. Through testing transcript a and transcript b in a series of human samples, we found that cyclin D1 transcript b is ubiquitously expressed as transcript a but in the lower abundance compared to transcript a. Epidemiological studies have reported that the cyclin D1 gene (CCND1) G870A polymorphism influences the risk for a variety of cancer. In this investigation, we examined the cyclin D1b levels in tumor samples with different genotypes and found that higher levels of cyclin D1b are expressed from the A allele than the G allele. Cyclin D1 is known as a cell cycle regulator facilitating the progression of the cell cycle from G1 to S phase in response to the mitogenic signals. It also interacts with several transcription factors and transcriptional coregulators to modulate their activities. It has been reported that cyclin D1a can substitute for estrogen to activate estrogen receptor α (ERα) mediated transcription and can induce the proliferation of estrogen responsive tissues. However the biological role of cyclin D1b in ERα transcriptional regulation has not been previously explored. In this study, we determined that cyclin D1b antagonizes the action of cyclin D1a on ERα mediated transcription. Cell proliferation assays provided the evidence that cyclin D1b negatively regulates estrogen responsive breast cancer cell growth. Taken together, our findings show that the CCND1 G870A polymorphism is correlated with increased levels of cyclin D1b and that cyclin D1b antagonizes the action of cyclin D1a on ERα mediated transcription providing evidence for the mechanism by which the CCND1 G870A polymorphism may be protective in certain types of breast cancer. ^

Mechanism of methylation gene reactivation in human cancer cells

Epigenetic silencing of tumor suppressor genes by DNA hypermethylation at promoter regions is a common event in carcinogenesis and tumor progression. Abrogation of methylation and reversal of epigenetic silencing is a very potent way in cancer treatment. However, the reactivation mechanisms are poorly understood. In this study, we first developed a cell line model system named YB5, derived from SW48 cancer cell line, which bears one copy of stably integrated EGFP gene on Chromosome 1p31.1 region. The GFP gene expression is transcriptionally silenced due to the hypermethylated promoter CMV. However, the GFP expression can be restored using demethylating agent 5-aza-2' deoxycytidine (DAC), and detected by FACS and fluorescent microscopy. Using this system, we observed the heterogeneous reactivation induced by DAC treatment. After flow sorting, GFP negative cells exhibited similar level of incomplete demethylation compared to GFP positive cells on repetitive LINE1 element, tumor suppressor genes such as P16, CDH13, and RASSF1a, and CMV promoter as well. However, the local chromatin of CMV-GFP locus altered to an open structure marked by high H3 lysine 9 acetylation and low H3 lysine 27 tri-methylation in GFP positive cells, while the GFP negative cells retained mostly the original repressive marks. Thus, we concluded that DAC induced DNA hypomethylation alone does not directly determine the level of re-expression, and the resetting of the local chromatin structure under hypomethylation environment is required for gene reactivation. Besides, a lentivirus vector-based shRNA screening was performed using the YB5 system. Although it is the rare chance that vector lands in the neighboring region of GFP, we found that the exogenous vector DNA inserted into the upstream region of GFP gene locus led to the promoter demethylation and reactivated the silenced GFP gene. Thus, epigenetic state can be affected by changing of the adjacent nucleic acid sequences. Further, this hypermethylation silenced system was utilized for epigenetic drug screening. We have found that DAC combined with carboplatin would enhance the GFP% yield and increase expression of other tumor suppressor genes than DAC alone, and this synergistic effect may be related to DNA repair process. In summary, these studies reveal that reversing of methylation silencing requires coordinated alterations of DNA methylation, chromatin structure, and local microenvironment. ^