Possible role of duration of PKC-induced ERK activation in the effects of agonists and phorbol esters on DNA synthesis in Panc-1 cells

Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) have been implicated in the effects of regulatory peptides on proliferation. We studied how ERK was activated by PKC following regulatory peptide or phorbol ester stimulation and we also investigated the effect of ERK activation on proliferation in Panc-1 cells. Panc-1 cells transfected with CCK1 receptors were treated with cholecystokinin (CCK), neurotensin (NT), or phorbol 12-myristate 13-acetate (PMA). DNA synthesis was studied by measuring tritiated thymidine incorporation. PKC isoforms were selectively inhibited with G?6983 and 200 nM Ro-32-0432, their translocation was detected by confocal microscopy and by subcellular fractionation followed by immunoblotting. ERK cascade activation was detected with phosphoERK immunoblotting and inhibited with 20 microM PD98059. PMA and CCK inhibited, NT stimulated DNA synthesis. These effects were inhibited by Ro-32-0432 but not by G?6983 suggesting the involvement of PKCepsilon in proliferation control. Confocal microscopy and subcellular fractionation demonstrated that PMA, CCK, and NT caused cytosol to membrane translocation of PKCepsilon and ERK activation that was inhibited by Ro-32-0432 but not by G?6983. ERK activation was prolonged following PMA and CCK, but transient after NT treatment. PMA, CCK, and NT all activated cyclinD1, while p21CIP1 expression was increased by only PMA and CCK, but not by NT; each of these effects is inhibited by PD98059. In conclusion, our results provide evidence for PKCepsilon-mediated differential ERK activation and growth regulation in Panc-1C cells. Identification of the mechanisms by which these key signaling pathways are modulated could provide a basis for the development of novel therapeutic interventions to treat pancreatic cancer.     

Metazoan origins of DNA replication: Regulation through dynamic chromatin structure

DNA replication in eukaryotes is initiated at multiple replication origins distributed over the entire genome, which are normally activated once per cell cycle. Due to the complexity of the metazoan genome, the study of metazoan replication origins and their activity profiles has been less advanced than in simpler genome systems. DNA replication in eukaryotes involves many protein–protein and protein–DNA interactions, occurring in multiple stages. As in prokaryotes, control over the timing and frequency of initiation is exerted at the initiation site. A prerequisite for understanding the regulatory mechanisms of eukaryotic DNA replication is the identification and characterization of the cis‐acting sequences that serve as replication origins and the trans‐acting factors (proteins) that interact with them. Furthermore, in order to understand how DNA replication may become deregulated in malignant cells, the distinguishing features between normal and malignant origins of DNA replication as well as the proteins that interact with them must be determined. Based on advances that were made using simple genome model systems, several proteins involved in DNA replication have been identified. This review summarizes the current findings about metazoan origins of DNA replication and their interacting proteins as well as the role of chromatin structure in their regulation. Furthermore, progress in origin identification and isolation procedures as well as potential mechanisms to inhibit their activation in cancer development and progression are discussed. J. Cell. Biochem. 106: 512–520, 2009. © 2009 Wiley‐Liss, Inc.     

Human Papillomaviruses Activate the ATM DNA Damage Pathway for Viral Genome Amplification upon Differentiation

Human papillomaviruses (HPV) are the causative agents of cervical cancers. The infectious HPV life cycle is closely linked to the differentiation state of the host epithelia, with viral genome amplification, late gene expression and virion production restricted to suprabasal cells. The E6 and E7 proteins provide an environment conducive to DNA synthesis upon differentiation, but little is known concerning the mechanisms that regulate productive viral genome amplification. Using keratinocytes that stably maintain HPV-31 episomes, and chemical inhibitors, we demonstrate that viral proteins activate the ATM DNA damage response in differentiating cells, as indicated by phosphorylation of CHK2, BRCA1 and NBS1. This activation is necessary for viral genome amplification, as well as for formation of viral replication foci. In contrast, inhibition of ATM kinase activity in undifferentiated keratinocytes had no effect on the stable maintenance of viral genomes. Previous studies have shown that HPVs induce low levels of caspase 3/7 activation upon differentiation and that this is important for cleavage of the E1 replication protein and genome amplification. Our studies demonstrate that caspase cleavage is induced upon differentiation of HPV positive cells through the action of the DNA damage protein kinase CHK2, which may be activated as a result of E7 binding to the ATM kinase. These findings identify a major regulatory mechanism responsible for productive HPV replication in differentiating cells. Our results have potential implications for the development of anti-viral therapies to treat HPV infections.     

Programs of Gene Expression During the Laying Down of Memory Formation as Revealed by DNA Microarrays

Many experiments in the past have demonstrated the requirement of de novo gene expression during memory formation. In contrast to the initial reductionistic view that genes relevant to learning and memory would be easily found and would provide a simple key to understand this brain function, it is becoming apparent that the genetic contribution to memory is complex. Previous approaches have been focused on individual genes or genetic pathways and failed to address the massively parallel nature of genome activities and collective behavior of the genes that ultimately control the molecular mechanisms underlying brain function. In view of the broad variety of genes and the cross talk of genetic pathways involved in this regulation, only gene expression profiles may reflect the complete behavior of regulatory pathways. In this review we illustrate how DNA microarray-based gene expression profiling may help to dissect and analyze the complex mechanisms involved in gene regulation during the acquisition and storage of memory in the mammalian brain.     

High resolution methylation analysis of the HoxA5 regulatory region in different somatic tissues of laboratory mouse during development

Homeobox genes encode a group of DNA binding regulatory proteins whose key function occurs in the spatial-temporal organization of genome during embryonic development and differentiation. The role of these Hox genes during ontogenesis makes it an important model for research. HoxA5 is a member of Hox gene family playing a central role during axial body patterning and morphogenesis. DNA modification studies have shown that the function of Hox genes is partly governed by the methylation-mediated gene expression regulation. Therefore the study aimed to investigate the role of epigenetic events in regulation of tissue-specific expression pattern of HoxA5 gene during mammalian development. The methodology adopted were sodium bisulfite genomic DNA sequencing, quantitative real-time PCR and chromatin-immunoprecipitation (ChIP). Methylation profiling of HoxA5 gene promoter shows higher methylation in adult as compared to fetus in various somatic tissues of mouse being highest in adult spleen. However q-PCR results show higher expression during fetal stages being highest in fetal intestine followed by brain, liver and spleen. These results clearly indicate a strict correlation between DNA methylation and tissue-specific gene expression. The findings of chromatin-immunoprecipitation (ChIP) have also reinforced that epigenetic event like DNA methylation plays important role in the regulation of tissue specific expression of HoxA5.     

Roles of the envelope proteins in the amplification of covalently closed circular DNA and completion of synthesis of the plus-strand DNA in hepatitis B virus

Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion.     

早期胚胎发育合子基因组激活调控

动物早期胚胎发育始于分化成熟的雌雄配子经受精后重编程为全能性合子。在胚胎发育的初期,合子基因组的转录水平处于静默状态,母源物质调控占据主导地位。随着胚胎发育的进行,母源物质会经历分阶段的降解,合子基因组开始逐渐激活转录,标志着早期胚胎发育从母源性调控向合子基因组调控的转变,也称为母源-合子转换（maternal-zygotic transition,MZT）。其中一个关键的转折性事件就是合子基因组激活（zygotic genome activation,ZGA）,ZGA的正确发生对于早期胚胎发育和细胞命运决定至关重要。然而,目前对于ZGA的调控因子和具体的分子机制仍知之甚少。研究表明,ZGA在不同物种中存在较大差异,可能受到DNA甲基化、组蛋白修饰、非编码RNA、染色质重塑以及ZGA相关因子等多种调控因素的影响。本文探讨了上述几种调控因素影响合子基因组激活的研究进展,对进一步研究早期胚胎ZGA的相关机制具有借鉴意义。     

甲基苯丙胺成瘾的表观遗传学机制

苯丙胺类兴奋剂是全世界第二大滥用程度的药物,甲基苯丙胺作为苯胺类兴奋剂中的主要药物,是中国滥用的“头号毒品”。而现有的研究对甲基苯丙胺成瘾机制尚不清晰,且临床上对药物成瘾的治疗依然存在无药可医的局面。因此,发现新的成瘾机制和治疗策略尤为迫切。甲基苯丙胺成瘾与额前叶皮质（mPFC）、中脑腹侧被盖区（VTA）和伏隔核（NAc）中的多巴胺（DA）、谷氨酸（Glu）、去甲肾上腺素（NE）和血清素（SNRIS）等神经递质的异常释放有关。研究表明,这些神经递质受到表观遗传机制中组蛋白乙酰化、甲基化、泛素化和非编码RNA等调节,某些基因的表达在甲基苯丙胺的诱导过程中增强或被抑制,导致甲基苯丙胺依赖性产生。本文将针对表观遗传学对甲基苯丙胺成瘾机制的影响进行着重论述,以期推进临床开发甲基苯丙胺戒断药物的研究。