细胞自噬与高迁移率族蛋白B-1(HMGB1)介导肿瘤耐药的研究进展

恶性肿瘤可通过多种细胞机制,产生对抗癌药物和放疗的抗性,即所谓耐药现象.细胞自噬是肿瘤细胞耐药的一个重要原因.高迁移率族蛋白B-1(HMGB1)是高度保守的非组蛋白DNA结合蛋白,在DNA结构、基因转录、基因重组、DNA损伤修复以及细胞存活等方面发挥重要的调控作用;HMGB1在细胞内的功能,与其氧化还原状态和细胞定位息...     

The lyase activity of the DNA repair protein beta-polymerase protects from DNA-damage-induced cytotoxicity

Small DNA lesions such as oxidized or alkylated bases are repaired by the base excision repair (BER) pathway. BER includes removal of the damaged base by a lesion-specific DNA glycosylase, strand scission by apurinic/apyrimidinic endonuclease, DNA resynthesis and ligation. BER may be further subdivided into DNA beta-polymerase (beta-pol)-dependent single-nucleotide repair and beta-pol-dependent or -independent long patch repair subpathways. Two important enzymatic steps in mammalian single-nucleotide BER are contributed by beta-pol: DNA resynthesis of the repair patch and lyase removal of 5'-deoxyribose phosphate (dRP). Fibroblasts from beta-pol null mice are hypersensitive to mono-functional DNA-methylating agents, resulting in increases in chromosomal damage, apoptosis and necrotic cell death. Here we show that only the dRP lyase activity of beta-pol is required to reverse methylating agent hypersensitivity in beta-pol null cells. These results indicate that removal of the dRP group is a pivotal step in BER in vivo. Persistence of the dRP moiety in DNA results in the hypersensitivity phenotype of beta-pol null cells and may signal downstream events such as apoptosis and necrotic cell death.     

Endonuclease G is an apoptotic DNase when released from mitochondria.

Nucleosomal fragmentation of DNA is a hallmark of apoptosis (programmed cell death), and results from the activation of nucleases in cells undergoing apoptosis. One such nuclease, DNA fragmentation factor (DFF, a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD)), is capable of inducing DNA fragmentation and chromatin condensation after cleavage by caspase-3 (refs 2,3,4). However, although transgenic mice lacking DFF45 or its caspase cleavage site have significantly reduced DNA fragmentation, these mice still show residual DNA fragmentation and are phenotypically normal. Here we report the identification and characterization of another nuclease that is specifically activated by apoptotic stimuli and is able to induce nucleosomal fragmentation of DNA in fibroblast cells from embryonic mice lacking DFF. This nuclease is endonuclease G (endoG), a mitochondrion-specific nuclease that translocates to the nucleus during apoptosis. Once released from mitochondria, endoG cleaves chromatin DNA into nucleosomal fragments independently of caspases. Therefore, endoG represents a caspase-independent apoptotic pathway initiated from the mitochondria.     

The Cdt1 protein is required to license DNA for replication in fission yeast

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.     

Novel role of PKR in inflammasome activation and HMGB1 release

The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1β, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1β, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.     

Dynamic changes of apoptosis in duck embryo fibroblasts induced by new type Gosling viral enteritis virus

The monolayer duck embryo fibroblast （DEF） cells were experimentally infected with new type Gosling viral enteritis virus （NGVEV） and the dynamic changes of apoptosis were detected at different time points after NGVEV infection by transmission electron microscopy （TEM）, DNA agarose gel electrophoresis and Annexin V-FITC/PI stained fluorescence-activated cell sorter （FACS）. The result shows that NGVEV can induce infected cells undergoing apoptosis and changing regularly. A series of characteristic apoptotic morphological changes including shrinkage of the cells, chromatin condensation and margination, as well as formation of apoptotic bodies, were observed by TEM. The typical ladder pattern of DNA fragmentation was demonstrated by agarose gel electrophoresis. And using flow cytometry analysis of Annexin V-FITC/PI staining, the dead, viable, apoptotic and necrotic cells could be analyzed quantitatively.     

Molecular identification of a danger signal that alerts the immune system to dying cells

In infections, microbial components provide signals that alert the immune system to danger and promote the generation of immunity. In the absence of such signals, there is often no immune response or tolerance may develop. This has led to the concept that the immune system responds only to antigens perceived to be associated with a dangerous situation such as infection. Danger signals are thought to act by stimulating dendritic cells to mature so that they can present foreign antigens and stimulate T lymphocytes. Dying mammalian cells have also been found to release danger signals of unknown identity. Here we show that uric acid is a principal endogenous danger signal released from injured cells. Uric acid stimulates dendritic cell maturation and, when co-injected with antigen in vivo, significantly enhances the generation of responses from CD8+ T cells. Eliminating uric acid in vivo inhibits the immune response to antigens associated with injured cells, but not to antigens presented by activated dendritic cells. Our findings provide a molecular link between cell injury and immunity and have important implications for vaccines, autoimmunity and inflammation.     

53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility

Double-strand breaks activate the ataxia telangiectasia mutated (ATM) kinase, which promotes the accumulation of DNA damage factors in the chromatin surrounding the break. The functional significance of the resulting DNA damage foci is poorly understood. Here we show that 53BP1 (also known as TRP53BP1), a component of DNA damage foci, changes the dynamic behaviour of chromatin to promote DNA repair. We used conditional deletion of the shelterin component TRF2 (also known as TERF2) from mouse cells (TRF2(fl/-)) to deprotect telomeres, which, like double-strand breaks, activate the ATM kinase, accumulate 53BP1 and are processed by non-homologous end joining (NHEJ). Deletion of TRF2 from 53BP1-deficient cells established that NHEJ of dysfunctional telomeres is strongly dependent on the binding of 53BP1 to damaged chromosome ends. To address the mechanism by which 53BP1 promotes NHEJ, we used time-lapse microscopy to measure telomere dynamics before and after their deprotection. Imaging showed that deprotected telomeres are more mobile and sample larger territories within the nucleus. This change in chromatin dynamics was dependent on 53BP1 and ATM but did not require a functional NHEJ pathway. We propose that the binding of 53BP1 near DNA breaks changes the dynamic behaviour of the local chromatin, thereby facilitating NHEJ repair reactions that involve distant sites, including joining of dysfunctional telomeres and AID (also known as AICDA)-induced breaks in immunoglobulin class-switch recombination.     

Translational control of InsP3-induced chromatin condensation during the early cell cycles of sea urchin embryos

The cycles of DNA synthesis and chromatin condensation in dividing cells are controlled by signals from the cytoplasm. Changes in the concentration of free calcium (Cai) in the cytoplasm control a variety of cellular functions and it has thus been suggested that observed variations in Cai during the cell cycle may be the cytoplasmic signal that co-ordinates nuclear and cytoplasmic division. We show here that increases in Cai induced by the calcium-releasing second messenger inositol 1,4,5-triphosphate (Ins(1,4,5)P3), or by calcium buffers, cause premature chromatin condensation and breakdown of the nuclear envelope in sea urchin (Lytechinus pictus) early embryos. Both natural and induced chromatin condensation are prevented by calcium chelators. The nucleus becomes sensitive to the Cai signal 45 min after fertilization, but remains insensitive if protein synthesis is prevented. Our experiments demonstrate that Cai regulates the behaviour of the nucleus during the cell cycle, suggest that Ins(1,4,5)P3 is a cell cycle messenger and indicate that there is an interaction between the protein and ionic signals that control the state of chromatin during the cell cycle.     

HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response

Minutes after DNA damage, the variant histone H2AX is phosphorylated by protein kinases of the phosphoinositide kinase family, including ATM, ATR or DNA-PK. Phosphorylated (gamma)-H2AX-which recruits molecules that sense or signal the presence of DNA breaks, activating the response that leads to repair-is the earliest known marker of chromosomal DNA breakage. Here we identify a dynamic change in chromatin that promotes H2AX phosphorylation in mammalian cells. DNA breaks swiftly mobilize heterochromatin protein 1 (HP1)-beta (also called CBX1), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). Local changes in histone-tail modifications are not apparent. Instead, phosphorylation of HP1-beta on amino acid Thr 51 accompanies mobilization, releasing HP1-beta from chromatin by disrupting hydrogen bonds that fold its chromodomain around H3K9me. Inhibition of casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair, suppresses Thr 51 phosphorylation and HP1-beta mobilization in living cells. CK2 inhibition, or a constitutively chromatin-bound HP1-beta mutant, diminishes H2AX phosphorylation. Our findings reveal an unrecognized signalling cascade that helps to initiate the DNA damage response, altering chromatin by modifying a histone-code mediator protein, HP1, but not the code itself.     

H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes

DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes. DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently. Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.     

依折麦布对高迁移率族蛋白1诱导血管内皮细胞激活的影响机制研究

目的探讨依折麦布对高迁移率族蛋白1(HMGB1)诱导血管内皮细胞激活影响的分子机制.方法分离、培养雄性SD大鼠胸主动脉内皮细胞,分为空白对照组、HMGB1刺激组、依折麦布组(HMGB1刺激前10μmol/L依折麦布预处理)、CLI-095组(HMGB1刺激前1μmol/L CLI-095预处理).荧光定量分析中性粒细胞与内皮细胞的黏附能力;RT-PCR和Western blot检测内皮细胞中TLR4,ICAM-1、可溶性E选择素mRNA和蛋白表达水平;EMSA法检测NF-κb-p65的DNA结合活性.结果 HMGB1活化的内皮细胞与中性粒细胞黏附活力均明显高于空白对照组,差异具有统计学意义(P0.05);依折麦布组、CLI-095组内皮细胞与中性粒细胞黏附活力均明显低于HMGB1诱导组,差异具有统计学意义(P0.05).HMGB1刺激组的内皮细胞TLR4,mRNA和蛋白表达量明显高于空白对照组,差异具有统计学意义(P0.05);依折麦布组和CLI-095组mRNA和蛋白表达量均明显低于HMGB1组,差异具有统计学意义(P0.05).HMGB1组内皮细胞NF-κb p65亚单位核位移明显强于空白对照组,差异具有统计学意义(P0.05);依折麦布组和CLI-095组较HMGB1组核位移明显减弱,差异具有统计学意义(P0.05).HMGB1组ICAM-1、可溶性E选择素表达水平明显高于空白对照组,差异具有统计学意义(P0.05).而经依折麦布、CLI-095干预后可明显降低ICAM-1、可溶性E选择素表达水平,与HMGB1组之间差异具有统计学意义(P0.05).结论依折麦布可通过调节黏附分子(ICAM-1、可溶性E选择素)的表达而有效抑制HMGB1诱导的血管内皮细胞活化效应,其机制与抑制TLR4的表达和NF-κb激活有关.     

Signal sequence directs localized secretion of bacterial surface proteins

All living cells require specific mechanisms that target proteins to the cell surface. In eukaryotes, the first part of this process involves recognition in the endoplasmic reticulum of amino-terminal signal sequences and translocation through Sec translocons, whereas subsequent targeting to different surface locations is promoted by internal sorting signals. In bacteria, N-terminal signal sequences promote translocation across the cytoplasmic membrane, which surrounds the entire cell, but some proteins are nevertheless secreted in one part of the cell by poorly understood mechanisms. Here we analyse localized secretion in the Gram-positive pathogen Streptococcus pyogenes, and show that the signal sequences of two surface proteins, M protein and protein F (PrtF), direct secretion to different subcellular regions. The signal sequence of M protein promotes secretion at the division septum, whereas that of PrtF preferentially promotes secretion at the old pole. Our work therefore shows that a signal sequence may contain information that directs the secretion of a protein to one subcellular region, in addition to its classical role in promoting secretion. This finding identifies a new level of complexity in protein translocation and emphasizes the potential of bacterial systems for the analysis of fundamental cell-biological problems.     

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.     

The RCAF complex mediates chromatin assembly during DNA replication and repair

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.     

Apoptotic cell death induced by c-myc is inhibited by bcl-2.

Apoptosis is a form of physiological cell death, characterized by chromatin condensation, cytoplasmic blebbing and DNA fragmentation, which often depends on RNA and protein synthesis by the dying cell. The c-myc proto-oncogene, usually implicated in cell transformation, differentiation and cell-cycle progression also has a central role in some forms of apoptosis. These opposing roles of myc in cell growth and death require that other gene products dictate the outcome of c-Myc expression on a cell. A candidate for such a modifying gene is bcl-2, whose product prolongs cell survival and blocks apoptosis in some systems. Here we demonstrate that Bcl-2 prevents apoptotic death induced by c-Myc, provide a mechanism whereby cells can express c-Myc without undergoing apoptosis, and give a possible explanation for the ability of Bcl-2 to synergize with c-Myc in cell transformation.     

Chromatin dynamics during epigenetic reprogramming in the mouse germ line

A unique feature of the germ cell lineage is the generation of totipotency. A critical event in this context is DNA demethylation and the erasure of parental imprints in mouse primordial germ cells (PGCs) on embryonic day 11.5 (E11.5) after they enter into the developing gonads. Little is yet known about the mechanism involved, except that it is apparently an active process. We have examined the associated changes in the chromatin to gain further insights into this reprogramming event. Here we show that the chromatin changes occur in two steps. The first changes in nascent PGCs at E8.5 establish a distinctive chromatin signature that is reminiscent of pluripotency. Next, when PGCs are residing in the gonads, major changes occur in nuclear architecture accompanied by an extensive erasure of several histone modifications and exchange of histone variants. Furthermore, the histone chaperones HIRA and NAP-1 (NAP111), which are implicated in histone exchange, accumulate in PGC nuclei undergoing reprogramming. We therefore suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The marked chromatin changes are intimately linked with genome-wide DNA demethylation. On the basis of the timing of the observed events, we propose that if DNA demethylation entails a DNA repair-based mechanism, the evident histone replacement would represent a repair-induced response event rather than being a prerequisite.     

XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.