Physical Regulation of Human Mesenchymal Stem Cells through Altered Calcium Dynamics

This article has no abstract.     

The ATR-Independent DNA Replication Checkpoint

No Abstract Available.     

Survival of the Replication Checkpoint Deficient Cells Requires MUS81-RAD52 Function

In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress.     

Microtubule-Driven Stress Granule Dynamics Regulate Inhibitory Immune Checkpoint Expression in T Cells

1. Download high-res image (225KB)
2. Download full-size image

     

Replication of Oral BK Virus in Human Salivary Gland Cells

BK polyomavirus (BKPyV) is the most common viral pathogen among allograft patients. Increasing evidence links BKPyV to the human oral compartment and to HIV-associated salivary gland disease (HIVSGD). To date, few studies have analyzed orally derived BKPyV. This study aimed to characterize BKPyV isolated from throat wash (TW) samples from HIVSGD patients. The replication potential of HIVSGD-derived clinical isolates HIVSGD-1 and HIVSGD-2, both containing the noncoding control region (NCCR) architecture OPQPQQS, were assessed and compared to urine-derived virus. The BKPyV isolates displayed significant variation in replication potential. Whole-genome alignment of the two isolates revealed three nucleotide differences that were analyzed for a potential effect on the viral life cycle. Analysis revealed a negligible difference in NCCR promoter activity despite sequence variation and emphasized the importance of functional T antigen (Tag) for efficient replication. HIVSGD-1 encoded full-length Tag, underwent productive infection in both human salivary gland cells and kidney cells, and expressed viral DNA and Tag protein. Additionally, HIVSGD-1 generated DNase-resistant particles and by far surpassed the replication potential of the kidney-derived isolate in HSG cells. HIVSGD-2 encoded a truncated form of Tag and replicated much less efficiently. Quantitation of infectious virus, via the fluorescent forming unit assay, suggested that HIVSGD BKPyV had preferential tropism for salivary gland cells over kidney cells. Similarly, the results suggested that kidney-derived virus had preferential tropism for kidney cells over salivary gland cells. Evidence of HIVSGD-derived BKPyV oral tropism and adept viral replication in human salivary gland cells corroborated the potential link between HIVSGD pathogenesis and BKPyV.     

Turning the Replication Checkpoint On and Off

The replication checkpoint monitors the progress of DNA replication forks during S phase, and delays the firing of later replication origins when active replication forks are stalled due to collisions with damaged or abnormally structured DNA. Key components of the replication checkpoint pathway are the apical protein kinase, ATR, and its downstream target kinase, Chk1. Defects in either ATR or Chk1 function result in loss of DNA replication fidelity and cell viability, even in the absence of extrinsic genotoxic stress. Moreover, several clinically important antitumor agents, such as the camptothecins (CPTs), exert their antitumor effects by interfering with DNA replication, and hence the therapeutic response to these drugs is intimately related to signaling through the replication checkpoint. A recent report from this laboratory adds a new facet to the regulatory mechanisms that control the function and duration of checkpoint signaling through the ATR-Chk1 pathway. The results indicate that replication stress induced by a variety of agents, including CPT and deep hypoxia, triggers the ubiquitin-dependent degradation of the checkpoint kinase Chk1 in both normal and transformed human cells. This review provides an overview of the study’s major findings, together with their implications for both replication checkpoint function and tumor responsiveness to CPT and related anticancer drugs.     

Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells

Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets.     

UV辐射所致HL－60细胞DNA不均一修复的研究

在特定基因水平检测了ＵＶ照射后ＨＬ－６０细胞的ＤＮＡ修复。结果显示活性转录的ｃ－ｍｙｃ基因的修复水平明显高于非活性转录的β珠蛋白基因和全基因组。而进一步应用链专一性ＲＮＡ探针检测,发现ｃ－ｍｙｃ基因中的转录链和非转录链的修复效率没有明显差异。上述结果表明,ＨＬ－６０细胞能够对活跃表达基因的损伤进行选择性高效修复,但不能对活跃表达基因中的转录链进行进一步的选择性修复。     

Genetic Control of Replication through N1-methyladenine in Human Cells

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.     

Dynamics of Human Immunodeficiency Virus Type 1 Replication in Vertically Infected Infants

Plasma human immunodeficiency virus type 1 (HIV-1) turnover and kinetics were studied in children aged 15 days to 2 years following the initiation of a triple antiretroviral drug regimen consisting of zidovudine, lamivudine, and nevirapine. HIV-1 turnover was at least as rapid as that previously described in adults; turnover rates were more rapid in infants and children aged 3 months to 2 years than in infants less than 3 months of age. These data confirm the central role of HIV-1 replication in the pathogenesis of vertical HIV-1 infection and reinforce the importance of early, potent combination therapies for the long-term control of HIV-1 replication.     

A Personal Reflection on the Replicon Theory: From R1 Plasmid to Replication Timing Regulation in Human Cells

Fifty years after the Replicon Theory was originally presented, detailed mechanistic insight into prokaryotic replicons has been obtained and rapid progress is being made to elucidate the more complex regulatory mechanisms of replicon regulation in eukaryotic cells. Here, I present my personal perspectives on how studies of model replicons have contributed to our understanding of the basic mechanisms of DNA replication as well as the evolution of replication regulation in human cells. I will also discuss how replication regulation contributes to the stable maintenance of the genome and how disruption of replication regulation leads to human diseases.     

Telomere Dynamics in Human Cells Reprogrammed to Pluripotency

Background

Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell “rejuvenation.”

Methodology/Principal Findings

We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres.

Conclusions/Significance

While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.     

A Role of hIPI3 in DNA Replication Licensing in Human Cells

The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.     

Herpes Simplex Virus and Human Cytomegalovirus Replication in WI-38 Cells I. Sequence of Viral Replication

A comparison, under standardized conditions, of herpes simplex virus (HSV) and human cytomegalovirus (CMV) revealed differences in viral morphology, in the timing of their infectious cycles, and in several morphological events during those cycles. Structural distinctions between the two viruses included the coating of unenveloped cytoplasmic CMV capsids, but not those of HSV, and a variation in the structure of their cores. Since the two cycles were carried out in the same host cell strain under conditions of one-step growth (input multiplicity = 10 PFU/cell), it was possible to construct time scales locating the major events of each cycle. Comparison of the two showed that HSV replicated and released progeny within 8 h postinfection, whereas CMV required 4 days. These results correlated well with those of concurrent plaque assays. Within the longer CMV cycle, most of the major events appeared retarded to a similar degree, and no obvious limiting step in particle production could be identified. Distinctions between the two cycles included the following: condensation of the chromatin in HSV- but not CMV-infected cells; the greater tendency of HSV to produce membrane alterations; and the appearance of cytoplasmic dense bodies in CMV- but not HSV-infected cells. Identification of these differences even under identical conditions of culture and infection strongly implies that they result from intrinsic differences in the nature of the viruses, and are not caused by variations in experimental conditions.     

Abrogation of the Postmitotic Checkpoint Contributes to Polyploidization in Human Papillomavirus E7-Expressing Cells

High-risk types of human papillomavirus (HPV) are considered the major causative agents of cervical carcinoma. The transforming ability of HPV resides in the E6 and E7 oncogenes, yet the pathway to transformation is not well understood. Cells expressing the oncogene E7 from high-risk HPVs have a high incidence of polyploidy, which has been shown to occur as an early event in cervical carcinogenesis and predisposes the cells to aneuploidy. The mechanism through which E7 contributes to polyploidy is not known. It has been hypothesized that E7 induces polyploidy in response to mitotic stress by abrogating the mitotic spindle assembly checkpoint. It was also proposed that E7 may stimulate rereplication to induce polyploidy. We have tested these hypotheses by using human epithelial cells in which E7 expression induces a significant amount of polyploidy. We find that E7-expressing cells undergo normal mitoses with an intact spindle assembly checkpoint and that they are able to complete cytokinesis. Our results also exclude DNA rereplication as a major mechanism of polyploidization in E7-expressing cells upon microtubule disruption. Instead, we have shown that while normal cells arrest at the postmitotic checkpoint after adaptation to the spindle assembly checkpoint, E7-expressing cells replicate their DNA and propagate as polyploid cells. Thus, abrogation of the postmitotic checkpoint leads to polyploidy formation in E7-expressing human epithelial cells. Our results suggest that downregulation of pRb is important for E7 to induce polyploidy and abrogation of the postmitotic checkpoint.An important hallmark of human cancers is aneuploidy, the state in which a cell has extra or missing chromosomes (12, 25). Polyploidy is the state in which cells have more than two equal sets of chromosomes and is thought to be an early event in multistep carcinogenesis that can lead to aneuploidy (1, 24), as exemplified in Barrett''s esophagus (11). Polyploidy has recently been shown to occur as an early event in cervical carcinogenesis and to predispose the cells to aneuploidy (26). Other recent studies have shown that tetraploid but not diploid mouse or human cells induce tumor formation in mice (3, 9). These studies highlight the potential importance of polyploidy in carcinogenesis.The cellular mechanisms responsible for this polyploidy formation are as of yet undetermined, but several models have been proposed. First, abrogation of the spindle assembly checkpoint followed by cleavage failure may lead to polyploidy formation (36, 40). A second proposed model is rereplication, a process of multiple rounds of DNA replication without an intervening mitosis. Third, cells that adapt to the mitotic spindle checkpoint halt in a G₁-like state with 4C DNA content. Abrogation of this postmitotic checkpoint allows the cells to replicate their 4C DNA content, leading to polyploidy formation. This has been shown in cells that express the human papillomavirus type 16 (HPV-16) E6 oncogene that degrades p53 (21). Finally, cleavage failure, which yields binucleate cells with 4C DNA content, is also a potential mechanism for polyploidy formation (31).The postmitotic checkpoint becomes activated when cells with an intact spindle assembly checkpoint become arrested during mitosis for a prolonged period of time and eventually adapt to the checkpoint, exit mitosis without cleavage, and progress into a G₁-like state with 4C DNA content (19, 22). The cells are prevented from continuing through the cell cycle and replicating their DNA by a proposed p53- and pRb-dependent postmitotic checkpoint (18, 19).High-risk types of HPV (of which HPV-16 is the most prevalent) are commonly associated with lesions that can progress to cervical carcinoma, which is one of the leading causes of cancer death in women worldwide (42). The transforming properties of high-risk HPVs primarily reside in the E6 and E7 oncogenes (reviewed in reference 7). The ability of high-risk HPV E6 and E7 proteins to promote the degradation of p53 and pRb, respectively, has been suggested as a mechanism by which HPV induces cellular transformation (6, 30). Expression of the high-risk HPV E6 and E7 oncogenes in human keratinocytes leads to polyploidy, which is enhanced by DNA damage and by activation of the spindle checkpoint through microtubule disruption (15, 27, 37, 38).Previously, it was thought but not directly shown that high-risk E6 and E7 induce polyploidy in response to microtubule disruption by abrogating the spindle checkpoint and that degradation of the tumor suppressor p53 by E6 is the mechanism by which E6 accomplishes this polyploidy formation (27, 37, 38). Others have proposed that E7 may play a role in stimulating DNA rereplication that occurs prior to mitosis initiation and polyploidy formation (20). Our recent studies demonstrate that E6 does not affect the mitotic spindle checkpoint (21). Instead, E6 abrogates the postmitotic checkpoint to induce polyploidy after microtubule disruption. Interestingly, E6 mutant proteins defective in inducing p53 degradation also induce polyploidy (21). The mechanism by which HPV E7 induces polyploidy remains to be determined. In this study, we investigate these possible mechanisms through which HPV-16 E7 induces polyploidy formation.     

流行性出血热病毒在人B淋巴母细胞中复制增殖

本文应用细胞培养、免疫荧光及电镜技术研究了B淋巴母细胞对EHF病毒的易感性。结果表明EHF病毒可在该细胞中增殖。感染细胞无明显细胞病变,在形态上与对照组无差别。虽然大部分感染细胞呈现明亮病毒抗原荧光,但在电镜下却难以找到完整的病毒颗粒,仅在扩张的囊泡中发现一些性质待定的微丝样物质。人B淋巴母细胞持续感染的建立,提示患者外周血中大量出现的异型淋巴细胞可能允许EHF病毒在其中复制。     

Dynamics of Human Replication Protein A Subunit Distribution and Partitioning in the Cell Cycle

Human replication protein A (RPA) is a three-subunit protein complex involved in DNA replication, repair, and recombination. To gain insight into the dynamics of subunit assembly, we examined the subcellular distribution of RPA subunits (p70, p34, and p11) during the cell cycle. All three subunits colocalized in G1 and S phases, showing a diffuse nuclear distribution in G1 but a dot-like nuclear pattern in S phase. During S phase, the subunits showed a pattern reminiscent of the replication granules/factories described by others as sites of replication machinery. In metaphase, p70 preferentially associated with the spindle poles, p34 was found on chromosomes, and p11 remained in the cytoplasm. In telophase, p70 and p34 appeared in the forming daughter nuclei; p11 remained in the cytoplasm until G1. Among the three subunits only p34 was associated with the nuclear matrix and this association persisted throughout the cell cycle. We conclude that (i) RPA complex assembly is differentially regulated, (ii) the replication machinery may be anchored to the nuclear matrix, and (iii) RPA subunits partition during mitosis and sort into daughter nuclei by different routes.