Ku70对HTLV-1阳性T细胞中HTLV-1病毒蛋白表达的影响

目的:探讨Ku70对人嗜T淋巴细胞病毒1(HTLV-1)阳性T细胞中HTLV-1病毒蛋白表达的影响。方法:Western blot实验检测不同的HTLV-1阳性T细胞系中Ku70的表达水平;构建Ku70基因沉默siRNA并通过Western blot实验检测其敲减Ku70表达的效率;采用siRNA在HTLV-1阳性的T细胞中敲减Ku70表达后,通过real-time PCR和Western blot实验分别检测HTLV-1病毒蛋白在mRNA和蛋白水平上的表达量,并通过real-time PCR实验检测干扰素和促炎因子的表达量。结果:HTLV-1阳性T细胞系MT2、MT4及C8166中的Ku70均呈高表达;采用siRNA敲减Ku70的表达后,MT2细胞及MT4细胞中的HTLV-1病毒蛋白表达量上升;采用siRNA敲减Ku70的表达后,MT2细胞及MT4细胞中干扰素α、干扰素γ及肿瘤坏死因子α的表达下降。结论:Ku70在HTLV-1阳性T细胞中高表达;在HTLV-1阳性T细胞中,Ku70促进干扰素和促炎因子的表达,抑制HTLV-1病毒蛋白的表达。     

DNMT1 represses p53 to maintain progenitor cell survival during pancreatic organogenesis

In the developing pancreas, self-renewal of progenitors and patterning of cell fates are coordinated to ensure the correct size and cellular makeup of the organ. How this coordination is achieved, however, is not clear. We report that deletion of DNA methyltransferase 1 (Dnmt1) in pancreatic progenitors results in agenesis of the pancreas due to apoptosis of progenitor cells. We show that DNMT1 is bound to the p53 regulatory region and that loss of Dnmt1 results in derepression of the p53 locus. Haploinsufficiency of p53 rescues progenitor cell survival and cellular makeup of the Dnmt1-deleted pancreas.     

Replication independent ATR signalling leads to G2/M arrest requiring Nbs1, 53BP1 and MDC1

Ataxia telangiectasia and Rad3-related (ATR) is a phosphoinositol-3-kinase like kinase (PIKK) that initiates a signal transduction response to replication fork stalling. Defects in ATR signalling have been reported in several disorders characterized by microcephaly and growth delay. Here, we gain insight into factors influencing the ATR signalling pathway and consider how they can be exploited for diagnostic purposes. Activation of ATR at stalled replication forks leads to intra-S and G2/M phase checkpoint arrest. ATR also phosphorylates gamma-H2AX at single-stranded (ss) DNA regions generated during nucleotide excision repair (NER) in non-replicating cells, but the critical analysis of any functional consequence has not been reported. Here, we show that UV irradiation of G2 phase cells causes ATR-dependent but replication-independent G2/M checkpoint arrest. This process requires the Nbs1 N-terminus encompassing the FHA and BRCT domains but not the Nbs1 C-terminus in contrast to ATM-dependent activation of G2/M arrest in response to ionizing radiation. Thus, Nbs1 has a function in ATR signalling in a manner distinct to any role at stalled replication forks. Replication-independent ATR signalling also requires the mediator proteins, 53BP1 and MDC1, providing direct evidence for their role in ATR signalling, but not H2AX. Finally, the process is activated in Cockayne's syndrome but not Xeroderma pigmentosum group A cells providing evidence that ssDNA regions generated during NER are the ATR-pathway-specific activating lesion. Replication-independent G2/M checkpoint arrest represents a suitable assay to specifically identify patients with defective ATR signalling, including Seckel syndrome, Nijmegen breakage syndrome and MCPH-1-dependent primary microcephaly.     

抑癌基因p53结合蛋白1基因突变与前列腺腺癌的关系

目的 检测抑癌基因p53结合蛋白1(53BP1)基因突变在前列腺腺癌和良性前列腺增生组织中的发生率,分析53BP1基因突变与前列腺腺癌的相关性.方法 采用基因组DNA抽提、聚合酶链反应(PCR)扩增及基因测序的方法 检测50例前列腺腺癌与10例良性前列腺增生组织中53BP1基因的突变情况.结果 50例前列腺腺癌组织中共检测到15种不同类型的53BP1基因异常,其中4种为正常组织中也检测到的单核苷酸多态性,11种不同类型的突变发生在12例前列腺腺癌组织中,总突变率达24.0%(12/50).11种53BP1基因突变中,7种为错义突变,但均未发生在重要结构域上,另外4种为同义突变,其中c.4760G>T突变位于53BP1 Tudor结构域.53BP1基因突变与前列腺腺癌患者多种临床病理参数均无明显相关性(P>0.05).结论 对前列腺腺癌患者的53BP1基因全长外显子进行突变检测发现有一定比例的基因突变,推测53BP1基因突变与前列腺腺癌的发生有关.

Abstract：

Objective To study the incidence of 53BP1 gene mutations in prostatic adenocarcinoma and benign prostatic hypertrophy, and to analyze the relationship between 53BP1 mutations and prostatic adenocarcinoma. Methods Genomic DNA extraction, PCR amplification and gene sequencing were used to detect the occurrence of 53BP1 gene mutations in 50 cases of prostatic adenocarcinoma. Ten cases of benign prostatic hypertrophy were included as controls. Results Amongst the 50 cases of prostatic adenocarcinoma studied, 15 showed genetic alterations of 53BP1, including 4 cases with single nucleotide polymorphism. The mutation rate was 24.0%(12/50). Seven of the 53BP1 mutations detected represented missense mutations and none of them were situated in functionally important domains. The other 4 were synonymous mutations, in which c.4760G>T was situated in Tudor domain. There was no obvious correlation between 53BP1 gene mutations and the various clinicopathologic parameters of prostate adenocarcinoma(P>0.05).ConclusionCertain percentage of prostatic adenocarcinoma harbors 53BP1 mutations which may be involved in the carcinogenesis.     

Enhanced intra-switch region recombination during immunoglobulin class switch recombination in 53BP1-/- B cells

Immunoglobulin class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single-stranded DNA. U:G mismatches created by AID are processed to produce lesions that recruit and activate DNA damage response proteins including Ataxia-telangiectasia mutated (ATM), histone H2AX, Nijmegen breakage syndrome 1 (Nbs1), and p53 binding protein 1 (53BP1). Among these proteins, absence of 53BP1 produces the most severe impairment of class switching. Here, we demonstrate that AID is targeted normally to switch region DNA and that intra-switch region recombination is enhanced in 53BP1-/- B cells. In addition, Smicro-Sgamma1 switch region junctions cloned from 53BP1-/- B cells show unusual insertions suggestive of failed class switching. Our data are consistent with a role for 53BP1 in stabilizing the synapsis of switch regions during CSR.     

Structure of the 53BP1 BRCT region bound to p53 and its comparison to the Brca1 BRCT structure

Brca1 C-terminal (BRCT) domains are a common protein-protein interaction motif in proteins involved in the DNA damage response and DNA repair. The DNA-damage response protein 53BP1 has two BRCT domains that bind to the DNA-binding domain of p53. The 53BP1 tandem-BRCT region is homologous to the tandem-BRCT region of Brca1, which is involved in double-strand break repair and homologous recombination and which binds BACH1, a member of the DEAH helicase family. Here we report the structures of a human 53BP1-p53 complex and of the rat Brca1 BRCT repeats. The 53BP1-p53 structure shows that the two BRCT repeats are arranged tandemly and pack extensively through an interface that also involves the inter-repeat linker. The first BRCT repeat and the linker together bind p53 on a region that overlaps with the DNA-binding surface of p53 and involves p53 residues that are mutated in cancer and are important for DNA binding. Comparison with the structure of the tandem-BRCT region of Brca1 shows a remarkable conservation of the repeat arrangement and of the inter-BRCT repeat interface. Analysis of human BRCA1 tumor-derived mutations and conservation identifies a potential protein-binding site that we show through mutagenesis is involved in BACH1 binding. The BACH1-binding region of Brca1 consists of a unique insertion in the first BRCT repeat and the inter-repeat linker and is analogous to the region of 53BP1 that binds p53.     

Galectin-1 supports survival of naive T cells without promoting cell proliferation

Naive T cells do not proliferate but remain alive in vivo. In contrast, naive T cells rapidly die in an in vitro culture, suggesting that some factors that are present at the sites of naive T cell circulation in vivo but missing in the bovine serum-containing culture medium, are necessary for their survival. The present study was designed to search for such factors. By functional screening of the cDNA library from murine lymph node-derived stromal cells (LNS) that effectively support the survival of naive T cells, we found that nascent polypeptide-associated complex (alpha-NAC) promoted T cell survival. A conditioned medium derived from culture supernatant of Cos7 cells transfected with alpha-NAC gene supported T cell survival, indicating that alpha-NAC induced production of soluble factor(s) that were secreted into the medium. By examining the products that were cloned from a functional screening of the cDNA library from alpha-NAC-transfected NIH3T3 cells but were not detected in that from control vector-transfected cells, galectin-1 was found as a soluble factor in the conditioned medium of the LNS. Our study demonstrates the novel role of galectin-1 as a soluble factor that functions to maintain naive T cell survival without inducing cell proliferation.     

Lymphotropic papovavirus transforms hamster cells without altering the amount or stability of p53.

Expression of the early regions of several primate polyomaviruses (SV40, BKV, JCV, and LPV) in hamster cells induces transformation, manifested by the ability to grow in soft agar. Hamster cells transformed by SV40 contain complexes between the SV40 T antigen and the cellular tumor suppressor protein p53. We detected analogous complexes between p53 and the BKV T antigen in hamster cells transformed by the BKV early region, where the half life of p53 increased 16-fold. However, neither a LPV-transformed hamster fibroblast cell line [LPV-HE (F); K. K. Takemoto and T. Kanda, 1984, J. Virol. 50, 100-105] nor BHK-21 cells transformed by the LPV early region contained detectable complexes between the LPV T antigen and p53, nor was the stability of p53 in LPV transformed BHK-21 cells altered. Association between hamster p53 and the LPV T antigen expressed as glutathione S-transferase fusion protein could not be detected in vitro. These data indicate that alteration of the amount or stability of p53 is not required for transformation of hamster cells by LPV. However, as viruses such as SV40 and BKV whose T antigens bind p53 are oncogenic in hamsters, whereas LPV is not, the alteration of p53 amount or stability may be required for tumorigenesis.     

Genetic polymorphisms of TP53-binding protein 1 (TP53BP1) gene and association with breast cancer risk

In the present study, we evaluated the association between the TP53BP1 Glu353Asp and T-885G polymorphisms and breast cancer risk as well as with the clinicopathological characteristics of the patients. Genotyping of these polymorphisms was performed on 387 breast cancer patients and 252 normal and healthy women who had no history of any malignancy using PCR-RFLP method in a hospital-based Malaysian population. Breast cancer risk was not observed among women who were heterozygous (OR(adj) = 0.887; 95% CI, 0.632-1.245) or homozygous (OR(adj) = 1.083; 95% CI, 0.595-1.969) for Asp allele, and those carriers of Asp allele (OR(adj) = 0.979; 95% CI, 0.771-1.243). Similarly, women who were TG heterozygotes (OR(adj) = 1.181; 95% CI, 0.842-1.658) or GG homozygotes (OR(adj) = 1.362; 95% CI, 0.746-2.486) and carriers of G allele (OR(adj) = 1.147; 95% CI, 0.903-1.458) were not associated with increased risk of breast cancer. Asp allele genotype was significantly associated with ER negativity (p = 0.0015) and poorly differentiated tumours (p = 0.008), but G allele genotype was not associated with the clinicopathological characteristics. In conclusion, Glu353Asp and T-885G polymorphic variants might not have an influence on breast cancer risk, thus might not be potential candidates for cancer susceptibility. Glu353Asp variant might be associated with tumour aggressiveness as defined by its association with ER negativity and poorly differentiated tumours.     

Demethylation of TNFSF7 contributes to CD70 overexpression in CD4+ T cells from patients with systemic sclerosis

The pathogenesis of systemic sclerosis (SSc) is still unclear. CD70, a B cell costimulatory molecule that interacts with CD27 during B–T cell contact, is overexpressed due to demethylation of its promoter regulatory elements in CD4+ T cells from patients with the following autoimmune diseases, namely systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus (SCLE) and primary Sjögren's syndrome (pSS). However, as an autoimmune disease, it is unknown whether aberrant expression and methylation of CD70 occur in SSc CD4+ T cells.     

Discoidin domain receptor 1 contributes to the survival of lung fibroblast in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF), characterized by fibroblast proliferation and accumulation of extracellular matrix, including collagen, is a chronic progressive disorder that results in lung remodeling and fibrosis. However, the cellular mechanisms that may make fibroblasts resistant to apoptosis have not been completely elucidated. Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase whose ligand is collagen, is expressed in vivo and contributes in vitro to leukocyte differentiation and nuclear factor (NF)-kappaB activation, which may play an important role in fibroblast survival. In this study, we examined in vivo and in vitro DDR1 expression and its role in cell survival using fibroblasts obtained from IPF and non-IPF patients. Immunohistochemically, fibroblasts present in fibroblastic foci expressed endogenous DDR1. The DDR1 expression level was significantly higher in fibroblasts from IPF patients, and the predominant isoform was DDR1b. In IPF patients, DDR1 activation in fibroblasts inhibited Fas ligand-induced apoptosis and resulted in NF-kappaB nuclear translocation. Suppression of DDR1 expression in fibroblasts by siRNA abolished these effects, and an NF-kappaB inhibitor abrogated the anti-apoptotic effect of DDR1 activation. We propose that DDR1 contributes to fibroblast survival in the tissue microenvironment of IPF and that DDR1 up-regulation may occur in other fibroproliferative lung diseases as well.     

Cisplatin induced cell killing and chromosomal aberrations in CHO cells: treated during G1 or S phase

Variation in sensitivity to cisplatin during the cell cycle was studied synchronous cells treated during G₁ or late S phase. The cells were assayed for cell killing, cell-cycle delay, and chromosomal aberrations after they were treated with cisplatin (1–12 μg/ml) for 1 h at 37°C. They were either plated for colony survival, or colcemid was added from 12 to 40 h after plating followed by fixation 4 h later for analysis of chromosal aberrations after the cells completed 1 or 2 cycles (i.e. first or second mitosis). Cells treated with 6 μg/ml exhibited about a 10-h delay during the first cycle after treatment during G₁ compared with about 3 h during the first cycle and 6 h during the second cycle after treatment during late S. In both cases, cells entering metaphase exhibited predominantly chromatid-type breaks and exchanges. For both cell killing and chromosomal aberrations, the cells in G₁ were 1.5–1.6 times more sensitive than those treated in late S, with 1 aberration per cell corresponding to about 37% survival. However, the exchanges and breaks were observed primarily in the first mitosis when cells were treated in G₁ compared with the second mitosis when cells were treated in late S. These results suggest that DNA replication opposite cisplatin cross-links in the DNA results in lethal chromosomal aberrations.     

Identification of a Saccharomyces cerevisiae mutation that allows cells to grow without chitin synthase 1 or 2

Chitin is a component of the yeast cell wall which is localized to the septum between mother and daughter cells. Previous work in Saccharomyces cerevisiae has shown that this organism possesses three chitin synthases, 1, 2, and 3. Disruption experiments have shown that loss of chitin synthase 2 has a more profound effect on cell viability than loss of either of the other two and is lethal in complete media. We report here the finding of an S. cerevisiae strain which does not require the chitin synthase 2 structural gene for viability. We present evidence that there is a gene in this strain which suppresses the lethality of disruption of the chitin synthase 2 structural gene and is genetically distinct from the structural genes for chitin synthase 1 and chitin synthase 2. We show that an S. cerevisiae mutant containing the suppressor and lacking both structural genes for chitin synthase 1 and 2 has normal amounts of chitin in its cell wall. We hypothesize that the suppressor gene encodes or controls the expression of chitin synthase 3.     

Significant association between 53 BP1 expression and grade of intraepithelial neoplasia of esophagus: Alteration during esophageal carcinogenesis

BackgroundAbnormal DNA damage response (DDR) leads to genomic instability and carcinogenesis. P53-binding protein 1 (53 BP1), a DDR molecule, is known to accumulate at the sites of DNA double-strand breaks. The aim of this study was to analyze the expression pattern of 53 BP1-nuclear foci (NF) in esophageal neoplasms in order to visualize the state of DDR in esophageal carcinogenesis and to clarify its significance in the molecular pathology of the disease.MethodsA total of 61 lesions from 22 surgically resected samples of esophageal cancer, including histologically normal squamous epithelium, low-grade intraepithelial neoplasia (LG-IN), high-grade intraepithelial neoplasia (HG-IN), carcinoma in situ (CIS), and invasive squamous cell carcinoma (SCC), were included in the study. 53 BP1 and Ki-67 expression were analyzed by double-labeled immunofluorescence.ResultsThe number of discrete 53 BP1-NF increased as the tumor progressed from normal epithelium through LG-IN, HG-IN, CIS, and SCC. 53 BP1-NF larger than 1 μm in diameter (large foci), indicating intensive DDR, also showed a stepwise increase during the progression of carcinogenesis. Of note, large foci of 53 BP1 were found in significantly higher numbers in HG-IN than in LG-IN. Furthermore, localization of 53 BP1-NF in Ki-67-positive cells, indicating the abnormal timing of DDR, also increased with malignancy progression.Conclusions53 BP1-NF accumulation increases during cancer progression from LG-IN to HG-IN to CIS to SCC. Detection of 53 BP1-NF by immunofluorescence, especially large foci, is a feasible method of estimating DNA instability and the malignant potential of esophageal intraepithelial neoplasia.     

Persistence during G1 of gamma ray- or mitomycin C-induced lesions eliciting SCE in murine salivary gland cells in vivo

The efficiency of mitomycin C or gamma rays to induce SCE in early or late G₁ was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation before S. The SCE frequencies induced by MMC in the first division (before BrdU incorporation) were significantly lower in the early G₁ compared to the late G₁, indicating some repair of SCE-inducing lesions. In the second division (after BrdU incorporation), there was no difference between SCE induced in early and late G₁, indicating that MMC-induced lesions in such conditions are very persistent and not repairable during G₁. The radio induced SCE frequency at early G₁ was significantly lower than that observed in late G₁, in cells irradiated after BrdU incorporation, suggesting that half of gamma ray-induced DNA lesions that elicit SCEs were repaired.     

HBcAg-induced upregulated 4-1BB ligand on B cells contributes to B-cell hyperactivation during chronic hepatitis B infection

During the natural history of chronic hepatitis B infection (CHB), the function of B cells is still obscure. Several limited studies have suggested that B cells are highly active in patients with CHB. In the present study, we reported that the 4-1BB ligand (4-1BBL) expression on B cells was significantly higher in patients with CHB than that in healthy subjects, meanwhile, the patients with CHB had higher serological IgG levels. Further, after being stimulated with sCD40L or hepatitis B core antigen (HBcAg), B cells had higher levels of 4-1BBL. After being cocultured with 4-1BBL+ B cells, the expressions of CD69 and 4-1BB on CD4+ T cells were significantly higher than that cocultured with 4-1BB− B cells. Cytokines including interleukin (IL)-2, IL-4, and IL-6 were significantly higher in the supernatant from 4-1BBL+ B cells coculture group than those from coculture group of 4-1BBL− B cell group, respectively; while IFN-γ and TNF-α in cocultured supernatant of 4-1BBL+ B cell group were significantly lower. Consistently, the total IgG levels in culture supernatant were significantly higher in 4-1BBL+ B cell group. Thus, hyperactive status of B cells in patients with CHB could be partially derived from the higher 4-1BBL expression on B cells triggered by HBcAg. 4-1BBL signaling pathway is involved in B cells activation, and further regulates B cell-T cell interaction by modulating the cytokines secretion, which might be critical in B cells dysfunction during CHB infection.