RADS1和BRCA1在结直肠癌中的表达及临床意义

目的观察结直肠癌中RAD51和BRCA1基因的表达,探讨二者与结直肠癌发生发展及治疗的关系。方法收集结直肠癌癌灶及癌旁正常组织各42例,采用免疫组织化学法及逆转录一聚合酶链反应（reverse transeription-PCR,RTPCR）检测标本组织中RAD51、BRCA1蛋白和mRNA的表达水平。分析RAD51、BRCA1在结直肠癌中的表达水平与临床病理特征的关系以及二者之间的相互关系。结果在结直肠癌组织中RAD51（33例,78．6％）、BRCA1（30例,71．4％）的表达较癌旁正常组织RAD51（7例,16．7％）、BRCA1（18例,42．9％）高（P〈0．05）;结直肠癌中RAD51mRNA（0．51±0．26）和BRCA1 mRNA（O．70±0．96）的值较两者在正常组织中mRNA（0．10±0．22）高（P〈0．01）;两者蛋白及mRNA的表达水平与性别、年龄、分化程度、TNM分期等均无统计学差异（P〉0．05）;BRCA1与RAD51在结直肠癌中的表达水平成明显正相关（蛋白：r=0．731,P〈0．01mRNA：r=0．572,P〈0．01）。结论BRCA1与RAD51在结直肠癌组织中高表达,且二者的表达水平呈明显正相关;BRCAl与RAD51的表达异常可能与结直肠癌的发生发展有关。     

BRCA1 interacts with dominant negative SWI/SNF enzymes without affecting homologous recombination or radiation-induced gene activation of p21 or Mdm2

BRCA1 is a tumor suppressor gene linked to familial breast and ovarian cancer. The BRCA1 protein has been implicated in a diverse set of cellular functions, including activation of gene expression by the p53 tumor suppressor and control of homologous recombination (HR) during DNA repair. Prior reports have demonstrated that BRCA1 can exist in cells in a complex with the BRG1-based SWI/SNF ATP-dependent chromatin remodeling enzymes and that SWI/SNF components contribute to p53-mediated gene activation. To investigate the link between SWI/SNF function and BRCA1 mediated effects on p53-mediated gene activation and on mechanisms of homologous recombination, we have utilized mammalian cells that inducibly express an ATPase-deficient, dominant negative SWI/SNF enzymes. Mutant SWI/SNF ATPases retain the ability to interact with BRCA1 in cells. We report that expression of dominant negative SWI/SNF enzymes does not affect p53-mediated induction of the p21 cyclin dependent kinase inhibitor or the Mdm2 E3 ubiquitin ligase that regulates p53 in cells exposed to UV or gamma irradiation. Similarly, integration of a reporter that monitors homologous recombination by gene conversion into these cells demonstrated no change in the recombination rate in the absence of functional SWI/SNF enzyme. We conclude that the SWI/SNF chromatin remodeling enzymes may contribute to but are not required for these processes.     

Primary structure-based function characterization of BRCT domain replicates in BRCA1

BRCA1 is a large protein that exhibits a multiplicity of functions in its apparent role in DNA repair. Certain mutations of BRCA1 are known to have exceptionally high penetrance with respect to familial breast and ovarian cancers. The structures of the N-terminus and C-terminus of the protein have been determined. The C-terminus unit consists of two alpha-beta-alpha domains designated BRCT. We predicated two homologous BRCT regions in the BRCA1 internal region, and subsequently produced and purified these protein domains. Both recombinant domains show significant self-association capabilities as well as a preferential tendency to interact with each other. These results suggest a possible regulatory mechanism for BRCA1 function. We have demonstrated p53-binding activity by an additional region, and confirmed previous results showing that two regions of BRCA1 protein bind p53 in vitro. Based on sequence analysis, we predict five p53-binding sites. Our comparison of binding by wild-type and mutant domains indicates the sequence specificity of BRCA1-p53 interaction.     

Chk1 suppresses bypass of mitosis and tetraploidization in p53-deficient cancer cells

Many cancer cells are unable to maintain a numerically stable chromosome complement. It is well established that aberrant cell division can generate progeny with increased ploidy, but the genetic factors required for maintenance of diploidy are not well understood. Using an isogenic model system derived by gene targeting, we examined the role of Chk1 in p53-proficient and -deficient cancer cells. Targeted inactivation of a single CHK1 allele in stably diploid cells caused an elevated frequency of mitotic bypass if p53 was naturally mutated or experimentally disrupted by homologous recombination. CHK1-haploinsufficient, p53-deficient cells frequently underwent sequential rounds of DNA synthesis without an intervening mitosis. These aberrant cell cycles resulted in whole-genome endoreduplication and tetraploidization. The unscheduled bypass of mitosis could be suppressed by targeted reversion of a p53 mutation or by exogenous expression of Cdk1. In contrast, the number of tetraploid cells was not increased in isogenic cell populations that harbor hypomorphic ATR mutations, suggesting that suppression of unscheduled mitotic bypass is a distinct function of Chk1. These results are consistent with a recently described role for Chk1 in promoting the expression of genes that promote cell cycle transitions and demonstrate how Chk1 might prevent tetraploidization during the cancer cell cycle.     

Chk1 suppresses bypass of mitosis and tetraploidization in p53-deficient cancer cells

Many cancer cells are unable to maintain a numerically stable chromosome complement. It is well established that aberrant cell division can generate progeny with increased ploidy, but the genetic factors required for maintenance of diploidy are not well understood. Using an isogenic model system derived by gene targeting, we examined the role of Chk1 in p53-proficient and -deficient cancer cells. Targeted inactivation of a single CHK1 allele in stably diploid cells caused an elevated frequency of mitotic bypass if p53 was naturally mutated or experimentally disrupted by homologous recombination. CHK1-haploinsufficient, p53-deficient cells frequently underwent sequential rounds of DNA synthesis without an intervening mitosis. These aberrant cell cycles resulted in whole-genome endoreduplication and tetraploidization. The unscheduled bypass of mitosis could be suppressed by targeted reversion of a p53 mutation or by exogenous expression of Cdk1. In contrast, the number of tetraploid cells was not increased in isogenic cell populations that harbor hypomorphic ATR mutations, suggesting that suppression of unscheduled mitotic bypass is a distinct function of Chk1. These results are consistent with a recently described role for Chk1 in promoting the expression of genes that promote cell cycle transitions and demonstrate how Chk1 might prevent tetraploidization during the cancer cell cycle.     

BRCA1在人类肿瘤细胞中的表达

人类乳癌易感基因1(BRCA1)是乳癌,卵巢癌和前列腺癌的危险因素之一,而且表现出许多的生物功能.采用WesternBlotting和半定量RT-PCR的方法,我们检测了内源性BRCA1蛋白质和mR-NA在从十一种人类肿瘤组织中建立的四十三种肿瘤细胞系的表达水平.在不同的肿瘤细胞中BR-CA1的表达水平是各不一样的.而且并没有发现BRCA1的表达和细胞的内源性p53基因状况有明显的相关性.通过采用细胞转染乳头状瘤病毒-E6致癌基因或采用畸变的p53基因(143Ala→Va1)而导致的p53基因功能失活并不对内源性BRCA1本底表达水平产生任何的影响,但两种与p53功能有关p21(-/-)和Gadd45基因剔除则轻微地增加BRCA1蛋白质的表达.因此,虽然我们目前还不清楚BRCA1在人类肿瘤细胞中不同表达的功能意义,但本文的结果为进一步研究BRCA1在不同肿瘤细胞系的生物功能提供了有价值的背景资料.     

RYBP stabilizes p53 by modulating MDM2

The mouse double minute 2 (MDM2)–p53 interaction regulates the activity of p53 and is a potential target for human cancer therapy. Here, we report that RYBP (RING1‐ and YY1‐binding protein), a member of the polycomb group (PcG), interacts with MDM2 and decreases MDM2‐mediated p53 ubiquitination, leading to stabilization of p53 and an increase in p53 activity. RYBP induces cell‐cycle arrest and is involved in the p53 response to DNA damage. Expression of RYBP is decreased in human cancer tissues compared with adjacent normal tissues. These results show that RYBP is a new regulator of the MDM2–p53 loop and that it has tumour suppressor activity.     

A BRCA1-interacting lncRNA regulates homologous recombination

Long non-coding RNAs (lncRNAs) are important players in diverse biological processes. Upon DNA damage, cells activate a complex signaling cascade referred to as the DNA damage response (DDR). Using a microarray screen, we identify here a novel lncRNA, DDSR1 (DNA damage-sensitive RNA1), which is induced upon DNA damage. DDSR1 induction is triggered in an ATM-NF-κB pathway-dependent manner by several DNA double-strand break (DSB) agents. Loss of DDSR1 impairs cell proliferation and DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect in the absence of DDSR1 is marked by aberrant accumulation of BRCA1 and RAP80 at DSB sites. In line with a role in regulating HR, DDSR1 interacts with BRCA1 and hnRNPUL1, an RNA-binding protein involved in DNA end resection. Our results suggest a role for the lncRNA DDSR1 in modulating DNA repair by HR.     

BRCA1 and BRCA2 protect against oxidative DNA damage converted into double-strand breaks during DNA replication

BRCA1 and BRCA2 mutation carriers are predisposed to develop breast and ovarian cancers, but the reasons for this tissue specificity are unknown. Breast epithelial cells are known to contain elevated levels of oxidative DNA damage, triggered by hormonally driven growth and its effect on cell metabolism. BRCA1- or BRCA2-deficient cells were found to be more sensitive to oxidative stress, modeled by treatment with patho-physiologic concentrations of hydrogen peroxide. Hydrogen peroxide exposure leads to oxidative DNA damage induced DNA double strand breaks (DSB) in BRCA-deficient cells causing them to accumulate in S-phase. In addition, after hydrogen peroxide treatment, BRCA deficient cells showed impaired Rad51 foci which are dependent on an intact BRCA1–BRCA2 pathway. These DSB resulted in an increase in chromatid-type aberrations, which are characteristic for BRCA1 and BRCA2-deficient cells. The most common result of oxidative DNA damage induced processing of S-phase DSB is an interstitial chromatid deletion, but insertions and exchanges were also seen in BRCA deficient cells. Thus, BRCA1 and BRCA2 are essential for the repair of oxidative DNA damage repair intermediates that persist into S-phase and produce DSB. The implication is that oxidative stress plays a role in the etiology of hereditary breast cancer.     

SAFB1 interacts with and suppresses the transcriptional activity of p53

A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity.

Structured summary

p53physically interacts with SRPK1a: shown by two hybrid (view interaction)p53physically interacts with SRPK1a: shown by pull down (view interaction)p53physically interacts with SRPK1a: shown by anti bait coimmunoprecipitation (view interaction)p53physically interacts with SRPK1a: shown by anti tag coimmunoprecipitation (view interaction)SAFB1physically interacts with p53: shown by pull down (view interactions 1, 2)SAFB1physically interacts with p53: shown by anti bait coimmunoprecipitation (view interactions 1, 2)SAFB1 and p53colocalize: shown by fluorescence microscopy (view interaction)SAFB2physically interacts with p53: shown by pull down (view interaction)     

Viral oncoprotein LMP1 disrupts p53-induced cell cycle arrest and apoptosis through modulating K63-linked ubiquitination of p53

Disruption of the gatekeeper p53 tumor suppressor is involved in various virus-associated tumorigeneses, with aberrant ubiquitination as the major cause of p53 abnormalities in virus-associated tumors. Of note, wild-type p53 is accumulated in Epstein-Barr virus (EBV)-associated tumors, especially in nasopharyngeal carcinoma (NPC). We have previously identified that p53 is accumulated and phosphorylated by EBV oncoprotein latent membrane protein 1 (LMP1) in NPC. Here, we further found that LMP1 promoted p53 accumulation via two distinct ubiquitin modifications. LMP1 promoted p53 stability and accumulation by suppressing K48-linked ubiquitination of p53 mediated by E3 ligase MDM2, which is associated with its phosphorylation at Ser20, while increasing the levels of total cellular ubiquitinated p53. LMP1 also induced K63-linked ubiquitination of p53 by interacting with tumor necrosis factor receptor-associated factor 2 (TRAF2), thus contributing to p53 accumulation. Furthermore, LMP1 rescued tumor cell apoptosis and cell cycle arrest mediated by K63-linked ubiquitination of p53. Collectively, these results demonstrate aberrant ubiquitin modifications of p53 and its biological functions by viral protein LMP1, which has broad implications to the pathogenesis of multiple EBV-associated tumors.     

Apak competes with p53 for direct binding to intron 1 of p53AIP1 to regulate apoptosis

The KRAB-type zinc-finger protein Apak was recently identified as a negative regulator of p53-mediated apoptosis. However, the mechanism of this selective regulation is not fully understood. Here, we show that Apak recognizes the TCTTN₂₋₃₀TTGT consensus sequence through its zinc-fingers. This sequence is specifically found in intron 1 of the proapoptotic p53 target gene p53AIP1 and largely overlaps with the p53-binding sequence. Apak competes with p53 for binding to this site to inhibit p53AIP1 expression. Upon DNA damage, Apak dissociates from the DNA, which abolishes its inhibitory effect on p53-mediated apoptosis.     

Histone chaperone ASF1 acts with RIF1 to promote DNA end joining in BRCA1-deficient cells

Replication timing regulatory factor 1 (RIF1) acts downstream of p53-binding protein 53BP1 to inhibit the resection of DNA broken ends, which plays critical roles in determining the DNA double-strand break repair pathway choice between nonhomologous end joining and homologous recombination (HR). However, the mechanism by which this choice is made is not yet clear. In this study, we identified that histone chaperone protein ASF1 associates with RIF1 and regulates RIF1-dependent functions in the DNA damage response. Similar to loss of RIF1, we found that loss of ASF1 resulted in resistance to poly (ADP-ribose) polymerase (PARP) inhibition in BRCA1-deficient cells with restored HR and decreased telomere fusion in telomeric repeat–binding protein 2 (TRF2)-depleted cells. Moreover, we showed that these functions of ASF1 are dependent on its interaction with RIF1 but not on its histone chaperone activity. Thus, our study supports a new role for ASF1 in dictating double-strand break repair choice. Considering that the status of 53BP1–RIF1 axis is important in determining the outcome of PARP inhibitor–based therapy in BRCA1- or HR-deficient cancers, the identification of ASF1 function in this critical pathway uncovers an interesting connection between these S-phase events, which may reveal new strategies to overcome PARP inhibitor resistance.