同源重组和重组酶Rad51的调控

同源重组是细胞非常重要的生命活动,参与维持基因组的完整性与稳定性,且与人类健康密切相关.同源重组的研究不断取得进步.本文讨论了同源重组的模式,重组酶RecA/Rad51的作用机制以及Rad51调节蛋白对Rad51入核及Rad51参与重组过程中的单链结合、同源配对、入侵及链交换阶段的调控,将有利于我们对同源重组的深入了解.     

征服癌症的双刃刀--Rad51 D蛋白

人类Rad5l蛋白是同源重组的关键酶,发挥着链转移或链交换活性,启动DNA同源配对的作用。Rad51D蛋白是Rad51蛋白的5种同源物之一,对细胞调节有正反两种作用机制一方面作为辅助因子参与DNA修复同源重组,维持正常细胞周期;另一方面又是诱发癌症病变,防止癌细胞衰老的因素之一。Rad51D蛋白对细胞的作用机制,是人类征服癌症的双刃刀,如果阻止癌细胞的Rad51D蛋白作用可以促进癌细胞的死亡;而同时Rad51D蛋白作用的减弱将使细胞发生周期紊乱,产生新的病变。本文将近年来有关Rad51D的研究成果进行了整理,主要包括Rad51D蛋白的生物学特征和生物学功能两部分,同时对Rad51D蛋白的研究方向提出了自己的看法。     

慢性疼痛治疗的研究进展

慢性疼痛是临床常见的病症,给患者和社会都带来极大的负担。其发病机制受生理、心理和社会等多种因素的影响较为复杂,因此,慢性疼痛的治疗一直是临床上的一大难题。单一的治疗手段往往不能取得令人满意的效果,目前常采用多手段联合的方式来治疗慢性疼痛,常见的包括药物治疗、心理治疗、介入治疗以及自我管理等。针对不同类型的慢性疼痛甚至同一类型的不同病人其治疗方案也不尽相同,近年来兴起的跨学科康复计划为慢性疼痛的治疗指明了方向。本文就慢性疼痛治疗的研究进展进行了综述,以期为临床实践提供更多参考和理论依据。     

硫化铜纳米粒子在肿瘤成像与治疗中的功能研究进展

硫化铜是一种二价铜的硫化物,可以作为半导体材料,化学式为CuS,呈黑褐色,溶解度极低。硫化铜纳米粒子(Copper sulfide nanoparticles, CuS NPs)是纳米尺度大小的硫化铜。近年来,CuS NPs因其结构的可塑性,良好的光热稳定性、生物相容性、突出的光热及光声转换性能,成为了当今纳米材料医学领域的研究热点,在肿瘤诊断和治疗领域中引起了广泛关注。CuS NPs本身可通过介质鳌合金属离子合成多功能纳米粒子,实现肿瘤多模式诊断,并且在光热治疗研究中体现出突出的治疗效果。本文综述了近几年CuS NPs在肿瘤诊断与治疗方面的研究进展,总结肿瘤治疗中的应用研究方法,对CuS NPs在生物医学领域应用中存在的问题进行分析,为解决实际操作过程所遇到的问题提供参考。     

综述: 肿瘤干细胞微环境与靶向干预策略

肿瘤干细胞具有自我更新和可塑性的潜能,能够维持肿瘤生长和异质性的能力．肿瘤干细胞是肿瘤产生、转移、耐药和复发的根源,肿瘤干细胞学说逐渐被肿瘤研究者所接受,因此,对肿瘤干细胞的深入理解有重大的科学和临床意义．肿瘤干细胞的微环境是肿瘤微环境的组成部分,包括细胞-细胞接触、分泌型因子等．肿瘤非干细胞和肿瘤干细胞本身都可以作为肿瘤干细胞的微环境．肿瘤干细胞的微环境可以维持肿瘤干细胞的可塑性,保护肿瘤干细胞免受免疫系统攻击,也可以促进其转移．肿瘤干细胞对其微环境的塑造、肿瘤干细胞的微环境对肿瘤干细胞自我更新的影响,以及针对肿瘤干细胞微环境的靶向干预等问题,已成为肿瘤干细胞研究的前沿问题．本文就肿瘤干细胞的发现、自我更新维持机制、肿瘤干细胞的微环境,及其肿瘤干细胞及微环境的干预策略等研究进展进行了综述．     

综述: 肿瘤微环境与免疫治疗研究进展

肿瘤的发生和发展包括了一系列复杂的生物学过程,其中与免疫系统的相互抗衡,直接决定了这场健康保卫战的成败．由“种子与土壤”学说展开的肿瘤微环境的研究,为解开肿瘤形成与侵袭转移之谜提供了新视角,为肿瘤免疫治疗的临床应用提供了理论依据．本文综述了肿瘤微环境的主要成分及其介导的免疫耐受,及肿瘤免疫治疗的研究现状．     

同源肿瘤克隆细胞蛋白表达和侵袭能力的异质性研究

摘要 目的：比较同源肿瘤细胞来源的不同单克隆表型差异。方法：采用极限稀释法,在悬浮培养条件下获取HCT116结肠癌细胞系的单个细胞,对每孔含单个的细胞进行扩增培养,获得子代单克隆,并以同样方法继续挑取单克隆,连续获得子三代克隆。根据单克隆形态特点,选取第三代的三株代表性的单克隆,采用Western blot和免疫荧光法比较其SOX2、EpCAM和Vimentin蛋白表达差异。采用放疗观察三株单克隆的Vimentin蛋白的动态变化,研究其放疗干预的时间异质性,Transwell体外侵袭实验比较克隆侵袭力的差异。结果：三株由单细胞扩增培养的同源第三代子克隆依然存在明显生物学差异。形态有明显区别的球形与不规则的克隆形态。不规则形态克隆更表现为SOX2低表达及Vimentin的高表达。并且在单个细胞水平上,同个单克隆群体内也存在个体细胞间蛋白的表达差异（Vimentin; EpCAM）。通过观察放疗前后Vimentin蛋白在不同时间点上的荧光强度,发现肿瘤单克隆细胞存在时间异质性。Transwell体外侵袭实验也显示三个同源克隆间存在明显的差异性。结论：同源的、连续单细胞扩增获得的第三代单克隆依然存在明显生物学差异,提示肿瘤内部异质性是其固有特征,并且在治疗干预下,也会引起肿瘤时间异质性的产生。     

间充质干细胞与肿瘤形成的研究进展

间充质干细胞(Mesenchymal stem cells,MSCs)具有独特的免疫调节作用、自我更新和跨胚层多向分化的潜能,存在于许多组织中并活跃地向组织损伤部位迁移,参与伤口修复。在对肿瘤的信号发生反应后,MSCs不断被招募并成为肿瘤微环境的成分。肿瘤相关MSCs(Tumor-associated MSCs, TA-MSCs)在肿瘤发生、促进、进展和转移中有重要作用。本文对MSCs在调节肿瘤细胞的存活、增殖、迁移、药物抵抗中如何发挥作用,以及MSCs对肿瘤微环境免疫状态的影响作一综述。我们强调了MSCs和其他肿瘤基质细胞之间的复杂关系,特别是炎症细胞可以改变肿瘤微环境的免疫状态,以期通过对TA-MSCs进一步的研究来取得对不同肿瘤类型和肿瘤进展不同阶段中肿瘤相关MSCs功能的更好的理解,并优化MSCs来得到更有效和安全的MSCs为基础的肿瘤治疗。MSCs已被有效用于治疗慢性炎性疾病和慢性损伤,因此,其机制方面的研究还有利于在其他疾病中合理利用MSCs从而达到疾病治疗的目的。     

巨噬细胞游走抑制因子在肿瘤中的作用研究进展

巨噬细胞游走抑制因子是一种具有多种生物学效应的糖蛋白,可以调节不同的下游信号如ERK/AKT、NF-κB等通路参与肿瘤细胞增殖、侵袭、转移、血管形成和自噬等生物学过程。临床相关研究表明巨噬细胞游走抑制因子与肿瘤发生发展关系密切,且在乳腺癌、肺癌、前列腺癌、甲状腺癌、结肠癌等多种肿瘤中高表达,因此以巨噬细胞游走抑制因子为靶点的相关肿瘤治疗逐渐受到重视。有关巨噬细胞游走抑制因子拓扑异构酶活性抑制剂及巨噬细胞游走抑制因子中和抗体在肿瘤治疗中的研究越来越多。本文对巨噬细胞游走抑制因子在肿瘤发生发展中的作用以及针对巨噬细胞游走抑制因子进行的肿瘤治疗研究作一综述。     

甲状腺激素受体β与肿瘤关系研究进展

甲状腺激素受体(thyroid hormone receptors,TRs)是一种配体依赖性转录因子,由TRα和TRβ基因编码。在哺乳动物中已发现多个不同的TR亚型,分别由TRα和TRβ基因由于转录起始位点的不同或选择性剪接而产生的若干同工体。近年来被越来越多的研究证实TRs除参与调控机体正常的发育和代谢平衡外,还具有对肿瘤发生的调节作用,尤其是TRβ亚型在肿瘤的发生、发展及转移等过程中发挥重要的生物学作用,表现出了明显的肿瘤抑制功能。在多种肿瘤组织中可检测到TRβ表达的降低甚至缺失。TRβ参与调控细胞内多个信号转导通路,与肿瘤的发生发展密切相关。对TRβ参与的细胞内调控机制的研究有助于在分子水平上对肿瘤的发生发展作更深入的了解,以发掘新的肿瘤治疗靶点。本文主要对TRβ与肿瘤关系的研究进展进行综述。     

Targeting human Rad51 by specific DNA aptamers induces inhibition of homologous recombination

Human Rad51 (HsRad51), a key element of the homologous recombination repair pathway, is related to the resistance of cancer cells to chemo- and radio-therapies. This protein is thus a good target for the development of anti-cancer treatments. We have searched for new inhibitors directed against HsRad51 using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach. We have selected three aptamers displaying strong effects on strand exchange activity. Analysis by circular dichroism shows that they are highly structured DNA molecules. Our results also show that they affect the first step of the strand exchange reaction by promoting the dissociation of DNA from the ATP/HsRad51/DNA complex. Moreover, these inhibitors bind only weakly to RecA, a prokaryotic ortholog of HsRad51. Both the specificity and the efficiency of their inhibition of recombinase activity offer an analytical tool based on molecular recognition and the prospect of developing new therapeutic agents.     

Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair

In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the Rad51-dependent recombination pathway. These paralogs form two complexes (Rad51C/Xrcc3 and Rad51B/C/D/Xrcc2), which play roles in somatic recombination, DNA repair and chromosome stability. However, little is known of their possible involvement in meiosis, due to the inviability of the corresponding knockout mice. We have recently reported that the Arabidopsis homolog of one of these Rad51 paralogs (AtXrcc3) is involved in DNA repair and meiotic recombination and present here Arabidopsis lines carrying mutations in three other Rad51 paralogs (AtRad51B, AtRad51C and AtXrcc2). Disruption of any one of these paralogs confers hypersensitivity to the DNA cross-linking agent Mitomycin C, but not to gamma-irradiation. Moreover, the atrad51c-1 mutant is the only one of these to show meiotic defects similar to those of the atxrcc3 mutant, and thus only the Rad51C/Xrcc3 complex is required to achieve meiosis. These results support conservation of functions of the Rad51 paralogs between vertebrates and plants and differing requirements for the Rad51 paralogs in meiosis and DNA repair.     

Role of RAD51AP1 in homologous recombination DNA repair and carcinogenesis

Homologous recombination (HR) serves to repair DNA double-strand breaks and damaged replication forks and is essential for maintaining genome stability and tumor suppression. HR capacity also determines the efficacy of anticancer therapy. Hence, there is an urgent need to better understand all HR proteins and sub-pathways. An emerging protein that is critical for RAD51-mediated HR is RAD51-associated protein 1 (RAD51AP1). Although much has been learned about its biochemical attributes, the precise molecular role of RAD51AP1 in the HR reaction is not yet fully understood. The available literature also suggests that RAD51AP1 expression may be relevant for cancer development and progression. Here, we review the efforts that led to the discovery of RAD51AP1 and elaborate on our current understanding of its biochemical profile and biological function. We also discuss how RAD51AP1 may help to promote cancer development and why it could potentially represent a promising new target for therapeutic intervention.     

RAD51AP1-deficiency in vertebrate cells impairs DNA replication

RAD51-associated protein 1 (RAD51AP1) is critical for homologous recombination (HR) by interacting with and stimulating the activities of the RAD51 and DMC1 recombinases. In human somatic cells, knockdown of RAD51AP1 results in increased sensitivity to DNA damaging agents and to impaired HR, but the formation of DNA damage-induced RAD51 foci is unaffected. Here, we generated a genetic model system, based on chicken DT40 cells, to assess the phenotype of fully inactivated RAD51AP1 in vertebrate cells. Targeted inactivation of both RAD51AP1 alleles has no effect on either viability or doubling-time in undamaged cells, but leads to increased levels of cytotoxicity after exposure to cisplatin or to ionizing radiation. Interestingly, ectopic expression of GgRAD51AP1, but not of HsRAD51AP1 is able to fully complement in cell survival assays. Notably, in RAD51AP1-deficient DT40 cells the resolution of DNA damage-induced RAD51 foci is greatly slowed down, while their formation is not impaired. We also identify, for the first time, an important role for RAD51AP1 in counteracting both spontaneous and DNA damage-induced replication stress. In human and in chicken cells, RAD51AP1 is required to maintain wild type speed of replication fork progression, and both RAD51AP1-depleted human cells and RAD51AP1-deficient DT40 cells respond to replication stress by a slow-down of replication fork elongation rates. However, increased firing of replication origins occurs in RAD51AP1-/- DT40 cells, likely to ensure the timely duplication of the entire genome. Taken together, our results may explain why RAD51AP1 commonly is overexpressed in tumor cells and tissues, and we speculate that the disruption of RAD51AP1 function could be a promising approach in targeted tumor therapy.     

The HsRAD51B–HsRAD51C stabilizes the HsRAD51 nucleoprotein filament

There are six human RAD51 related proteins (HsRAD51 paralogs), HsRAD51B, HsRAD51C, HsRAD51D, HsXRCC2, HsXRCC3 and HsDMC1, that appear to enhance HsRAD51 mediated homologous recombinational (HR) repair of DNA double strand breaks (DSBs). Here we model the structures of HsRAD51, HsRAD51B and HsRAD51C and show similar domain orientations within a hypothetical nucleoprotein filament (NPF). We then demonstrate that HsRAD51B–HsRAD51C heterodimer forms stable complex on ssDNA and partially stabilizes the HsRAD51 NPF against the anti-recombinogenic activity of BLM. Moreover, HsRAD51B–HsRAD51C stimulates HsRAD51 mediated D-loop formation in the presence of RPA. However, HsRAD51B–HsRAD51C does not facilitate HsRAD51 nucleation on a RPA coated ssDNA. These results suggest that the HsRAD51B–HsRAD51C complex plays a role in stabilizing the HsRAD51 NPF during the presynaptic phase of HR, which appears downstream of BRCA2-mediated HsRAD51 NPF formation.     

The Caenorhabditis elegansRAD51 homolog is transcribed into two alternative mRNAs potentially encoding proteins of different sizes

In prokaryotes, the RecA protein plays a pivotal role in homologous recombination, catalyzing the transfer of a single DNA strand into an homologous molecule. Structural homologs of the bacterial RecA protein, called Rad51, have been found in different eukaryotes (from yeast to man), suggesting a certain level of conservation in recombination pathways among living organisms. We have cloned the homolog of RAD51 in Caenorhabditis elegans. The CeRAD51 gene is transcribed into two alternative mRNAs and potentially codes for two proteins of 395 and 357 amino acids in length, respectively. We discuss the evolutionary implications of these findings. Received: 26 May 1998 / Accepted: 18 August 1998     

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.     

The interaction profile of homologous recombination repair proteins RAD51C,RAD51D and XRCC2 as determined by proteomic analysis

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.     

Over-expression of RAD51 or RAD54 but not RAD51/4 enhances extra-chromosomal homologous recombination in the human sarcoma (HT-1080) cell line

RAD51 and RAD54, members of the RAD52 epistasis group, play key roles in homologous recombination (HR). The efficiency of homologous recombination (HR) can be increased by over-expression of either of them. A vector that allows co-expression of RAD51 and RAD54 was constructed to investigate interactions between the two proteins during extra-chromosomal HR. The efficiency of extra-chromosomal HR evaluated by GFP extra-chromosomal HR was enhanced (110-245%) in different transfected Human sarcoma (HT-1080) cell colonies. We observed that RAD51 clearly promotes extra-chromosomal HR; however, the actions of RAD54 in extra-chromosomal HR were weak. Our data suggest that RAD51 may function as a universal factor during HR, whereas RAD54 mainly functions in other types of HR (gene targeting or intra-chromosomal HR), which involves interaction with chromosomal DNA.