Perspectives of gene combinations in phenotype presentation

Cells exhibit a variety of phenotypes in different stages and diseases. Although several markers for cellular phenotypes have been identified, gene combinations denoting cellular phenotypes have not been completely elucidated. Recent advances in gene analysis have revealed that various gene expression patterns are observed in each cell species and status. In this review, the perspectives of gene combinations in cellular phenotype presentation are discussed. Gene expression profiles change during cellular processes, such as cell proliferation, cell differentiation, and cell death. In addition, epigenetic regulation increases the complexity of the gene expression profile. The role of gene combinations and panels of gene combinations in each cellular condition are also discussed.     

Using induced pluripotent stem cells as a tool for modelling carcinogenesis

Cancer is a highly heterogeneous group of diseases that despite improved treatments remain prevalent accounting for over 14 million new cases and 8.2 million deaths per year. Studies into the process of carcinogenesis are limited by lack of appropriate models for the development and pathogenesis of the disease based on human tissues. Primary culture of patient samples can help but is difficult to grow for a number of tissues. A potential opportunity to overcome these barriers is based on the landmark study by Yamanaka which demonstrated the ability of four factors;Oct4, Sox2, Klf4, and c-Myc to reprogram human somatic cells in to pluripotency. These cells were termed induced pluripotent stem cells(i PSCs) and display characteristic properties of embryonic stem cells. This technique has a wide range of potential uses including disease modelling, drug testing and transplantation studies. Interestingly i PSCs also share a number of characteristics with cancer cells including self-renewal and proliferation, expression of stem cell markers and altered metabolism. Recently, i PSCs have been generated from a number of human cancer cell lines and primary tumour samples from a range of cancers in an attempt to recapitulate the development of cancer and interrogate the underlying mechanisms involved. This review will outline the similarities between the reprogramming process and carcinogenesis, and how these similarities have been exploited to generate i PSC models for a number of cancers.     

DNA damage responses in cancer stem cells: Implications for cancer therapeutic strategies

The identification of cancer stem cells(CSCs) that are responsible for tumor initiation, growth, metastasis, and therapeutic resistance might lead to a new thinking on cancer treatments. Similar to stem cells,CSCs also display high resistance to radiotherapy and chemotherapy with genotoxic agents. Thus, conventional therapy may shrink the tumor volume but cannot eliminate cancer. Eradiation of CSCs represents a novel therapeutic strategy. CSCs possess a highly efficient DNA damage response(DDR) system, which is considered as a contributor to the resistance of these cells from exposures to DNA damaging agents. Targeting of enhanced DDR in CSCs is thus proposed to facilitate the eradication of CSCs by conventional therapeutics. To achieve this aim, a better understanding of the cellular responses to DNA damage in CSCs is needed. In addition to the protein kinases and enzymes that are involved in DDR, other processes that affect the DDR including chromatin remodeling should also be explored.     

PLAC1特异性TCR基因修饰T细胞对乳腺癌的抗肿瘤作用

目的研究胎盘特异性基因1（PLAC1）特异性T细胞受体（TCR）基因修饰T细胞对乳腺癌的抗肿瘤作用。 方法磁珠分选人类白细胞抗原分型为A2（HLA-A2）的志愿者外周血单个核细胞（PBMC）中的CD8⁺ T细胞,流式检测CD8⁺ T细胞的表型。通过慢病毒载体构建、包装,将可识别乳腺癌肿瘤抗原PLAC1的HLA-A2限制性的TCR基因导入CD8⁺ T细胞（称为TCR-T细胞）,以慢病毒空载体包装、感染的CD8⁺ T细胞（NC-T细胞）作为对照细胞,通过流式细胞术检测PLAC1特异性TCR的表达效率。免疫荧光和流式细胞术检测乳腺癌细胞MCF-7和MDA-MB-231（三阴性乳腺癌细胞）的PLAC1和HLA-A2血清型的表达。WST-1法检测不同效靶比（5?:?1、10?:?1和20?:?1）TCR-T细胞或NC-T细胞与乳腺癌细胞MCF-7或MDA-MB-231作用后的细胞毒性,并通过ELISA检测共培养后T细胞IFN-γ的释放量。通过裸鼠皮下人乳腺癌移植瘤模型检测TCR-T细胞以及NC-T细胞的抗肿瘤作用。采用单因素方差分析及独立t检验进行统计学分析。 结果磁珠分选出的CD8⁺ T细胞CD3⁺ CD8⁺比例达到（98.89±0.30）%。经慢病毒感染、五聚体检测,TCR-T细胞中PLAC1特异性TCR的正确表达率为（24.58±0.82）%,NC-T细胞不表达PLAC1特异性TCR。免疫荧光和流式结果显示乳腺癌细胞MCF-7和MDA-MB-231为HLA-A2和PLAC1双阳性表达细胞。其中流式检测结果显示,MCF-7和MDA-MB-231细胞中HLA-A2的表达效率分别为（93.04±1.36）%和（98.72±0.12）%。在效靶比为20?:?1时,TCR-T细胞对MCF-7杀伤率为（51.5±1.37）%,高于NC-T细胞对MCF-7的杀伤率（5.93±2.40）%,t = 15.507,P < 0.01;TCR-T细胞对MDA-MB-231杀伤率为（44.34±2.20）%,高于NC-T细胞对MDA-MB-231杀伤率（5.15±2.40）% （t?= 10.694,P < 0.01）。在相同效靶比情况下,TCR-T细胞对MCF-7或MDA-MB-231细胞的细胞毒性高于NC-T细胞,且随着效靶比的增加杀伤效果增强。在效靶比为20?:?1时,与MCF-7共培养后TCR-T细胞IFN-γ的分泌水平[（347.49±4.10）pg/ml]高于NC-T细胞[（18.14±6.22）pg/ml]（t = -76.638,P < 0.01）;与MDA-MB-231共培养后TCR-T细胞IFN-γ的分泌水平为（255.25±6.85）pg/ml,高于NC-T细胞[（14.70±6.38）pg/ml] （t = -44.526,P < 0.01）,且随着效靶比的增加分泌量升高。在裸鼠皮下人乳腺癌移植瘤实验中,生理盐水组和NC-T细胞移植组小鼠的肿瘤生长迅速,TCR-T细胞治疗组小鼠肿瘤生长相对缓慢,在移植后第35天,生理盐水组、NC-T细胞组和TCR-T细胞组小鼠肿瘤的平均体积分别为（5?636.96±2?879.55）mm³、（5?522.12±3?391.48）mm³和（1?403.85±1?394.31）mm³,TCR-T细胞治疗组小鼠肿瘤体积明显小于生理盐水组（F = 0.1813,P < 0.05）和NC-T细胞组（F = 0.1307,P?< 0.05）。 结论PLAC1特异性TCR基因修饰T细胞对乳腺癌细胞具有较强的抗肿瘤作用,PLAC1可作为乳腺癌治疗的潜在靶标;PLAC1特异性TCR基因修饰T细胞治疗是PLAC1表达阳性的乳腺癌治疗的新策略。     

Review of metabolic pathways activated in cancer cells as determined through isotopic labeling and network analysis

Cancer metabolism has emerged as an indispensable part of contemporary cancer research. During the past 10 years, the use of stable isotopic tracers and network analysis have unveiled a number of metabolic pathways activated in cancer cells. Here, we review such pathways along with the particular tracers and labeling observations that led to the discovery of their rewiring in cancer cells. The list of such pathways comprises the reductive metabolism of glutamine, altered glycolysis, serine and glycine metabolism, mutant isocitrate dehydrogenase (IDH) induced reprogramming and the onset of acetate metabolism. Additionally, we demonstrate the critical role of isotopic labeling and network analysis in identifying these pathways. The alterations described in this review do not constitute a complete list, and future research using these powerful tools is likely to discover other cancer-related pathways and new metabolic targets for cancer therapy.     

Analysis of mitochondrial metabolism in situ: Combining stable isotope labeling with selective permeabilization

To date, it is well-established that mitochondrial dysfunction does not only play a vital role in cancer but also in other pathological conditions such as neurodegenerative diseases and inflammation. An important tool for the analysis of cellular metabolism is the application of stable isotope labeled substrates, which allow for the tracing of atoms throughout metabolic networks. While such analyses yield very detailed information about intracellular fluxes, the determination of compartment specific fluxes is far more challenging. Most approaches for the deconvolution of compartmented metabolism use computational models whereas experimental methods are rare. Here, we developed an experimental setup based on selective permeabilization of the cytosolic membrane that allows for the administration of stable isotope labeled substrates directly to mitochondria. We demonstrate how this approach can be used to infer metabolic changes in mitochondria induced by either chemical or genetic perturbations and give an outlook on its potential applications.     

异常激活的葡萄糖-6-磷酸脱氢酶通过上调周期蛋白D1表达促进肾透明细胞癌增殖

葡萄糖6-磷酸脱氢酶（glucose 6-phosphate dehydrogenase,G6PD）为磷酸戊糖途径的调节酶。研究表明,G6PD与多种恶性肿瘤的发生密切相关。然而,G6PD在肾透明细胞癌（clear cell renal cell carcinoma, ccRCC）中的功能及其作用机制却鲜有报道。本研究通过TCGA数据分析发现,G6PD在肾透明细胞癌TNM Ⅲ/Ⅳ期mRNA表达水平显著升高,与患者的性别、原发肿瘤直径、淋巴结转移、远端转移、病灶一侧的偏重性、病理分级以及TNM临床分期密切相关。并且,G6PD异常激活有可能成为评价肾透明细胞癌患者不良预后的分子。细胞系检测结果提示,与对照293T细胞及恶性程度较低的786-O细胞相比,恶性程度较高的Caki-1细胞中的G6PD表达及活性明显增加。基因稳定转染结合CCK8分析结果显示,G6PD过表达或异常激活可显著提高293T及786-O细胞的增殖能力,并且促进786-O细胞中周期蛋白D1基因表达上调。综上,本研究通过TCGA数据库分析和稳定细胞系检测及CCK8分析,结果显示,G6PD在肾透明细胞癌中异常激活,并可上调细胞周期蛋白D1表达,进而促进肿瘤细胞增殖。该研究为进一步揭示肾透明细胞癌分子发病机制以及开发有效的靶向治疗方案提供了借鉴。