Tumor-specific lytic path “hyperploid progression mediated death”: Resolving side effects through targeting retinoblastoma or p53 mutant

A major advance was made to reduce the side effects of cancer therapy via the elucidation of the tumor-specific lytic path “hyperploid progression-mediated death” targeting retinoblastoma (Rb) or p53-mutants defective in G1 DNA damage checkpoint. The genetic basis of human cancers was uncovered through the cloning of the tumor suppressor Rb gene. It encodes a nuclear DNA-binding protein whose self-interaction is regulated by cyclin-dependent kinases. A 3D-structure of Rb dimer is shown, confirming its multimeric status. Rb assumes a central role in cell cycle regulation and the “Rb pathway” is universally inactivated in human cancers. Hyperploidy refers to a state in which cells contain one or more extra chromosomes. Hyperploid progression occurs due to continued cell-cycling without cytokinesis in G1 checkpoint-defective cancer cells. The evidence for the triggering of hyperploid progression-mediated death in RB-mutant human retinoblastoma cells is shown. Hence, the very genetic mutation that predisposes to cancer can be exploited to induce lethality. The discovery helped to establish the principle of targeted cytotoxic cancer therapy at the mechanistic level. By triggering the lytic path, targeted therapy with tumor specificity at the genetic level can be developed. It sets the stage for systematically eliminating side effects for cytotoxic cancer therapy.     

超声微泡介导EGFP 质粒对视网膜母细胞瘤细胞转染效率的实验研究

目的探讨不同浓度的超声微泡造影剂，在不同声强的超声辐照下，介导DNA质粒转染视网膜母细胞瘤（RB）细胞的效率及可行性，为实现外源基因高效、定向的转移奠定基础。方法将培养的RB细胞分别予以超声条件为0．25，0．5，0．75，1．0，1．25W／cm^2，60S的连续波辐照，微泡造影剂浓度为1％，100．4，20％，30％，以筛选出对RB细胞活性无明显抑制的最适超声声强、辐照时间和微泡浓度。根据以上筛选条件，转染EGFP基因入RB细胞，24～48h后，在荧光显微镜下观察EGFP表达情况，并用RT-PCR对EGFPmRNA进行半定量检测。结果声强〈0．75W／cm^2（60s），以及微泡浓度〈20％时，对RB细胞的活性无明显抑制。当微泡浓度10％，超声声强为0．5W／cm^2或0．75W／cm^2时，介导的DNA质粒对RB细胞转染具有较高的转染效率，明显高于其他实验组。超声声强为0．5W／cm^2或0．75W／cm^2介导的转染效率，在统计学上差异无显著性意义。结论浓度适当的微泡在优化的声强条件下，能够有效地提高DNA质粒在RB细胞中的转染效率。     

Blood flow velocity in monocular retinoblastoma assessed by color doppler

OBJECTIVE:

To analyze the flow of retrobulbar vessels in retinoblastoma by color Doppler imaging.

METHODS:

A prospective study of monocular retinoblastoma treated by enucleation between 2010 and 2014. The examination comprised fundoscopy, magnetic resonance imaging, ultrasonography and color Doppler imaging. The peak blood velocities in the central retinal artery and central retinal vein of tumor-containing eyes (tuCRAv and tuCRVv, respectively) were assessed. The velocities were compared with those for normal eyes (nlCRAv and nlCRVv) and correlated with clinical and pathological findings. Tumor dimensions in the pathological sections were compared with those in magnetic resonance imaging and ultrasonography and were correlated with tuCRAv and tuCRVv. In tumor-containing eyes, the resistivity index in the central retinal artery and the pulse index in the central retinal vein were studied in relation to all variables.

RESULTS:

Eighteen patients were included. Comparisons between tuCRAv and nlCRAv and between tuCRVv and nlCRVv revealed higher velocities in tumor-containing eyes (p<0.001 for both), with a greater effect in the central retinal artery than in the central retinal vein (p=0.024). Magnetic resonance imaging and ultrasonography measurements were as reliable as pathology assessments (p=0.675 and p=0.375, respectively). A positive relationship was found between tuCRAv and the tumor volume (p=0.027). The pulse index in the central retinal vein was lower in male patients (p=0.017) and in eyes with optic nerve invasion (p=0.0088).

CONCLUSIONS:

TuCRAv and tuCRVv are higher in tumor-containing eyes than in normal eyes. Magnetic resonance imaging and ultrasonography measurements are reliable. The tumor volume is correlated with a higher tuCRAv and a reduced pulse in the central retinal vein is correlated with male sex and optic nerve invasion.     

Aging with ING: a comparative study of different forms of stress induced premature senescence

Cell senescence contributes to organismal aging and is induced by telomere erosion and an ensuing DNA damage signal as cells reach the end of their replicative lifespan in vitro or in vivo. Stresses induced by oncogene or tumor suppressor hyperactivation, oxidative stress, ionizing radiation and other DNA damaging agents result in forms of stress induced premature senescence (SIPS) that show similarities to replicative senescence. Since replicative senescence and SIPS occur over many days and many population doublings of the mass cultures of primary cells used to study senescence, the sequence of events that occur downstream of senescence signaling can be challenging to define. Here we compare a new model of ING1a-induced senescence with several other forms of senescence. The ING1a epigenetic regulator synchronously induces senescence in mass cultures several-fold faster than all other agents, taking 24 and 36 hours to activate the Rb/ p16^INK4a, but not the p53 tumor suppressor axis to efficiently induce senescence. ING1a induces expression of intersectin 2, a scaffold protein necessary for endocytosis, altering the stoichiometry of endocytosis proteins, subsequently blocking growth factor uptake leading to activation of Rb signaling to block cell growth. ING1a acts as a novel link in the activation of the Rb pathway that can impose senescence in the absence of activating p53-mediated DNA damage signaling, and should prove useful in defining the molecular events contributing to Rb-induced senescence.     

MiR-433 inhibits retinoblastoma malignancy by suppressing Notch1 and PAX6 expression

Retinoblastoma (RB) is the most frequent primary intraocular cancer. It has been demonstrated by previous studies that retinoblastoma is initiated primarily by the inactivation of the retinoblastoma Rb1 gene in retinal cells. However, additional genetic alterations than Rb1 mutation could play important roles in the process of transforming benign retinal cells into retinoblastoma tumor cells. In this study, we identified that microRNA miR-433 is one of such genetic factors. We found that the expression levels of miR-433 were downregulated in RB tissues. We also determined that miR-433 negatively regulated RB cell proliferation, migration and invasion, and induced cell cycle arrest and apoptosis of RB cells. We used bioinformatics method to predict and confirmed that Notch1 and PAX6 were miR-433 target genes in RB cells. Importantly, we demonstrated that restoration of Notch1 and PAX6 expression partially rescued the inhibition of cell proliferation and metastasis induced by miR-433 overexpression, suggesting that miR-433 regulates RB cell proliferation and metastasis through suppressing the expression of Notch1 and PAX6.     

视网膜母细胞瘤组织中DNA甲基转移酶1、DNA甲基转移酶3a、3b的表达

目的 观察视网膜母细胞瘤(RB)组织中DNA甲基转移酶(DNMT)1、DNMT3a和DN MT3b的表达.方法 62例RB肿瘤组织标本及6例正常视网膜组织标本纳入研究.其中,低分化组织标本17例,高分化组织标本45例；侵袭性肿瘤16例,非侵袭性肿瘤46例.采用免疫组织化学染色方法检测RB肿瘤组织标本中DNMT1、DNMT3a、DNMT3b的表达.以细胞核棕色染色为阳性表达,蓝色为阴性表达.以DNMT1、DNMT3a及DNMT3b阳性细胞分别≥65％、60％及40％为DNMT1、DNMT3a、DNMT3b高表达,≥1％但＜65％、60％及40％为低表达.同时分析DNMT1、DNMT3a、DNMT3b表达和MIB-1标记指数之间的相关性.结果 光学显微镜观察发现,正常视网膜组织中DNMT1、DNMT3a和DNMT3b均为阴性表达.肿瘤视网膜组织中均可见DNMT1、DNMT3a和DNMT3b表达,其阳性表达率分别为100％、98％、92％.高低分化组织标本比较,DNMT1、DNMT3a高表达率(x2=12.57、10.54)及阳性细胞计数(U=179、198)间差异均有统计学意义(P＜0.05)；DNMT3b高表达率(x2=1.5)和阳性细胞计数(U=307)间差异均无统计学意义(P＞0.05).侵袭性及非侵袭性肿瘤组织标本比较,DNMT1高表达率(x2=4.72)及阳性细胞计数(U=236)间差异均有统计学意义(P＜0.05)；DNMT3a、DNMT3b高表达率(x2=3.53、0.84)及阳性细胞计数(U=338、257)间差异均无统计学意义(P＞0.05).相关性分析结果显示,MIB-1标记指数与RB肿瘤组织中DNMT1、DNMT3a及DNMT3b表达呈正相关(R2=0.554、0.376、0.219,P＜0.05).结论 RB肿瘤视网膜组织中DNMT1、DNMT3a和DNMT3b均呈高表达.     

视网膜母细胞瘤高迁移率族蛋白A、MIB-1标记指数和let-7的表达及其相关性分析

目的 观察分析视网膜母细胞瘤(RB)肿瘤组织中高迁移率族蛋白A (HMGA)1和HMGA2、MIB-1标记指数(LI)及let-7的表达及其相关性.方法 44例RB肿瘤组织标本纳入研究.其中,低分化组织标本11例,高分化组织标本33例.侵袭性肿瘤组织标本8例,非侵袭性肿瘤组织标本36例.采用免疫组织化学染色方法检测RB肿瘤组织标本中HMGA1、HMGA2及MIB-1 LI的表达.HMGA1、HMGA2表达判定标准:以0为无表达,1％～10％为低度表达,11％～50％为中度表达,＞50％为高度表达；MIB-1 LI表达判定标准:以0为无表达,1％～40％为低度表达,＞40％为高度表达.采用逆转录聚合酶联反应检测let-7的表达；以≥80％为无明显下降,60％～79％为中度下降,＜60％为高度下降.结果 44例标本中,HMGA1无表达14例,占32％；低度表达11例,占25％；中度表达10例,占23％;高度表达9例,占20％.低分化组织标本的HMGA1表达率高于高分化组织标本,差异有统计学意义(x2=11.3,P＜0.01).侵袭性与非侵袭性肿瘤组织标本的HMGA1表达率比较,差异无统计学意义(x2=5.9,P＞0.05).HMGA2无表达11例,占25％；低度表达11例,占25％；中度表达9例,占20％；高度表达13例,占30％.低分化组织标本的HMGA2表达率高于高分化组织标本,差异有统计学意义(x2=20.9,P＜0.05).侵袭性肿瘤组织标本的HMGA2表达率高于非侵袭性肿瘤组织标本,差异有统计学意义(x2=8.7,P＜0.05).MIB-1 LI无表达4例,占9％；低度表达18例,占41％；高度表达22例,占50％.低分化组织标本的MIB-1 LI表达水平高于高分化组织标本,差异有统计学意义(t=5.2,P＜0.05).侵袭性肿瘤组织标本的MIB-1 LI表达水平高于非侵袭性肿瘤组织标本,但差异无统计学意义(t=-1.1,P＞0.05).let-7表达无明显下降27例,占61％,中度下降8例,占18％,高度下降9例,占21％.相关性分析结果显示,MIB-1 LI表达与HMGA1和HMGA2表达呈显著正相关(r=0.327、0.602,P＜0.05)；let-7表达与HMGA1、HMGA2表达及MIB-1 LI表达均呈负相关(r=-0.247、-0.310、-0.392,P＜0.05).结论 在RB肿瘤组织中HMGA1、HMGA2和MIB-1 LI均呈现高表达,let-7表达下降.let-7有可能抑制HMGA1和HMGA2的表达.     

白藜芦醇对视网膜母细胞瘤细胞多药耐药性相关因子表达的影响

目的 观察白藜芦醇对视网膜母细胞瘤(RB)细胞多药耐药性(MDR)因子表达的影响.方法 体外培养RB细胞,取对数生长期细胞进行实验.实验分为实验组及对照组进行.采用浓度为6.25、12.50、25.00、50.00、100.00 μmol/L的白藜芦醇处理RB细胞,作用24、48 h后,噻唑蓝(MTT)比色法检测不同浓度组RB细胞的吸光度[A,旧称光密度(OD)]值,以此表示RB细胞活性.采用浓度为50.00 μmol/L的白藜芦醇处理RB细胞48 h,逆转录聚合酶链反应(RT-PCR)和蛋白质免疫印迹法(Western blot)分别检测MDR-1、环氧化酶(COX)-2、多药耐药相关蛋白(MRP)-1、谷胱苷肽转移酶(GST)-π的m RNA和蛋白表达.3种检测均以加入等体积0.5％二甲基亚砜细胞培养液为对照组.结果 MTT比色法检测结果显示,与对照组比较,6.25、12.50、25.00、50.00 μmol/L实验组A值呈剂量依赖性降低,差异有统计学意义(F=4.782,P＜0.05).50.00、100.00 μmol/L实验组A值间差异无统计学意义(F=6.351,P＞0.05).RT-PCR检测结果显示,实验组MDR-1、MRP1、COX-2、GST-π mRNA表达较对照组显著降低,差异均有统计学意义(t=9.170、5.758、4.152、4.638,P＜0.05).Western blot检测结果显示,实验组MDR-1、MRP1、COX-2、GST-π蛋白表达较对照组显著降低,差异均有统计学意义(t=3.848、5.955、4.541、3.514,P＜0.05).结论 白藜芦醇可降低RB细胞MDR因子的表达.