DcR3与肿瘤研究新进展

DcR3又称肿瘤坏死因子受体(TNFR)6B,是新近发现的TNFR超家族成员,它具有抗凋亡和细胞调节的生物学特性.DcR3与肿瘤的发生发展密切相关,可为肿瘤的诊断、治疗、疗效观察和预后判断提供一种新的思路.现综述DcR3的生物学特性及其在肿瘤方面的研究进展.     

胆囊癌中MRP3的表达及其与预后的关系

目的:探讨multidrug resistance-related protein 3(MRP3)在胆囊癌中的表达及其与病理指标及预后的关系.方法:应用免疫组化SP法检测50例胆囊癌中MRP3的表达.结果:MRP3呈散在分布的淡黄色或棕褐色颗粒,主要定位于细胞浆中,散见于细胞核中,强阳性表达率为72%.随着转移程度、Nevin分级以及分化程度的增加,强阳性表达率增加,且具有统计学差异(P＜0.05).本组病人总生存期为16.42个月,强阳性表达组病人的总生存期为13.25个月,弱阳性表达组病人的总生存期为22.64个月,两者具有显著性差异(P＜0.05).单因素分析显示总生存期与MRP3的表达、Nevin分级、转移及分化程度有关(P＜0.05),多因素分析显示MRP3的表达强弱以及转移是总生存期的独立预后因子(P＜0.05).结论:高表达的MRP3参与了胆囊癌的发生发展,MRP3与胆囊癌的分化、转移有关,可作为判断预后的指标.     

Foxp3在成神经细胞瘤细胞株SK-N-SH中的表达及其对化疗的敏感性

摘 要　目的：研究成神经细胞瘤细胞株SK-N-SH 中Foxp3的表达及其对化疗药物环磷酰胺（cyclophosvnamide,CTX）和吡柔比星（pirarubicin, THP）的敏感性。方法：体外培养SK-N-SH细胞,流式细胞术检测Foxp3在SK-N-SH细胞中的表达。MTT法检测化疗药物CTX、THP对SK-N-SH细胞的敏感剂量;流式细胞术及real-time PCR检测CTX、THP对SK-N-SH细胞中Foxp3表达的影响。结果：流式细胞术检测结果显示,SK-N-SH细胞高表达Foxp3分子。CTX作用于SK-N-SH细胞的敏感剂量为6 mmol/L,THP作用于SK-N-SH细胞的敏感剂量为80 ng/ml。6 mmol/L CTX或80 ng/ml THP以及两者的联合不能抑制SK-N-SH细胞中Foxp3的表达（P>0.05）;real-time PCR结果也证实,CTX或THP以及两者的联合不能抑制SK-N-SH细胞中Foxp3 mRNA的表达。结论：成神经细胞瘤细胞株SK-N-SH高表达Foxp3蛋白,但其表达对化疗药物CTX和THP不敏感。     

Galectin-3蛋白表达与结直肠腺癌临床病理特征的关系

目的:探讨结直肠腺癌组织中Galectin-3蛋白表达及其与临床病理参数的关系.方法:采用微波-EliVisionTM免疫组化染色方法检测60例结直肠腺癌组织中Galectin-3的表达情况并分析其与结直肠腺癌浸润转移等的关系.结果:Galectin-3阳性表达主要在细胞质.60例结直肠腺癌组织中Galectin-3蛋白阳性表达率为68.3%(41/60).在结直肠腺癌浆膜浸润、淋巴结转移和TNMⅢ+Ⅳ期中明显高于无浆膜浸润、无淋巴结转移及TNM Ⅰ+Ⅱ期(P<0.05),不同分化肿瘤之间也有显著差异(P<0.05).结论:Galectin-3蛋白的高表达与结直肠腺癌高侵袭能力、淋巴结转移等有一定相关性,可作为潜在预测大肠癌浸润转移的指标.     

PCA3: from basic molecular science to the clinical lab

Prostate cancer is the second leading cause of cancer deaths in men in the United States. Use of the serum prostate specific antigen (PSA) test to screen men for prostate cancer since the late 1980s has improved the early detection of prostate cancer, however low specificity of the test translates to numerous false positive results and many unnecessary biopsies. New biomarkers to aid in prostate cancer diagnosis are emerging and prostate cancer gene 3 (PCA3) is one such marker. PCA3 is a noncoding RNA that is highly over-expressed in prostate cancer tissue compared to benign tissue. A non-invasive test for PCA3 was developed using whole urine collected after a digital rectal exam (DRE). Numerous clinical studies have demonstrated the utility of PCA3 for the diagnosis of prostate cancer and some studies suggest that PCA3 may also have prognostic value. The use of PCA3 in combination with serum PSA and other clinical information enhances the diagnostic accuracy of prostate cancer detection and will enable physicians to make more informed decisions with patients at risk for prostate cancer.     

Compensatory ErbB3/c-Src signaling enhances carcinoma cell survival to ionizing radiation

Summary EGFR and ErbB2 are two members of the ErbB family of receptor Tyr Kinases identified as therapeutic targets for treating carcinomas. Breast carcinoma cells express different complements and variable proportions of ErbB receptor Tyr kinases, which activate unique and redundant signaling cascades that are essential for cell survival. Previously it was shown that a COOH-terminal truncation mutant of the EGFR (EGFR-CD533) blocks EGFR dependent signals and radiosensitizes breast carcinoma cells. In this study the effects of EGFR-CD533 and an analogous truncation mutant of ErbB2 (ErbB2-CD572) on ErbB receptor family dimerization and signaling are further investigated. Using adenoviral vectors in breast carcinoma cell lines with variable ErbB expression profiles, we demonstrate different effects for each deletion mutant. EGFR-CD533 blocks ligand stimulation of EGFR, ErbB2, and ErbB4, but is associated with a compensatory Tyr kinase activity resulting in phosphorylation of ErbB3. In contrast, ErbB2-CD572 produces a weaker, non-specific pattern of ErbB receptor family inhibition, based upon the ErbB expression pattern of the cell type. Investigation of the compensatory Tyr kinase activity associated with EGFR-CD533 expression identified an ErbB3/c-Src signaling pathway that regulates expression of anti-apoptotic Bcl family proteins. This signaling is active in the T47D cell line, which inherently over-express ErbB3, absent in MDA-MB231 cells, which have low ErbB3 expression levels, and is restored in a MDA-MB231 cell line engineered to over-express ErbB3. Furthermore we demonstrate that ErbB3/c-Src signaling is radio-protective, and that its elimination through pharmacologic inhibition of c-Src enhances radiation-induced apoptosis. In summary, these studies identify a novel ErbB3/c-Src survival signal and point to ErbB3 expression levels as an important variable in therapeutic targeting of ErbB receptors in breast carcinoma cells. ^†(Rupert K. Schmidt-Ullrich) Deceased December 20, 2004 Address for offprints and correspondence: Joseph N. Contessa, Department of Radiation Oncology, University of Michigan, 1500 East Center Drive, UH B2 C490, Box 0010, Ann Arbor, MI 48109; E-mail: jcontess@med.umich.edu     

Autophagy contributes to resistance of tumor cells to ionizing radiation

Background and purpose

Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used.

Materials and methods

Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS-PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3.

Results

LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells.

Conclusion

Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance.     

Notch3-siRNA增强结肠癌细胞对托泊替康的化疗敏感性

目的：探讨下调Notch3表达是否可增强结肠癌SW620细胞对化疗药托泊替康（topotecan）的敏感性。方法：将Notch3siRNA转染到SW620细胞中,Westernblotting检测转染后SW620细胞中Notch3的表达水平。转染后不同时间加入托泊替康,MTr法检测SW620细胞的增殖;Hoechst33342染色和流式细胞仪检测SW620细胞的凋亡;Caspase-3活化试剂盒检测SW620细胞中caspase-3的活化。结果：Notch3siRNA转染可明显抑制SW620细胞中Notch3蛋白的表达;托泊替康作用于Notch3siRNA转染组细胞的IC50较对照CtrlsiRNA转染组显著降低（P〈0．05）;流式细胞仪检测显示沉默Notch3的表达可显著增强托泊替康诱导的结肠癌细胞凋亡（P〈0．05）和caspase-3活化（P〈0．05）。结论：siRNA沉默Notch3的表达可增强SW620细胞对托泊替康的化疗敏感性。     

伊马替尼对人卵巢癌细胞SKOV-3增殖及顺铂、紫杉醇敏感性的影响

目的:探讨伊马替尼对人卵巢癌细胞SKOV-3增殖和对顺铂、紫杉醇敏感性的影响及其作用机制.方法:四甲基偶氮唑蓝(MTT)快速比色法检测细胞增殖率、Hoechst33342染色检测细胞凋亡;采用免疫荧光测定伊马替尼处理前后人卵巢癌细胞株SKOV-3 p-PDGFRβ的表达情况.结果:伊马替尼可以诱导SKOV-3细胞凋亡,对其增殖抑制作用与对照组相比差异有统计学意义(P<0.05),并显著提高了SKOV-3细胞对顺铂、紫杉醇的敏感性(P<0.05); PDGF-BB可促进PDGFRβ发生磷酸化,伊马替尼在体外阻断了SKOV-3细胞p-PDGFRβ的表达,p-PDGFRβ可能是伊马替尼的主要作用靶点.结论:伊马替尼对SKOV-3细胞有明显杀伤作用,并显著增强该细胞株对顺铂、紫杉醇的敏感性;伊马替尼可在体外阻断SKOV-3细胞PDGFRβ的磷酸化,这可能是其影响细胞增殖的主要途径之一.     

STAT3基因RNAi诱导肝癌细胞凋亡及对STAT3信号转导相关基因表达的影响

背景与目的:探讨STAT3基因RNAi对人肝癌细胞SMMC7721凋亡的影响,及对STAT3信号转导相关基因表达的影响,为STAT3信号通路的肿瘤靶向治疗提供理论依据.材料与方法:采用RNAi技术特异性沉默SMMC7721细胞中STAT3的表达,通过Westem blot技术检测经RNAi作用后SMMC7721细胞STAT3蛋白表达水平的变化.采用透射电镜和流式细胞术检测经RNAi沉默SMMC7721细胞中STAT3的表达后,对细胞凋亡的影响.通过半定量RT-PCR法检测细胞凋亡相关因子bcl-2和survivin基因表达的变化. 结果:STAT3基因RNAi可有效沉默SMMC7721细胞中STAT3的表达(P<0.05).STAT3的表达被RNAi沉默后,SMMC7721细胞凋亡率较对照组明显增加(P<0.01).且SMMC7721细胞bel-2和survivin mRNA的表达水平均受到明显抑制,与对照组间的差异均具有统计学意义(P<0.01). 结论:STAT3基因RNAi可有效沉默SMMC7721细胞中STAT3的表达,诱导SMMC7721细胞凋亡,下调SMMC7721细胞bcl-2、survivin mRNA的表达.     

Evaluating the role of mitochondrial DNA variation to the genetic predisposition to radiation-induced toxicity

Background and purpose

Mitochondrial DNA common variants have been reported to be associated with the development of radiation-induced toxicity. Using a large cohort of patients, we aimed to validate these findings by investigating the potential role of common European mitochondrial DNA SNPs (mtSNPs) to the development of radio-toxicity.

Material and methods

Overall acute and late toxicity data were assessed in a cohort of 606 prostate cancer patients by means of Standardized Total Average Toxicity (STAT) score. We carried out association tests between radiation toxicity and a selection of 15 mtSNPs (and the haplogroups defined by them).

Results

Statistically significant association between mtSNPs and haplogroups with toxicity could not be validated in our Spanish cohort.

Conclusions

The present study suggests that the mtDNA common variants analyzed are not associated with clinically relevant increases in risk of overall radiation-induced toxicity in prostate cancer patients.     

Novel Non-tumorigenic Cell Variants Showing Potentially Different Susceptibility to v-src-induced Metastasis

Two non-tumorigenic variant cells were isolated from UV-irradiated Balb/c 3T3 cells on the basis of their different responsiveness in phorbol ester-induced morphological change (rounding formation). They showed marked differences of lung metastatic potentials after intravenous injections of their v- src transformants into nude mice; phorbol ester-resistant variant TR4 cells transformed by v- src were hypermetastatic, whereas v- src transformants of phorbol ester-sensitive variant TR5 cells were not metastatic at all. These different metastatic responses were not observed in v-K- ras -induced transformants of the variants. These non-tumorigenic variant cells may pre-acquire the genetic alteration of certain src -specific and metastasis-associated factors. This system may be useful for genetic analysis of the induction of metastasis.     

Variant Translocation of the BCL6 Gene to Immunoglobulin k Light Chain Gene in B-Cell Lymphoma

A lymphoma cell line with a variant type of translocation, t(2;3)(pll;q27), was established from a patient who had received liver transplantation. To elucidate the molecular mechanism of the t(2;3)(pll;q27) chromosomal translocation, we compared the structures of both derivative (der) chromosomal breakpoints with those of their germline predecessors. We noted that the BCL6 gene on chromosome 3 was juxtaposed with the immunoglobulin k light chain (Ig k ) gene on chromosome 2 in a head-to-head configuration. The breakpoint of the BCL6 gene was within a previously reported breakpoint cluster region. The breakpoint on chromosome 2 was within the intron between the leader (L) and variable (V) sequences of one of the V k genes, which was fused to the J k 3 (J=joining) segment. At chromosomal junctures, a direct repeat duplication of chromosome 3 sequences and a deletion of chromosome 2 sequences were discovered. These results are consistent with a translocation model with illegitimate pairing of staggered double-stranded DNA breaks at 3q27 and 2pll, repair, and ligation to generate der(3) and der(2) chromosomes.     

New methods to control neuroblastoma growth

Downstream of growth factor receptors, signaling by the phosphoinositide 3 kinase (PI3K) pathway is known to play an important role in the growth and survival of many tumor types. The PI3K pathway simplistically comprises PI3K itself, followed by PDK-1, then AKT and finally glycogen synthase kinase 3 (GSK3).¹ PI3K/AKT signaling promotes increased GSK3 phosphorylation, that is associated with reduced GSK3 activity. There are two isoforms of GSK3, GSK3α and GSK3β, which have a high degree of sequence homology.² GSK3 plays a role not only in the regulation of glycogen synthase activity but in the expression of multiple other proteins that play a role in cancer biology, including cyclins and anti-apoptotic proteins.^3,4     

单链抗体融合重构型人caspase-3蛋白的靶向抑瘤作用研究

目的：探讨分泌表达的抗ErbB2单链抗体与重构型人caspase3融合蛋白对ErbB2抗原阳性肿瘤细胞的靶向杀伤作用。 方法：将重构型人caspase3基因亚克隆入pCMVe23scFvPEⅡPEⅢ相应位点，构建表达分泌型的抗ErbB2单链抗体与重构型人caspase3融合蛋白基因的真核表达载体pCMVe23scFvPEⅡrevcasp3，转染人T淋巴瘤细胞系Jurkat，筛选并建系，ELISA检测培养上清中融合蛋白的分泌表达。用含有该融合蛋白的培养介质培养人宫颈癌细胞Hela、乳腺癌细胞SKBr3以及人卵巢癌细胞SKOV3等研究该蛋白对细胞生长的抑制作用；将转染建系的Jurkat细胞尾静脉注射SKBr3荷瘤裸鼠研究其对肿瘤的抑制作用，间接免疫荧光检测重构型人caspase3蛋白在瘤体的靶向分布。结果：融合蛋白基因可通过Jurkat细胞分泌表达并杀伤ErbB2抗原阳性的SKBr3和SKOV3细胞而对ErbB2抗原阴性的Hela细胞生长无明显影响，尾静脉注射该细胞可靶向抑制荷瘤裸鼠肿瘤生长。结论：分泌表达的抗ErbB2单链抗体与重构型人caspase3融合蛋白靶向诱导ErbB2抗原阳性肿瘤细胞死亡。     

Effects of administration routes on the uptake of uridine and 5-fluorouridine into an adenocarcinoma transplanted to rat liver

In a model of secondary liver cancer in Wistar rats, the effect of different administration routes on the uptake of 3H-uridine into tumor and several normal tissues was studied. The rats were inoculated with a tumor cell suspension in the central liver lobe. Ten days later, they were distributed into four groups with a catheter placed in the gastroduodenal artery, the portal vein, one of the femoral veins, or in the peritoneal cavity. 3H-uridine was injected 46 h later and after an additional 90 minutes the animals were anesthetised and pieces of liver tumor and normal tissues were removed and frozen. The incorporation into the acid-soluble fraction and RNA was analyzed. In a separate experiment, 3H-fluorouridine was administered by the gastroduodenal artery and a comparison was made with the uptake of 3H-uridine. A significantly higher amount of uridine was incorporated into the tumor by the arterial route. The intraportal and intraperitoneal routes were comparable, while a somewhat higher incorporation was found by the systemic route. The consequences of using the different routes upon the incorporation into RNA of tumor and dose-limiting organs are demonstrated. With the aid of this experimental model, it is possible to evaluate further the effect by different manipulations on different drugs regarding the administration route.