EYA4 gene functions as a prognostic marker and inhibits the growth of intrahepatic cholangiocarcinoma

Background: The molecular prognostic markers and carcinogenesis of intrahepatic cholangiocarcinoma (ICC) have not been well documented. The purpose of this study was to investigate the prognostic value of the eyes absent homolog 4 (EYA4) gene in ICC and its biological effects on ICC growth in vitro and in vivo. Methods: One hundred twelve patients with ICC who underwent hepatectomy were enrolled in the study. EYA4 mRNA and EYA4 protein levels in ICC and adjacent non?tumoral tissues were evaluated using real?time quantitative polymerase chain reaction and immunohistochemical staining, respectively. EYA4 protein levels in ICC cells were determined using western blot analysis. The associations between EYA4 expression and clinicopathologic features of ICC were analyzed. To identify independent prognostic factors, univariate and multivariate analyses were performed. The biological effects of EYA4 on ICC cells were evaluated by establishing stable EYA4?overexpressing transfectants in vitro, and EYA4’s effects on tumor growth were evaluated by intra?tumoral injection of EYA4?expressing plasmids in a NOD/SCID murine model of xenograft tumors. Results: ICC tissues had signiifcantly lower EYA4 mRNA and protein levels compared with adjacent non?tumoral tis?sues (both P<0.001). Univariate and multivariate analyses showed that EYA4 protein level, tumor number, adjacent organ invasion, lymph node metastasis, and tumor differentiation were independent prognostic factors for disease?free survival and overall survival (all P<0.05). In vitro, EYA4 overexpression inhibited tumor cell growth, foci formation, and cell invasiveness. In vivo, intra?tumoral injection of EYA4?expressing plasmids signiifcantly inhibited ICC growth in the murine xenograft model compared with the control group (P<0.05). Conclusion: EYA4 gene functioned as a molecular prognostic marker in ICC, and its overexpression inhibited tumor growth in vitro and in vivo.     

Relationship between epidermal growth factor receptor gene mutation and copy number in Chinese patients with non-small cell lung cancer

Epidermal growth factor receptor(EGFR) gene mutation and copy number are useful predictive markers that guide the selection of non-small cell lung cancer(NSCLC) patients for EGFR-targeting therapy.This study aimed to investigate the correlation between EGFR gene mutation and copy number and clinicopathologic characteristics of Chinese patients with NSCLC.NSCLC specimens collected from 205 patients between November 2009 and January 2011 were selected to detect EGFR gene mutations with real-time polymerase chain reaction(RT-PCR) and to detect EGFR gene copy number with fluorescence in situ hybridization(FISH).EGFR mutations primarily occurred in females,non-smokers,and patients with adenocarinomas(all P < 0.001).Tissues from 128(62%) patients were FISH-positive for EGFR,including 37(18%) with gene amplification and 91(44%) with high polysomy.EGFR gene mutation was correlated with FISH-positive status(R = 0.340,P < 0.001).Multivariate analysis showed that not smoking(OR = 5.910,95% CI = 2.363-14.779,P < 0.001) and having adenocarcinoma(OR = 0.122,95% CI = 0.026-0.581,P = 0.008) were favorable factors for EGFR gene mutation.These results show a high frequency of EGFR FISH positivity in NSCLC tissues from Chinese patients and a significant relevance between EGFR gene mutations and FISH-positive status.Among the FISH-positive samples,EGFR gene mutation occurred more frequently in samples with gene amplification compared to those with high polysomy,suggesting that EGFR mutation and gene amplification should be used as clinical decision parameters to predict response to EGFR-targeting therapy.     

Is there diversity among UGT1A1 polymorphism in Japan?

AIM: To investigate into the diversity of UGT1A1 polymorphism across three different districts in Japan and highlight genetic differences among the population in Japan. METHODS: We enrolled 50 healthy volunteers from each of the Yamaguchi (western part of Japan), Kochi(southern part of Japan) and Akita (northern part of Japan) prefectures. Blood samples (7 mL) were collected from each participant and stored in EDTA for subsequent genotyping by fragment size analysis, direct sequencing and TaqMan assay of UGT1A1*28, UGT1A7*3/UGT1A9*22 and UGT1A1*93/UGT1A1*6/ UGT1A1*27/UGT1A1*60/UGT1A7 (-57), respectively. RESULTS: The only statistically significant differences in allele polymorphisms among the group examined were for UGT1A1*6. The Akita population showed more UGT1A1*6 heterozygosity (P = 0.0496). CONCLUSION: Our study revealed no regional diversity among UGT1A1, UGT1A7 or UGT1A9 polymorphisms in Japan.     

TET2 Expression in Bone Marrow Mononuclear Cells of Patients with Myelodysplastic Syndromes and Its Clinical Significances

Objective To investigate the expression of TET2 mRNA and protein in the bone marrow mononuclear cells(BMMNC) of patients with myelodysplastic syndrome(MDS) and its clinical significance. Methods The expression of TET2 mRNA and protein in bone marrow mononuclear cells(BMMNC) of 32 patients with MDS and 20 healthy donors was examined by qPCR and Western blot. Results The expression of TET2 mRNA in BMMNC was down-regulated in MDS patients compared with the donor group [(0.41±0.28)%vs.(1.07±0.56)%](P<0.001).Compared with lower expression group(TET2<0.4)[(6.53±6.17)%],patients with higher expression of TET2(≥0.4) presented significantly lower proportion of bone marrow blasts[(1.21±1.56)%](P<0.05).The expression of TET2 mRNA in BMMNC of MDS patients was inversely correlated with malignant clone burden(r=-0.398,P<0.05) and IPSS(r=-0.412, P<0.05).The expression of TET2 protein was down-regulated in MDS patients compared with that in the donor group. Conclusions The mRNA and protein expression of TET2 in BMMNC of MDS patients is decreased,which might be useful as an important parameter for the evaluation of MDS clone burden.     

Application of mesenchymal stem cells as a vehicle to deliver replication-competent adenovirus for treating malignant glioma

Although gene therapy was regarded as a promising approach for glioma treatment,its therapeutic efficacy was often disappointing because of the lack of efficient drug delivery systems.Mesenchymal stem cells(MSCs) have been reported to have a tropism for brain tumors and thus could be used as delivery vehicles for glioma therapy.Therefore,in this study,we attempted to treat glioma by using MSCs as a vehicle for delivering replication-competent adenovirus.We firstly compared the infectivity of type 3,type 5,and type 35 fiber-modified adenoviruses in MSCs.We also determined suitable adenovirus titer in vitro and then used this titer to analyze the ability of MSCs to deliver replication-competent adenovirus into glioma in vivo.Our results indicated that type 35 fiber-modified adenovirus showed higher infectivity than did naked type 3 or type 5 fiber-modified adenovirus.MSCs carrying replication-competent adenovirus significantly inhibited tumor growth in vivo compared with other control groups.In conclusion,MSCs are an effective vehicle that can successfully transport replication-competent adenovirus into glioma,making it a potential therapeutic strategy for treating malignant glioma.     

Tumor suppressor genes on frequently deleted chromosome 3p in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is among the most common malignancies in southern China.Deletion of genomic DNA,which occurs during the complex pathogenesis process for NPC,represents a pivotal mechanism in the inactivation of tumor suppressor genes (TSGs).In many circumstances,loss of TSGs can be detected as diagnostic and prognostic markers in cancer.The short arm of chromosome 3 (3p) is a frequently deleted chromosomal region in NPC,with 3p21.1-21.2 and 3p25.2-26.1 being the most frequently deleted minimal regions.In recent years,our research group and others have focused on the identification and characterization of novel target TSGs at 3p,such as RASSF1A,BLU,RBMS3,and CHL1,in the development and progression of NPC.In this review,we summarize recent findings of TSGs at 3p and discuss some of these genes in detail.A better understanding of TSGs at 3p will significantly improve our understanding of NPC pathogenesis,diagnosis,and treatment.     

Efficacy of Recombinant Adenoviral Human p53Gene in Treatment of Malignant Pleural or Peritoneal Effusions

Background and objective Once the malignant pleural or peritoneal effusion is developed it is difficult to control.This report presents a new method for controlling the malignant effusions.Methods Forty-eight patients,29 males and 19 females with an average age of 61.2 years old,who were satisfied with the study inclusion criteria,were recruited in this study.Twenty-seven and 21 patients had a malignant pleural and peritoneal effusion,respectively.After draining most of fluids,these patients received intra-cavity infusion of rAd-p53 once per week for 4 weeks,at dose of 2×1012 viral particles(VP) diluted into 200 mL of saline solution for pleural effusions,and 4×1012 VP diluted into 500 mL of saline solution for peritoneal effusions.Results Participants were followed up for a median time of 13.6 month.A total of 11 cases,7 with pleural effusions and 4 with peritoneal effusions achieved a complete response(CR),and 20 cases(12 pleural effusions and 8 peritoneal effusions) had a partial response(PR).The overall response rate is 64.6%.Patients’ quality of life,assessed by using Karnofsky performance scale(KPS) scores,was improved by an average of 26.4.The one-year of overall survival rate was 54.2% with a median survival time of 12.5 months.There were no serious side effects observed except for self-limited fever found in 79.8% of the cases.Conclusions Intra-cavity infusion of rAd-p53 is an effective and safe treatment for the patients with malignant pleural or peritoneal effusions,especially for those patients who can’t tolerate the standard treatments.     

逆转录病毒载体介导的双耐药基因转染小鼠骨髓细胞的实验研究

目的:将耐药基因以逆转录病毒为载体转染骨髓细胞以解决大剂量化疗所造成的骨髓抑制。方法:以逆转录病毒为载体,将双突变的二氢叶酸还原酶基因(DH-FR)和胞苷脱氨基酶基因(CD)转染小鼠骨髓细胞,观察该细胞耐MTX及Ara-C的CFU-GM生成情况;RT-PCR检测转基因的表达情况。结果:转基因的小鼠骨髓细胞有耐药克隆形成(14%);基因转染后小鼠骨髓细胞对MTX和Ara-C的耐受明显增加,P<0·005;RT-PCR显示转基因细胞有转染的基因条带。结论:双耐药基因通过逆转录病毒转染小鼠骨髓细胞并且获得表达,提高了骨髓细胞对MTX和Aar-C的耐药性。     

双突变二氢叶酸还原酶基因对小鼠的化疗保护作用

目的 探讨人双突变的二氢叶酸还原酶（DHFR）基因对小鼠化疗保护作用。方法 以反转录病毒为载体,将DHFR基因转染入小鼠骨髓干细胞,观察氨甲喋呤（MTX）处理后的骨髓细胞中粒细胞-巨噬细胞克隆形成单位（CFU-GM）的生成情况;观察大剂量MTX化疗后转基因小鼠血象、体重及生存率的变化;用RT-PCR检测转基因小鼠骨髓细胞耐药基因的表达。结果 转染SFG-F／S-NeoR耐药基因的骨髓细胞有耐药克隆的形成,供体小鼠为15.8％,受体小鼠为18．0％,对照组为0;大剂量化疗后,含耐药基因组小鼠血象、体重逐渐恢复正常,生存率为83．3％（第40天）,对照组为0;转基因小鼠骨髓细胞经RT-PCR检测,显示有F／S基因条带（400bp）。结论 DHFR耐药基因可导入小鼠骨髓细胞并获得表达,提高了骨髓细胞对MTX的耐药性。     

PRELIMINARY STUDY OF RETROVIRAL MEDIATED TRANSFER OF THE HUMAN mdr-1 GENE INTO MURINE AND HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS

ＰＲＥＬＩＭＩＮＡＲＹＳＴＵＤＹＯＦＲＥＴＲＯＶＩＲＡＬＭＥＤＩＡＴＥＤＴＲＡＮＳＦＥＲＯＦＴＨＥＨＵＭＡＮｍｄｒ１ＧＥＮＥＩＮＴＯＭＵＲＩＮＥＡＮＤＨＵＭＡＮＨＥＭＡＴＯＰＯＩＥＴＩＣＳＴＥＭ／ＰＲＯＧＥＮＩＴＯＲＣＥＬＬＳＦｅｎｇＫａｉ冯凯Ｐｅ...     

Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene

Hematopoietic toxicity produced by most anticancer drugs limits their potential for curative therapy. We have shown previously that the human cytidine deaminase (CD) gene can confer drug resistance in murine bone marrow cells (BMCs) to the nucleoside analog, cytosine arabinoside (ARA-C). In the present study, as the first objective we showed that the CD gene can also render drug resistance in BMCs to related analogs, 2',2'-difluorodeoxycytidine (dFdC) and 5-azadeoxycytidine (5-AZA-CdR). As a second objective, we investigated the potential of ex vivo selection with cytosine nucleoside analogs of CD-transduced BMC. The goal of this approach was to enrich the fraction of CD-transduced BMCs so as to increase the transgene expression and level of drug resistance before transplantation. This strategy may have the potential to circumvent the problem in clinical gene therapy of low level of gene transfer and adequate long-term gene expression. Using a bicistronic retroviral vector containing the CD and the green fluorescent protein (CDiGFP), we transduced murine L1210 leukemic cells. All three analogs, ARA-C, dFdC, and 5-AZA-CdR were demonstrated in vitro to enrich (>95%) the population of leukemic cells expressing the GFP transgene. However, with CD-transduced primary murine BMCs cultivated at high cell density we observed that in vitro selection with ARA-C was not possible due to release of CD into the culture medium at amounts that were sufficient to inactivate the analog. The CD-containing medium produced a chemoprotective effect on mock BMCs as shown by lack of significant growth inhibition in the presence of ARA-C. However, at low cell density in a cell mixture containing CD-transduced cells, the mock BMCs showed marked drug sensitivity to ARA-C as determined by clonogenic assay. Selection with ARA-C was shown to significantly increase the CD enzyme activity in transduced BMC. These results suggest that CD gene has the potential to be a good selectable marker and a possible tool for chemoprotection in cancer gene therapy.     

Retroviral transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to 5-fluorouracil in murine hematopoietic progenitor cells and human CD34+-enriched peripheral blood progenitor cells.

Severe 5-fluorouracil (5-FU) toxicity has been reported among patients lacking dihydropyrimidine dehydrogenase (DPD) enzymatic activity. DPD is the principal enzyme involved in the degradation of 5-FU to 5'-6'-dihydrofluorouracil, which is further metabolized to fluoro-beta-alanine. We demonstrate here that overexpression of human DPD confers resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and in human CD34+-enriched hematopoietic progenitor cells. An SFG-based dicistronic retroviral vector containing human DPD cDNA, an internal ribosomal entry site (IRES), and the neomycin phosphotransferase (Neo) gene was constructed (SFG-DPD-IRES-Neo). Transduced NIH3T3 cells demonstrated a 2-fold (ED50) increase in resistance to a 4-hour exposure of 5-FU in comparison to nontransduced cells. Expression of DPD was confirmed by Northern and Western blot analyses, and DPD enzyme activity was detectable only in transduced cells. Infection of mouse bone marrow cells with this retroviral construct resulted in an increased number of 5-FU-resistant CFU-GM colonies, compared to mock-transduced bone marrow in both 4-hour and 12- to 14-day exposures. Infection of human CD34+-enriched cells with this construct and incubation with 5-FU (10(-6) M) for 14 days also resulted in an increased number of 5-FU-resistant colonies. Retroviral transduction of human hematopoietic progenitor cells with a cDNA-expressing human DPD conferred resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and human CD34+-enriched cells. These results encourage the use of this gene as a method to protect patients from 5-FU myelotoxicity.     

Modulation of the effect of 1-beta-D-arabinofuranosylcytosine based on changes of cytidine deaminase activity in HL60 cells

The effect of changes in cytidine deaminase activity on the growth inhibitory effect of 1-beta-D-arabinofuranosylcytosine (Ara-C) has been investigated in the human promyelocytic cell line, HL60. 1,25 Dihydroxy vitamin D3 (vit. D3), which is an inducer of differentiation, caused an increase of cytidine deaminase in HL60 cells simultaneous with an inhibition of the effect of Ara-C. On the other hand, retinoic acid, which also induces differentiation had no influence on either the activity of cytidine deaminase or the effect of Ara-C. An atoxic inhibitor of cytidine deaminase tetrahydrouridine, THU, enhanced the effect of Ara-C. THU also enhanced the effect of Ara-C, even if the Ara-C effect was inhibited by deoxycytidine. These results show that it is possible to modulate the effect of Ara-C by changing the activity of cytidine deaminase. Furthermore, changes in the activity of cytidine deaminase and the effect on the Ara-C growth inhibition vary dependent on the differentiating inducer used.     

Coexpression of rat glutathione S-transferase A3 and human cytidine deaminase by a bicistronic retroviral vector confers in vitro resistance to nitrogen mustards and cytosine arabinoside in murine fibroblasts

The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc1) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to nitrogen mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6- and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of nitrogen mustards and cytosine nucleoside analogs.     

Modulation of the effect of l-ß-D-Arabinofuranosylcytosine based on changes of Cytidine Deaminase activity in Hl60 Cells

The effect of changes in cytidine deaminase activity on the growth inhibitory effect of 1-ß-D-arabinofuranosylcytosine (Ara-C) has been investigated in the human promyelocytic cell line, HL60. 1,25 Dihydroxy vitamin D₃ (vit. D₃), which is an inducer of differentiation, caused an increase of cytidine deaminase in HL60 cells simultaneous with an inhibition of the effect of Ara-C. On the other hand, retinoic acid, which also induces differentiation had no influence on either the activity of cytidine deaminase or the effect of Ara-C. An atoxic inhibitor of cytidine deaminase tetrahydrouridine, THU, enhanced the effect of Ara-C. THU also enhanced the effect of Ara-C, even if the Ara-C effect was inhibited by deoxycytidine. These results show that it is possible to modulate the effect of Ara-C by changing the activity of cytidine deaminase. Furthermore, changes in the activity of cytidine deaminase and the effect on the Ara-C growth inhibition vary dependent on the differentiating inducer used.     

Drug resistance to 5-aza-2′-deoxycytidine, 2′,2′-difluorodeoxycytidine, and cytosine arabinoside conferred by retroviral-mediated transfer of human cytidine deaminase cDNA into murine cells

Purpose: The hematopoietic toxicity produced by the cytosine nucleoside analogs is a critical problem that limits their effectiveness in cancer therapy. One strategy to prevent this dose-limiting toxicity would be to insert a gene for drug resistance to these analogs into normal bone marrow cells. Cytidine (CR) deaminase can deaminate and thus inactivate 5-aza-2^′-deoxycytidine (5-AZA-CdR), 2^′,2^′-difluorodeoxycytidine (dFdC) and cytosine arabinoside (ARA-C). The aim of this study was to determine if gene transfer of CR deaminase into murine fibroblast cells confers drug resistance to these cytosine nucleoside analogs and if this resistance can be prevented by the CR deaminase inhibitor, 3,4,5,6-tetrahydrouridine (THU). Methods: NIH 3T3 murine fibroblast cells were transduced with retroviral particles containing the human CR deaminase cDNA. Assays measuring CR deaminase activity as well as the inhibitory action of 5-AZA-CdR, dFdC and ARA-C on colony formation, were performed in the presence of different concentrations of THU. Results: Retroviral-mediated transfer of the CR deaminase gene into 3T3 fibroblasts produced a considerable increase in CR deaminase activity. The transduced cells also showed significant drug resistance to 5-AZA-CdR, dFdC and ARA-C, as demonstrated by a clonogenic assay. This drug resistance phenotype and elevated CR deaminase activity were reversed by THU. Conclusions: These findings indicate that the CR deaminase gene can potentially be used in cancer gene therapy for protecting normal cells against the cytotoxic actions of different cytosine nucleoside analogs. In addition, the CR deaminase-transduced cells can be used as a model for screening different CR deaminase inhibitors in an intact cellular system. Received: 30 September 1997 / Accepted: 16 January 1998     

多药耐药基因增强骨髓细胞对抗癌药物毒性的抵御能力

目的　探讨多药耐药基因 (mdr1基因 )的骨髓细胞转染增强骨髓细胞对抗癌药的抵御能力。方法　我们用mdr1基因表达质粒pHaMDR1/A ,通过脂质体介导 ,转染小鼠和人骨髓细胞 ,并采用骨髓移植建立小鼠动物模型。结果　成功的将mdr1基因导入了小鼠和人骨髓细胞并获得了表达 ,其细胞已对阿霉素、足叶乙甙 (Vp 16 )、长春新碱和秋水仙碱等产生了明显的抗性。同时 ,转染mdr1基因小鼠骨髓细胞的移植 ,在受体小鼠重建造血功能 ,受体小鼠骨髓细胞亦对抗癌药具有抗性。结论　从小鼠骨髓细胞、人骨髓细胞和小鼠体内模型 3个水平证实了mdr1基因的骨髓细胞转染可增强骨髓细胞对抗癌药的抵御能力