蛇毒半胱氨酸蛋白酶抑制剂基因转染对小鼠黑色素瘤B16F1细胞基因表达谱的影响

背景与目的:本实验室已证明蛇毒半胱氨酸蛋白酶抑制剂(snake venom cystatin, sv-cystatin)具有抗肿瘤侵袭转移作用.为了进一步阐明其分子机制,本研究利用高通量的基因芯片技术探讨sv-cystatin基因转染对小鼠黑色素瘤细胞(B16F1)基因表达谱的影响.方法:分别将pcrDNA3.1/sv-cystatin及空载体pcDNA3.1转染B16Fl细胞,建立稳定转染的B16Fl/sv-cystatin及B16F1/pcDNA3.1细胞株,提取总mRNA.应用高通量的基因芯片技术检测差异表达基因,半定量RT-PCR法分别验证5个上调和5个下调的差异表达基因.结果:B16F1细胞经转染sv-cystatin后.共检测了1218个基因,其中有45个差异表达基因,21个基因表达上调(Ratio>3),24个基因表达下调(Ratio<0.5),这些基因的功能涉及细胞粘附迁移、细胞免疫调节、增殖分化与凋亡、基因转录和细胞内信号转导等方面.10个差异表达基因的RT-PCR验证结果与基因芯片分析结果相符.结论:Sv-cystatin不仅具有抑制细胞外基质的作用,而且还具有细胞免疫调节、增殖分化与凋亡、基因转录和细胞内信号转导等多种生物学功能.     

三氧化二砷与HMBA对白血病K562细胞增殖抑制作用及其分子机制的研究

目的:探讨诱导分化荆对K562细胞增殖抑制的作用与分子机制.方法:通过MTT试验和细胞形态学观察K562细胞增殖的改变和分化情况.通过流式细胞仪实验检测细胞周期改变,RT-PCR检测EⅥ1、TGF-β1和WT1基因表达的变化.结果:K562细胞经六亚甲基二乙酰胺(HMBA)和(或)三氧化二砷(As2O3)作用后增殖明显受到抑制,并向不同方向分化,细胞被阻滞在G1期.K562细胞的增殖抑制作用与EVI1和WT1基因表达下调,TGF-β1基因表达上调一致.EⅥ1/β-aetin从0.925 3渐降至0.137 9,WT1/β-actin从0.995 3降至0.197 8.TGF-β1/β-actin从0.331 5增至1.245 2.结论:HMBA、As2O3及其联合抑制K562细胞增殖作用与EⅥ1和WT1基因表达下调,TGF-β1基因表达上调有关.     

表没食子儿茶素没食子酸酯对两株不同肝癌耐药细胞株基因表达谱的影响

背景与目的:绿茶中的儿茶索单体表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)在体内外能逆转肝癌细胞多药耐药性.鉴于多药耐药机制的复杂性,本研究拟采用高通量基因芯片技术进一步探讨EGCG逆转人肝癌细胞BEL7404/ADM、BEL7402/5-FU多药耐药的可能机制.方法:MTT法检测药物敏感性.基因芯片技术分析EGCG作用于两株不同肝癌耐药细胞的基因表达谱差异.RT-PCR和Western blot法分别检测相同处理条件下两株肝癌耐药细胞的基因MDR1、LRP和Cyclin G1蛋白表达变化以验证基因芯片技术结果.结果:EGCG对BEL7404/ADM、BELT402/5-FU细胞的IC10分别为24.76 mg/L、20.60 mg/L.20 mg/L EGCG联合0.05 mg/L ADM、100 μmol/L 5-FU对BEL7404/ADM、BEL7402/5-FU的逆转倍数分别为9.66、2.36倍.EGCG作用BEL7404/ADM双荧光通路显著差异基因210个,上调表达38个,下调表达172个.与耐药机制相关的可能基因有ABCB10(MDR/TAP)、TOP2A、TOP2B、CCNG1上调,ABCB1、MVP、ARHD、HDAC5、GSS、GSTPI、HSPA1B、HSPB7、CDKN1A、RAB11B、RAB9P40下调.作用BEL7402/5-FU双荧光通路显著差异基因179个,上调表达31个,下调表达148个.与耐药机制相关的可能基因有ABCG(BCRP)、CCNG2、GADD34、RB1、RBBP4上调,DTYMK、GPX1、USP5、BAX、BAK1、HSPA1L下调等.相同处理条件下两组肝癌耐药细胞的MDR1、LRP基因表达均减少,Cyclin G1蛋白表达均增加.结论:EGCG对肝癌多药耐药细胞BEL7404/ADM、BEL7402/5-FU具逆转作用,但作用于不同肝癌多药耐药细胞的基因表达谱变化不完全相同.     

靶向沉默核干因子对前列腺癌PC-3细胞全基因组表达谱变化的影响

目的 探讨核干因子(NS)基因在前列腺癌恶性增殖中的作用机制.方法 采用表达谱基因芯片技术,分析前列腺癌PC-3细胞NS基因表达下调后全基因组表达谱的变化,筛选与NS相互作用的差异表达基因及信号通路.采用实时荧光定量聚合酶链反应对重要的差异表达基因进行验证.结果 共筛选出219个差异表达的基因,这些基因涉及到细胞周期、细胞增殖、信号转导、细胞凋亡和细胞分化等多个方面.NS基因表达下调后主要引起周期素依赖激酶4抑制因子(INK4)家族基因(p15、p16和p18)的上调,以及cyclin D1和HDACI基因的下调,其主要作用点位于CDK4/6-cyclinD和pRb-E2F1复合体上.结论 在前列腺癌PC-3细胞中,NS基因可能主要通过抑制p15、p16和p18等INK4家族的肿瘤抑制基因的表达,从而促进肿瘤的发生和发展;NS基因是细胞周期G1/S期检查点的重要调控因子.     

下调PPP2R5C表达对Jurkat细胞GSK-3β、MDM2、pTEN、TP53通路相关调控基因表达谱的影响

目的 利用基因芯片技术研究RNA干扰下调PPP2R5C对Jurkat细胞中GSK-3β、MDM2、pTEN、TP53通路相关基因表达的影响。方法 利用核转染技术将PPP2R5C-siRNA799转导Jurkat细胞,培养48 h后,提取细胞总RNA,纯化mRNA后反转录制备目标序列,与Affymetrix基因表达谱芯片3’IVT杂交,以Affymetrix GeneChip Scanner 3000扫描仪扫描得到原始图像数据,并应用GeneSpringGX 11.0软件进行差异表达谱分析。结果 基因芯片分析GSK-3β、MDM2、pTEN、TP53信号通路相关的47个基因,发现全部基因发生了差异表达,其中上调基因有22个,下调基因有25个。明显上调的基因有SNAI1、APC、CDKN1A、ATM、MDM2和pTEN,而明显下调的基因只有GSK-3β。结论 下调Jurkat细胞PPP2R5C基因时,在一定程度上选择性调节涉及细胞增殖和凋亡相关的GSK-3β、MDM2、pTEN和TP53信号通路基因的表达,从而抑制Jurkat细胞增殖。     

基因芯片检测Ⅰ期低分化肺腺癌基因表达谱

目的 用基因芯片技术分析Ⅰ期低分化肺腺癌的异常基因表达。方法 提取肺癌组织及正常肺组织的mRNA,分别用Cy5-dUTPCy3-dUTP标记,再与8192点基因芯片杂交,用ScanArray4000扫描仪及GenePix3．0分析软件处理杂交信号获取结果。结果 肺癌组织与正常肺组织表达差异基因有580条,涉及到细胞生长调节、细胞间信息转导等多个方面。其中癌组织中上调基因为405条,下调基因175条;差异极显著性基因66条,表达上调35条,下调24条,另外有7条为未知基因。结论 多基因参与Ⅰ期低分化肺腺癌发病,基因芯片技术是疾病基因筛选的有效方法。     

RhoA小干扰RNA对胃癌AGS细胞基因表达谱的影响

目的:应用RhoA小干扰RNA(small interfering RNA,siRNA)转染胃癌细胞系AGS细胞,采用高通量基因芯片技术,筛选基因表达谱的变化,为进一步研究RhoA信号转导通路奠定基础.方法:用pSilencer3.1载体构建RhoA/siRNA重组质粒.Effectene转染试剂转染AGS细胞,空载体pSilencer3.1转染对照组.应用G418筛选转染细胞系,获得稳定转染细胞;美国Agilent基因芯片检测细胞基因表达谱变化.结果:基因芯片检测全基因组,发现表达上调的有1151个基因,表达下调的有1079个基因,涉及信号转导、细胞周期、细胞凋亡等相关信号分子.生物信息学分析显示,与细胞凋亡有关的caspase家族,以及细胞周期调控基因存在明显的差异表达.结论:基因芯片结果提示干扰RhoA表达后,与凋亡和增殖有关的分子在RhoA介导的肿瘤细胞异常增殖信号转导中起着重要的调控作用,为下一步研究其信号调控网络机制提供重要的信息.     

宝藿苷-I联合5-FU对食管癌Eca-109细胞增殖及凋亡的影响

目的：研究宝霍苷-I与5-FU联合应用对食管癌Eca-109细胞增殖和凋亡的影响。方法：实验设联合用药组（12.5mg/L宝霍苷-I＋12.5mg/L5-FU）、阴性对照组（0.1%DMSO）、25mg/L宝藿苷-I组和12.5mg/L5-FU组,共4组,每组均设3个复孔,每孔100μl（含Eca-109细胞数为1×10~4个）。作用24、48、72h时,采用四甲基偶氮唑盐（MTT）法分别检测各组Eca-109细胞的增殖;并在作用48h后,用流式细胞术检测Eca-109细胞凋亡率;用Western blot技术检测Bcl-2、Bax蛋白的表达。结果：宝霍苷-I、5-FU单用及其联合应用在24、48和72h时均可明显抑制Eca-109细胞的增殖,与阴性对照组比较,差异均有统计学意义（P均〈0.05）,联合应用的抑制作用显著优于单独应用（P〈0.05）。宝霍苷-I、5-FU单独及其联合作用48h后,均可诱导Eca-109细胞凋亡,均可下调Bcl-2蛋白的表达,上调Bax蛋白的表达,与对照组相比差异均有统计学意义（P均〈0.05）,且联合用药组较单独用药组作用明显（P〈0.05）。结论：宝霍苷-I与5-FU联合应用对Eca-109细胞的增殖抑制和凋亡诱导有协同作用,可能与下调Bcl-2蛋白的表达、上调Bax蛋白的表达有关。     

曲古霉素A和淫羊藿甙诱导HL-60细胞分化过程中nm23和c-myc及G-CSF表达变化的研究

目的:研究曲古抑菌素A(TSA)、淫羊藿甙(ICA)诱导急性非淋巴细胞白血病HL-60细胞株分化过程中nm23基因的表达变化及其作用机制.方法:通过MTT试验检测HL-60增殖分化变化,观察细胞形态,分析表面标志和硝基四氮唑蓝(NBT)还原反应,观察HL-60细胞分化水平变化情况;通过流式细胞仪检测细胞周期的改变,RT-PCR检测nm23-H1、nm23-H2、G-CSF、c-myc基因表达的变化.结果:HL-60细胞经TSA、ICA作用后增殖受抑制,24、48和72h抑制率分别为(12.73±1.17)%、(38.15±3.32)%和(58.45±3.06)%,向成熟粒细胞系分化,细胞被阻滞在G1期,nm23、c-myc基因的表达下调,G-CSF基因表达上调.结论:HL-60细胞分化过程中c-myc基因的表达下调,G-CSF基因表达上调与下调nm23基因的表达有关.     

shRNA沉默PLCε基因表达对肾癌细胞增殖的抑制及作用机制

目的:探讨经短发夹RNA(short hairpin RNA,shRNA)干扰技术沉默磷脂酶Cε(phospholipase C epsilon,PLCε)基因表达后对肾癌786-0细胞增殖的抑制及其作用机制.方法:脂质体介导重组质粒(pGenesil-PLCε)和空质粒(pGenesil-NP)转染786-0细胞48 h后,RT-PCR检测PLCε mRNA的表达;MTT法检测转染24、48和72 h后细胞增殖的抑制率;转染48 h后采用FCM方法分析细胞周期;RT-PCR和免疫细胞化学法分析转染48 h后p27和Ki67的表达情况.结果:转染重组质粒后可明显抑制PLCεmRNA的表达,其抑制率为69.7%;转染24、48和72 h后细胞增殖抑制率分别为21.2%,31.6%和32.7%;FCM结果显示转染重组质粒后细胞周期的分布发生了改变,G0/G1期细胞增多,S期和G2/M期细胞减少,细胞阻滞于G0/G1期,并出现了亚二倍体"凋亡峰".RT-PCR和免疫细胞化学结果均表明p27表达上调,而Ki67表达下调.结论:RNA干扰技术阻断PLCε基因表达后可抑制肾癌786-0细胞增殖,其作用机制可能是诱导p27表达上调和Ki67表达下调.     

Influence of dose on regeneration of murine hematopoietic stem cells after total body irradiation and 5-fluorouracil.

The regeneration of murine bone marrow stem cells after treatment with 5-fluorouracil (5-FU) and total body irradiation (TBI) was investigated by means of the spleen colony assay. 5-FU was given intraperitoneally in doses ranging from 75 to 225 mg/kg body weight. TBI was administrated as a single fraction in the dose range 0.75-3.50 Gy. The frequency of stem cells declined rapidly and reached a minimum (nadir) on day 1-2 after either of the 2 treatments. Their number returned to near-pretreatment values by day 7-10. Increasing the dose of either 5-FU or TBI resulted in a lower nadir in stem cell survival and a higher regeneration rate of the stem cells. The maximal regeneration rate corresponded to a doubling time of 19 h. The time to complete regeneration was constant and neither influenced by the treatment modality nor by the dose. Both this work and previous studies on cyclophosphamide and combined 5-FU and TBI may indicate that the regeneration rate of bone marrow stem cells after a cytotoxic assault is influenced primarily by the stem cell surviving the treatment rather than the treatment modality.     

Effect of combined 5-fluorouracil and radiation on murine hematopoietic tissue

The interaction of 5-fluorouracil (5-FU) and radiation in hematopoietic tissue was assessed as the survival of hematopoietic stem cells (CFUs) by means of the spleen colony assay. 5-FU was given intraperitoneally in the dose range 50-500 mg/kg body weight. In this dose range, stem cell survival decreased exponentially as a function of 5-FU dose. After 150 mg/kg of 5-FU alone (i.e. the maximum tolerated dose, MTD), the stem cell survival rapidly decreased, reaching a minimum after 1-2 days. The decrease was followed by a regeneration phase with a doubling time of about 28 h, with return to pretreatment values on day 7, and with an overshoot of survival on day 10-28. A similar regeneration was observed after 0.75 Gy radiation alone, but there was no evidence of an overshoot of stem cell number. 5-FU given 15 min before whole-body irradiation resulted in a pronounced reduction in stem cell survival due to an increase in the slope of the radiation survival curve by a factor of 2.1. After combined 5-FU and radiation, the survival rapidly decreased to a minimum at day 1, and it showed only a slight increase within the next 7 days. After this delay, the stem cells regenerated with a doubling time of about 30 h, reaching pretreatment values on day 15. The delayed stem cell regeneration was not seen following 3.5 Gy radiation alone or 225 mg/kg 5-FU alone, which resulted in the same nadir of CFUs survival as found after the combined treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The poison oligonucleotide F10 is highly effective against acute lymphoblastic leukemia while sparing normal hematopoietic cells

F10 is an oligonucleotide based on the thymidylate synthase (TS) inhibitory 5-fluorouracil (5-FU) metabolite, 5-fluoro-2''-deoxyuridine-5''-O-monophosphate. We sought to determine the activity of F10 against preclinical models of acute lymphoblastic leukemia (ALL). F10 treatment resulted in robust induction of apoptosis that could not be equaled by 100 fold more 5-FU. F10 was more potent than Ara-C and doxorubicin against a panel of murine and human ALL cells with an average IC₅₀ value of 1.48 nM (range 0.07 to 5.4 nM). F10 was more than 1000 times more potent than 5-FU. In vivo, F10 treatment significantly increased survival in 2 separate syngeneic ALL mouse models and 3 separate xenograft models. F10 also protected mice from leukemia-induced weight loss. In ALL cells made resistant to Ara-C, F10 remained highly active in vitro and in vivo. Using labeled F10, uptake by the ALL cell lines DG75 and SUP-B15 was rapid and profoundly temperature-dependent. Both cell lines demonstrated increased uptake compared to normal murine lineage- depleted marrow cells. Consistent with this decreased uptake, F10 treatment did not alter the ability of human hematopoietic stem cells to engraft in immunodeficient mice.     

Fluorouracil selectively enriches stem-like cells in the lung adenocarcinoma cell line SPC

Most adult stem cells are in the G0 or quiescent phase of the cell cycle and account for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. This study sought to enrich cancer stem cells and explore cancer stem-like cell clones using 5-fluorouracil (5-FU) in the lung adenocarcinoma cell line, SPC. Proliferation inhibition was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, according to which half maximal inhibitory concentration values were calculated. Expression levels of stem cell markers after treatment with 5-FU were examined using immunofluorescence and Western blotting. Additionally, side population (SP) cells were sorted using FACS. Properties of SP cells were evaluated by using Transwell, colony-forming assays, and tumor formation experiments. 5-FU greatly inhibits proliferation, especially of cells in S phase. SP cells possess greater invasive potential, higher clone-forming potential, and greater tumor-forming ability than non-SP cells. Treatment with 5-FU enriches the SP cells with stem cell properties in human lung adenocarcinoma cell lines.     

3-Deazaneplanocin A is a promising therapeutic agent for the eradication of tumor-initiating hepatocellular carcinoma cells

Recent advances in stem cell biology have identified tumor-initiating cells (TICs) in a variety of cancers including hepatocellular carcinoma (HCC). Polycomb group gene products such as BMI1 and EZH2 have been characterized as general self-renewal regulators in a wide range of normal stem cells and TICs. We previously reported that Ezh2 tightly regulates the self-renewal and differentiation of murine hepatic stem/progenitor cells. However, the role of EZH2 in tumor-initiating HCC cells remains unclear. In this study, we conducted loss-of-function assay of EZH2 using short-hairpin RNA and pharmacological inhibition of EZH2 by an S-adenosylhomocysteine hydrolase inhibitor, 3-deazaneplanocin A (DZNep). Both EZH2-knockdown and DZNep treatment impaired cell growth and anchorage-independent sphere formation of HCC cells in culture. Flow cytometric analyses revealed that the two approaches decreased the number of epithelial cell adhesion molecule (EpCAM)(+) tumor-initiating cells. Administration of 5-fluorouracil (5-FU) or DZNep suppressed the tumors by implanted HCC cells in non-obese diabetic/severe combined immunodeficient mice. Of note, however, DZNep but not 5-FU predominantly reduced the number of EpCAM(+) cells and diminished the self-renewal capability of these cells as judged by sphere formation assays. Our findings reveal that tumor-initiating HCC cells are highly dependent on EZH2 for their tumorigenic activity. Although further analyses of TICs from primary HCC would be necessary, pharmacological interference with EZH2 might be a promising therapeutic approach to targeting tumor-initiating HCC cells.     

Enforced expression of cytosolic 5′-nucleotidase I confers resistance to nucleoside analogues in vitro but systemic chemotherapy toxicity precludes in vivo selection

Purpose: Retroviral transfer of cDNA sequences that confer drug resistance can be used to protect against chemotherapy-induced hematopoietic toxicity and for the selective expansion of gene-modified cells. To successfully expand genetically engineered cells in vivo, an appropriate balance must be achieved between systemic toxicity induced by the selecting agent and the expansion of modified cells. Method: In this study, we investigate retroviral transfer of cytosolic 5′-nucleotidase I (cN-I) for protection and selection of gene-modified cells when treated with 2-chloro-2′-deoxyadenosine (2-CdA) and 5-fluorouracil (5-FU) alone and in combination. We also attempt to design a treatment strategy for the potential in vivo selection of cN-I-modified cells by administering 5-FU to mice prior to 2-CdA treatment. Results: Our results show that cN-I can be transferred by recombinant retroviruses, and that enforced expression of cN-I protects murine fibroblast and hematopoietic progenitor cells from the cytotoxic effects of 2-CdA and/or 5-FU. Furthermore, we show that the combined administration of 5-FU and 2-CdA potentiates hematopoietic stem cell toxicity. However, the treatment also results in severe myelosuppression. Conclusion: These results show that while cN-I provides both protective and selective benefits to gene-modified cells in vitro, selection requires a treatment strategy that is likely too toxic to consider cN-I as an in vivo selectable marker Tasha Gray, Erin L. Morrey contributed equally to this work     

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

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UPRT/5-FU前药转换酶系统对小鼠胃癌治疗的实验研究

目的　探讨UPRT/ 5 FU前药转换酶系统对小鼠胃癌细胞MFC的杀伤效应。方法　采用PCR技术从大肠杆菌K12基因组中扩增UPRT基因 ,并构建pLXSN逆转录病毒表达载体 ,进一步转染MFC细胞。采用MTT法检测转导UPRT基因前后 ,MFC对 5 FU的敏感性的变化。建立 6 15小鼠胃癌皮下移植瘤模型 ,腹腔内给于 5 FU ,观察治疗效果。结果　体外MTT法检测结果显示 ,转导UPRT基因后可使MFC对 5 FU的敏感性提高约 17.2 6倍。动物实验的结果表明 ,转导UPRT基因不影响MFC的致瘤性 ;经 5 FU治疗后 ,转UPRT基因的肿瘤 ,生长明显受抑 (P <0 .0 0 0 5 ) ,抑瘤率为 89.6 2 %。结论　UPRT基因修饰小鼠胃癌细胞株MFC ,能明显提高MFC对 5 FU杀伤的敏感性 ,增强 5 FU的抗瘤效应。     

Toxicity of 5-fluorouracil for aerobic and hypoxic cells in two murine tumours

Summary Chronically hypoxic cells in solid tumours are reported to be more slowly proliferating than aerobic cells situated closer to blood vessels. Cycle-active drugs such as 5-fluorouracil (5-FU) might be expected to have greater activity against rapidly proliferating aerobic cells. The effect of 5-FU was therefore assessed against aerobic and hypoxic cells in murine tumours; this was done by giving the drug alone or 1 h before irradiation of the tumour under aerobic or hypoxic conditions. Aerobic and hypoxic cells appeared to have equal sensitivity to 5-FU in the KHT and 16/C tumours, and the effect of a single dose of 5-FU was additive to that of radiation.Supported by a research grant from the National Cancer Institute of Canada and by grant CA 36913 from the National Cancer Institute, NIH, USA     

Uridine phosphorylase (-/-) murine embryonic stem cells clarify the key role of this enzyme in the regulation of the pyrimidine salvage pathway and in the activation of fluoropyrimidines

We have reported the elevation of uridine phosphorylase (UPase) in many solid tumors and the presence of a variant phosphorolytic activity in breast cancer tissues (M. Liu et al., Cancer Res., 58: 5418-5424, 1998). To better understand the biological and pharmacological significance of these findings, we have developed an UPase gene knockout embryonic stem (ES) cell model by specific gene targeting techniques. In this cellular model, we establish the critical role of UPase as an important anabolic enzyme in 5-fluorouracil (5-FU) activation and pyrimidine salvage pathway regulation. It has long been known that UPase regulates the plasma concentration of uridine; however, little is known of the role of UPase in the activation and metabolism of 5-FU and its derivatives, mainly because of the lack of an appropriate model system. The experimental data indicate that the disruption of UPase activity in murine ES cells leads to a 10-fold increase in 5-FU IC(50) and a 2-3-fold reduction in its incorporation into nucleic acids, whereas no differences in toxicity is seen with other pyrimidine nucleoside analogues such as 5-fluorouridine, 2'-deoxy-5-fluorouridine, and 1-beta-D-arabinofuranosylcytosine compared with WT (wild-type) ES cells. Benzylacyclouridine can specifically prevent the WT ES cells from the sensitivity of 5-FU. Our data also shows the effect of UPase on the cytotoxicity of 5'-deoxy-5-fluorouridine (5'DFUR), a 5-FU prodrug. The IC(50) is increased almost 16-fold in the knockout cells compared with the wild type cells, demonstrating the role of UPase in catalyzing the conversion of 5'DFUR to 5-FU. These findings additionally elucidate the tumor-specific selectivity of capecitabine, the oral fluoropyrimidine prodrug approved for the treatment of metastatic breast and colorectal cancers. Not only do the knockout cells present a decreased incorporation of 5-FU into nucleic acids but also an increased reliance on the pyrimidine salvage pathway. The reduced dependence of UPase knockout cells on the pyrimidine de novo synthesis is reflected in the apparent resistance to phosphonacetyl-L-aspartic acid, a specific inhibitor of pyrimidine pathway, with a 5-fold elevation in its IC(50) in UPase-nullified cells compared with WT. In summary, we have successfully generated an UPase gene knockout cell model that presents reduced sensitivity to 5-FU, 5'DFUR, and phosphonacetyl-L-aspartic acid, although it does not affect the basic cellular physiology under normal tissue culture conditions. Considering the role of UPase in 5-FU metabolism and the elevated expression of this protein in cancer cells compared with paired normal tissues, additional investigation should be warranted to firmly establish the clinical role of UPase in the tumor selective activation of 5-FU and capecitabine.