ADENOVIRUS-MEDIATED P53 GENE TRANSFER INCREASES THE THERMOSENSITIVITY OF HUMAN GASTRIC CARCINOMA CELL LINES(IN VITRO AND IN VIVO)

Objective: To evaluate the effect of adenovirus-mediated p53 gene (Adp53) on apoptosis andradiosensitivity of human gastric carcinoma cell lines.Methods: Recombinant adenovirus expressing wild-type p53 gene was transferred into four human gastric carcinoma cell lines with different p53 genetic status. p53 proteinexpression was detected by immunohistochemistry assayand western blot assay. Cell survival was assessed using a clonogenic assay. TUNEL assay was used in determination of apoptosis. Four human gastric carcinoma cells infectedwith Adp53 were irradiated with 4Gy and cell cycle distribution and Sub-G1 peak were assayed by flowcytometry. Results: G2/M arrest, apoptosis and inhibition of tumor cell proliferation were induced by infection atAdp53 at 100 MOI which caused high transfer rate ofwild-type p53 and strong expression of p53 protein in four human gastric carcinoma cells. The radio-enhancement ratio of Adp53 at 4Gy were 3.0 for W cell, 3.6 for M cell, 2.2 for neo cell and 2.5 for 823 cell in vitro. Conclusion: Thisstudy demonstrated that Adp53 transfer increased cellularapoptosis and radiosensitivity of human gastric carcinoma cell lines in vitro independently on cellular intrinsic p53status thus supporting the combination of p53 gene therapy with radiotherapy in clinical trials.

重组P_(53)基因在HepG-2中的表达及作用

为进一步探索正常Ｐ５３ 对肿瘤细胞增殖的抑制作用，本文用磷酸钙ＤＮＡ共沉淀法，将本室构建的ＰＸＴ１Ｐ５３ 真核表达细胞转移至ＨｅｐＧ２ 肝癌细胞系内，经斑点杂交技术和间接免疫荧光技术证实，外源性Ｐ５３ 基因已在ＨｅｐＧ２ 细胞中稳定表达，导入的Ｐ５３基因亦能抑制ＨｅｐＧ２ 肝癌细胞系增殖并诱导其凋亡。实验结果提示正常Ｐ５３ 基因可成为治疗不同肿瘤的重要靶分子之一。

p53基因治疗对鼻咽癌患者CD34标记的微血管密度和血小板计数的影响

目的观察放化疗联合重组人p53腺病毒注射液(rAd-p53)治疗鼻咽癌后对原发灶中CD34标记的微血管密度(CD34-MVD)及血小板(PLT)计数的影响,探讨其与预后的关系。方法 63例中晚期鼻咽癌患者随机分为两组,基因组32例给予rAd-p53瘤内注射+同步放化疗;常规组31例仅给予同步放化疗。采用免疫组化二步法检测两组患者癌组织中CD34-MVD水平,并检测PLT。结果治疗后基因组鼻咽癌原发灶CD34-MVD明显低于治疗前(P<0.05),而常规组治疗前后CD34-MVD比较差异无统计学意义(P>0.05);治疗后两组PLT均显著下降(P均<0.05),并且基因组下降更明显(P<0.05)。随访3年,基因组和常规组局部复发、远处转移率比较差异无统计学意义(P>0.05);但CD34-MVD表达增加组远处转移率(38.5%)明显高于表达下降组(13.5%)(P<0.05),3年总生存率、无瘤生存率低于表达下降组(P<0.05);PLT增加组与PLT下降组的局部复发率、远处转移率、3年总生存率、无瘤生存率差异均无统计学意义(P均>0.05)。结论放化疗联合基因治疗中晚期鼻咽癌,可使CD34-MVD、PLT计数明显下降。CD34-MVD过度表达可作为评估远处转移和预后不良的参考指标。

重组P_(53)真核表达载体的构建

为进一步探索Ｐ５３基因与机体发育、肿瘤发生的关系及其与细胞周期中其它调控因子的相互作用，我们采用粘性末端连接法构建了重组野生型Ｐ５３－ＰＸＴ１真核表达载体，并成功地转化Ｃａｃｌ２处理的ＲＲ１细胞，经多种方法鉴定表明其连接率为６９％，转化率为３９×１０３／μｇＤＮＡ。该重组载体的构建无疑将为肿瘤。

Synergistic anticancer effect of exogenous wild-type p53 gene combined with 5-FU in human colon cancer resistant to 5-FU in vivo

AIM To investigate the anticancer effect of a recombinant adenovirus-mediated p53(r Ad-p53) combined with 5-fluorouracil(5-FU) in human colon cancer resistant to 5-FU in vivo and the mechanism of r Ad-p53 in reversal of 5-FU resistance.METHODS nude mice bearing human colon cancer SW480/5-FU(5-FU resistant) were randomly assigned to four groups(n = 25 each): control group, 5-FU group, r Ad-p53 group, and r Ad-p53 + 5-FU group. At 24 h, 48 h, 72 h, 120 h and 168 h after treatment, 5 mice were randomly selected from each group and sacrificed using an overdose of anesthetics. The tumors were removed and the protein expressions of p53, protein kinase C(PKC), permeability-glycoprotein(P-gp) and multidrug resistance-associated protein 1(MRP1)(Western blot) and apoptosis(TUNEL) were determined.RESULTS The area ratios of tumor cell apoptosis were larger in the r Ad/p53 + 5-FU group than that in the control, 5-FU and r Ad/p53 groups(P < 0.05), and were larger in the r Ad/p53 group than that of the control group(P < 0.05) and the 5-FU group at more than 48 h(P < 0.05). The p53 expression was higher in the r Ad/p53 and the r Ad/p53 + 5-FU groups than that of the control and 5-FU groups(P < 0.05), and were higher in the r Ad/p53 + 5-FU group than that of the r Ad/p53 group(P < 0.05). Overexpression of PKC, P-gp and MRP1 was observed in the 5-FU and control groups. In the r Ad/p53 + 5-FU group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups(P < 0.05), and the expression of PKC was lower than that of the control, 5-FU and r Ad/p53 groups at more than 48 h(P < 0.05). In the r Ad/p53 group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups at more than 48 h(P < 0.05), and the expression of PKC was lower than that of the control and 5-FU groups at more than 120 h(P < 0.05).CONCLUSION5-FU combined with r Ad-p53 has a synergistic anticancer effect in SW480/5-FU(5-FU resistance), which contributes to reversal of 5-FU resistance.

Regulation of DNA double-strand break repair pathway choice:a new focus on 53BP1

Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break(DSB)signaling.P53-binding protein 1(53BP1)plays a critical role in coordinating the DSB repair pathway choice and promotes the non-homologous end-joining(NHEJ)-mediated DSB repair pathway that rejoins DSB ends.New insights have been gained into a basic molecular mechanism that is involved in 53BP1 recruitment to the DNA lesion and how 53BP1 then recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination(HR)signaling.This review focuses on the up-and downstream pathways of 53BP1 and how 53BP1 promotes NHEJ-mediated DSB repair,which in turn promotes the sensitivity of poly(ADP-ribose)polymerase inhibitor(PARPi)in BRCA1-deficient cancers and consequently provides an avenue for improving cancer therapy strategies.

Functional analysis of the acetylation of human p53 in DNA damage responses

As a critical tumor suppressor, p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore, it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However, the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination, we introduced various combinations of missense mutations （lysine to arginine） into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells （hESCs）. By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives, we demonstrate physiological importance of the acetylation events within the core domain （Kt20 and K164） and at the C-terminus （K370/372/373/381/382/ 386） in regulating human p53 responses to DNA damage.

DCAF1 (VprBP): emerging physiological roles for a unique dual-service E3 ubiquitin ligase substrate receptor

Cullin-RING ligases(CRLs)comprise a large group of modular eukaryotic E3 ubiquitin ligases.Within this family,the CRL4 ligase(consisting of the Cullin4[CUL4]scaffold protein,the Rbxl RING finger domain protein,the DNA damage-binding protein 1[DDB1],and one of many DDBl-associated substrate receptor proteins)has been intensively studied in recent years due to its involvement in regulating various cellular processes,its role in cancer development and progression,and its subversion by viral accessory proteins.Initially discovered as a target for hijacking by the human immunodeficiency virus accessory protein r,the normal targets and function of the CRL4 substrate receptor protein DDBl-Cul4-associated factor 1(DCAF1;also known as VprBP)had remained elusive,but newer studies have begun to shed light on these questions.Here,we review recent progress in understanding the diverse physiological roles of this DCAF1 in supporting various general and cell type-specific cellular processes in its context with the CRL4 E3 ligase,as well as another HECT-type E3 ligase with which DCAF1 also associates,called EDD/UBR5.We also discuss emerging questions and areas of future study to uncover the dynamic roles of DCAF1 in normal physiology.

Twenty years of Gendicine?rAd-p53 cancer gene therapy:The first-in-class human cancer gene therapy in the era of personalized oncology

Genetic mutations in TP53 contribute to human malignancies through various means.To date,there have been a variety of therapeutic strategies targeting p53,including gene therapy to restore normal p53 function,mutant p53 rescue,inhibiting the MDM2-p53 interaction,p53-based vaccines,and a number of other approaches.This review focuses on the functions of TP53 and discusses the aberrant roles of mutant p53 in various types of cancer.Recombinant human p53 adenovirus,trademarked as Gendicine,which is the first anti-tumor gene therapy drug,has made tremendous progress in cancer gene therapy.We herein discuss the biological mechanisms by which Gendicine exerts its effects and describe the clinical re-sponses reported in clinical trials.Notably,the clinical studies suggest that the combination of Gendicine with chemotherapy and/or radiotherapy may produce more pronounced efficacy in slowing tumor growth and progression than gene therapy/chemotherapy alone.Finally,we summarize the methods of administration of recombinant human p53 adenovirus for different cancer types to provide a reference for future clinical trials.