HSV-1 amplicon vector-mediated expression of ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and radiation sensitivity. Previous research has shown that it is possible to correct the hereditary deficiency A-T by DNA transfection in cell culture, but the large size of the ATM cDNA (9 kb) limits the use of many vector types for gene replacement. HSV-1 amplicon vectors provide a means to deliver large genes to cells efficiently and without toxicity. In this study, the FLAG-tagged cDNA for human ATM was inserted into an HSV-1 amplicon under control of the CMV promoter (designated as HGC-ATM). FLAG-ATM expression was confirmed in 293T/17 cells and human A-T fibroblasts (GM9607) after transduction, by immunoprecipitation, Western analysis, and immunocytochemistry. Functional recovery was assessed by two independent assays. First, in vitro kinase assay showed that vector-derived ATM in GM9607 cells could successfully phosphorylate wt p53 using recombinant GST-p53(1-101). Second, in A-T cells infected with the HGC-ATM vector, the extent of accumulation in G2/M phase at 24 h postirradiation was similar to that observed in cells with wild-type endogenous ATM and lower than that observed in A-T cells infected with a control vector. Thus, these vectors provide a tool to test the feasibility of HSV-amplicons as gene therapy vectors for A-T.     

p38MAPK对肿瘤细胞ATM、ATR和p53信号通路的影响

目的 研究p38MAPK对细胞周期信号转导通路中重要蛋白激酶ATM、ATR、P53的影响并探讨其可能的机制.方法 采用人类结肠癌细胞株HT-29和肺癌细胞株SPAC-1进行研究,分别使用紫外线（UV）刺激和p38MAPK特异性抑制剂SB203580抑制肿瘤细胞中的p38MAPK,通过Western blot技术检测ATM、ATR、P53蛋白磷酸化情况,同时用real-time PCR技术检测细胞中相应基因的表达情况.结果 紫外线能上调ATM、ATR、p53和p38MAPK基因的表达,使用p38MAPK特异抑制剂SB203580能够使ATM、ATR和p53的表达降低.结论 P38MAPK可以调控ATM、ATR和p53的表达,ATM-Chk2信号通路和p38MAPK信号通路之间可能存在交互联系.     

Effects of cisplatin, 5-fluorouracil, and radiation on cell cycle regulation and apoptosis in the hypopharyngeal carcinoma cell line

In head and neck cancer including hypopharyngeal cancer, cisplatin and 5-fluorouracil (5-FU) usually have been used as neoadjuvant chemotherapeutic agents. We investigated the effects of cisplatin, 5-FU and radiation on p53 protein expression and cell responses (cell cycle arrest and/or apoptosis) in the hypopharyngeal carcinoma cell line (PNUH-12; mutant type p53). PNUH-12 cells were treated with cisplatin, 5-FU and radiation. The changes in the cells were assessed by a cell cytotoxicity assay, Western blotting (p53 and p21(WAF1/CIP1) proteins), a DNA fragmentation assay, propidium iodide (PI) staining and DNA flow cytometry. The expression of p53 protein was increased after treatment with cisplatin and 5-FU, but not radiation. The expression of p21(WAF1/CIP1) protein was increased only after treatment with 5-FU, not cisplatin or radiation. With cisplatin and radiation, we observed apoptosis both by DNA fragmentation and PI staining and increased S phase in cisplatin and G2 phase in radiation by DNA flow cytometry. But, with 5-FU, we could not observe apoptosis by DNA fragmentation and PI stain but only an increased G1 phase by DNA flow cytometry. In PNUH-12, radiation induced p53-independent apoptosis and p21(WAF1/CIP1)-independent G2-phase cell cycle arrest. Cisplatin induced p53-dependent apoptosis and p21(WAF1/CIP1)-independent S-phase cell cycle arrest and 5-FU induced p53 and p21(WAF1/CIP1)-dependent G1-phase cell cycle arrest, not apoptosis. Cisplatin and 5-FU induced p53-dependent pathways, but radiation p53-independent pathway. The cell responses by cisplatin, 5-FU and radiation were all different pathways.     

p53和ATM基因缺失在慢性淋巴细胞白血病患者中的预后价值

目的 探讨p53和ATM基因缺失在慢性淋巴细胞白血病患者(CLL)中的预后价值.方法 采用间期荧光原位杂交(FISH)技术和p53、ATM基因的序列特异性DNA探针对80例CLL患者的染色体标本进行p53和ATM基因缺失的检测,分析p53和ATM基因缺失与外周血淋巴细胞绝对计数、Binet分期、血清乳酸脱氢酶(LDH)水平、β2微球蛋白(β2-MG)水平、ZAP-70表达、CD38表达、含氟达拉滨治疗疗效之间的相关性,单因素生存分析采用Kaplan-Meier法绘制生存曲线和Log-rank检验,多因素生存分析采用COX多元回归分析.结果 80例CLL患者中14例(17.5%)伴有p53基因缺失,9例(11.3%)伴有ATM基因缺失,其中3例(3.8%)同时伴有p53和ATM基因缺失.p53和ATM基因缺失与患者性别、年龄、Binet分期、外周血淋巴细胞绝对计数、血清LDH及β2-MG、ZAP-70表达水平无明显相关性;CD38高表达组中,p53和ATM基因缺失的发生率均明显高于CD38低表达组,且差异有统计学意义(P=0.025和P=0.001).41例患者接受含氟达拉滨方案治疗,32例不伴有p53和(或)ATM基因缺失的患者中,12例(37.5%)获得完全缓解(CR),9例伴有p53和(或)ATM基因缺失的患者均未获得CR(P=0.047).单因素生存分析显示,伴有p53基因缺失组的生存期明显较不伴有缺失组短,经Log-rank检验差异有统计学意义(P＜0.01);而伴有与不伴有ATM基因缺失组的生存期差异无统计学意义(P=0.556).COX多元回归分析显示,p53基因缺失(P:0.014)和CD38表达水平(P=0.017)是判断CLL预后的独立因素.结论 伴有p53和(或)ATM基因缺失的患者,接受含氟达拉滨方案治疗疗效差,FISH检测p53和ATM基因缺失在CLL预后判断和指导临床治疗中具有重要价值.     

Tumor-targeted p53-gene therapy enhances the efficacy of conventional chemo/radiotherapy.

A long-standing goal in gene therapy for cancer is a stable, low toxic, systemic gene delivery system that selectively targets tumor cells, including metastatic disease. Progress has been made toward developing non-viral, pharmaceutical formulations of genes for in vivo human therapy, particularly cationic liposome-mediated gene transfer systems. Ligand-directed tumor targeting of cationic liposome-DNA complexes (lipoplexes) is showing promise for targeted gene delivery and systemic gene therapy. Lipoplexes directed by ligands such as folate, transferrin or anti-transferrin receptor scFv, showed tumor-targeted gene delivery and expression in human breast, prostate, head and neck cancers. The two elements, ligand/receptor and liposome composition, work together to realize the goal of functional tumor targeting of gene therapeutics. The tumor suppressor gene, p53, has been shown to be involved in the control of DNA damage-induced apoptosis. Loss or malfunction of this p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy or radiation. The systemically delivered ligand-liposome-p53 gene therapeutics resulted in efficient expression of functional wild-type p53, sensitizing the tumors to chemotherapy and radiotherapy. This is a novel strategy combining current molecular medicine with conventional chemotherapy and radiotherapy for the treatment of cancer. The systemic delivery of normal tumor suppressor gene p53 by a non-viral, tumor-targeted delivery system as a new therapeutic intervention has the potential to critically impact the clinical management of cancer.     

A multifunctional PEI-based cationic polyplex for enhanced systemic p53-mediated gene therapy

We recently reported a novel coupling strategy involving salicylhydroxamic acid and phenyl(di)boronic acid molecules to attach the CNGRC peptide to PEI/DNA for CD13 targeting in tumors. Here, we doubly coupled Simian Virus (SV) 40 peptide-(nuclear localization signal)) and oligonucleotide-based (DNA nuclear targeting signal) nuclear signals to the same vector using peptide nucleic acid chemistry. This vector, CNGRC/PEG/PEI/DNA-betagal/NLS/DNTS, was predominantly localized in the cell nucleus, yielding about 200-fold higher betagal gene expression in vitro, more than 20-fold increase in tumor-specific gene delivery, and a robust betagal gene expression as demonstrated in stained tumor sections. For gene therapy purposes, we further engineered a similar targeting polyplex, CNGRC/PEG/PEI/DNA-p53/NLS/DNTS, with EBV-based episomal vector for sustained p53 gene expression. A distribution of vector DNA and apoptosis in p53-containing tumors was observed, yielding a significant tumor regression and 95% animal survival after 60 days. This multicomponent vector also co-targeted tumor and tumor-associated endothelial cells but not normal cells, and had more efficient therapeutic index than each vector administered as a single modality. The use of an efficient coupling strategy without compromising the vector's integrity for DNA condensation and endosomal escape; nuclear import; tumor-specific and persistent p53 gene expression clearly provides a basis for developing a single combinatorial approach for non-viral gene therapy.     

p53R2 inhibits the proliferation of human cancer cells in association with cell-cycle arrest

Deregulation of the expression of p53R2, a p53-inducible homologue of the R2 subunit of ribonucleotide reductase, has been found in various human cancer tissues; however, the roles p53R2 plays in cancer progression and malignancy remain controversial. In the present study, we examined changes in gene expression profiles associated with p53R2 in cancer cells, using the analysis of cDNA microarray. Gene set enrichment analysis identified that the gene set regulating cell-cycle progression was significantly enriched in p53R2-silencing human oropharyngeal carcinoma KB cells. Attenuation of p53R2 expression significantly reduced p21 expression and moderately increased cyclin D1 expression in both wild-type p53 cancer cells (KB and MCF-7) and mutant p53 cancer cells (PC3 and MDA-MB-231). Conversely, overexpression of p53R2-GFP resulted in an increase in the expression of p21 and decrease in the expression of cyclin D1, which correlated with reduced cell population in S-phase in vitro and suppressed growth in vivo. Furthermore, the MAP/ERK kinase inhibitor PD98059 partially abolished modulation of p21 and cyclin D1 expression by p53R2. Moreover, under the conditions of nonstress and adriamycin-induced genotoxic stress, attenuation of p53R2 in KB cells significantly increased phosphorylated H2AX, which indicates that attenuation of p53R2 may enhance DNA damage induced by adriamycin. Overall, our study shows that p53R2 may suppress cancer cell proliferation partially by upregulation of p21 and downregulation of cyclin D1; p53R2 plays critical roles not only in DNA damage repair but also in proliferation of cancer cells.     

Adenovirus-mediated wild-type p53 radiosensitizes human tumor cells by suppressing DNA repair capacity

Functional inactivation of the p53 gene and robust DNA repair capacity may be among the salient causes of radioresistance in tumor cells. We expressed the wild-type (wt) p53 gene in a p53-mutant human epidermoid carcinoma cell line, A431, using an adenoviral vector [adenovirus-p53 (Ad-p53), INGN 201], examined its radiosensitivity, and correlated p53 status and radiosensitivity with cellular repair functions. Using clonogenic survival assays and the terminal deoxynucleotidyl transferase-mediated nick end labeling assay for apoptosis, we demonstrated that preirradiation treatment with Ad-p53 significantly increased the radiosensitivity of A431 cells over controls. Induction of p53 expression using a construct where p53 expression was under the control of an inducible promoter also significantly increased radiosensitivity of H1299 lung tumor cells, which are otherwise null for p53. These results did not correlate with radiation-induced apoptosis but did correlate with functional impairment of DNA repair and suppressed expression of several repair-related genes, such as Ku70, DNA-dependent protein kinase, ataxia telangiectasia mutated, and X-ray-sensitive complementation group 4. Normal human fibroblast MRC-9 cells showed no impairment in the repair capability due to Ad-p53 despite the suppression of some repair genes. Expression of Ku70, which is known to mediate diverse cellular functions, correlated with the differential effects of p53 on radiosensitivity in the normal and tumor cells.     

Histone deacetylase inhibitor FK228 enhances adenovirus-mediated p53 family gene therapy in cancer models

Therapeutic replacement of the wild-type p53 gene has been pursued as a potential gene therapy strategy in a variety of cancer types; however, some cancer models are resistant to p53 in vivo and in vitro. Therefore, to improve p53 gene therapy, it is important to overcome the resistance to p53-mediated apoptosis. Histone deacetylase inhibitors are a novel class of chemotherapeutic agents that are able to reverse the malignant phenotype of transformed cells. A natural histone deacetylase inhibitor, FK228, is reported to enhance adenovirus infection due in part to the up-regulation of coxsackievirus adenovirus receptor expression. In this study, preclinical experiments were done to establish a mechanistic rationale for the combination of adenovirus-mediated p53 family gene transfer and FK228 pretreatment in future clinical trials. Pretreatment with FK228 enhanced apoptosis in human cancer cells through enhanced transduction of Ad-p53. FK228 also induced hyperacetylation of the p53 protein and specifically enhanced p53-mediated Noxa expression. Additionally, the combination of FK228 and Ad-p53 induced Bax translocation to the mitochondria. The double knockdown of Bax and Noxa expression by small interfering RNA antagonized the synergistic effect of Ad-p53 and FK228 on apoptosis induction. In human cancer xenograft models, FK228 significantly increased the therapeutic effectiveness of p53 as well as p63 gene therapy. These results provide a strong rationale for combining p53 gene therapy and FK228 pretreatment in cancer therapy.     

Adenovirus p53 gene therapy

To date, dysfunctional tumour suppressor genes are the most common genetic lesions identified in human cancers. Functional copies of tumour suppressor genes can be introduced into cancer cells by gene transfer using adenoviral vectors. This approach has been extensively studied in the clinic with intratumoural injection of a replication-defective adenovirus that expresses p53 (Ad-p53). Overexpression of p53 in cancer cells induces growth arrest and apoptosis. Ad-p53 injections have an excellent safety profile, and have mediated tumour regression and growth arrest as monotherapy, or have overcome resistance or increased the effectiveness of radiation therapy and chemotherapy. Expression of the p53 transgene has occurred at high levels and is associated with the activation of other genes in the p53 pathway. These studies indicate proof-of-principle for tumour suppressor gene therapy and represent a new paradigm in targeted therapy.     

Functional expression of ATM gene carried by HSV amplicon vector in vitro and in vivo

Ataxia-telangiectasia (AT) is a human autosomal recessive disease with a pleiotropic phenotype characterized by cerebellar degeneration, immunodeficiency, premature aging, cancer predisposition, and radiation sensitivity. The gene mutated in AT, ATM (for AT-mutated), had been cloned and found to have ionizing radiation and oxidative stress-inducible kinase activity. No treatment can stop the progression of the disease. In this study, the complete open-reading frame of ATM cDNA was cloned into a Herpes simplex virus type-1 (HSV-1) amplicon vector (pTO-ATM), and the transduction of cultured AT cells was demonstrated by immunohistochemistry and Western blot analysis. Functional gene expression was evaluated by cell colony-forming assays following exposure to oxidative stress. The survival of AT cells with ATM gene transduction was about 100% higher compared to nontransduced cells after t-butyl hydroperoxide treatments. Next, the normal ATM gene expression in different regions of the rat brain was studied. Immunohistochemistry staining demonstrated weak endogenous ATM protein expression in neurons of the caudate-putamen, with significantly higher levels of expression detected in neurons in other brain regions. Exogenous ATM gene expression from pTO-ATM after viral transduction in the caudate-putamen of the adult rat was examined. At 3 days after injection of the pTO-ATM viral vector, abundant positive ATM staining of the neurons was found at the injection sites, in comparison to the controls. These data demonstrate that the relatively large ATM cDNA can be transduced and expressed in vitro and in vivo from an HSV amplicon viral vector. These data provide initial evidence that the replacement of the ATM gene into the cells of AT patients might be possible some day.     

Transferrin-liposome-mediated systemic p53 gene therapy in combination with radiation results in regression of human head and neck cancer xenografts

The use of cationic liposomes as nonviral vehicles for the delivery of therapeutic molecules is becoming increasingly prevalent in the field of gene therapy. We have previously demonstrated that the use of the transferrin ligand (Tf) to target a cationic liposome delivery system resulted in a significant increase in the transfection efficiency of the complex [Xu, L., Pirollo, K.F., and Chang, E.H. (1997). Hum. Gene Ther. 8, 467-475]. Delivery of wild-type (wt) p53 to a radiation-resistant squamous cell carcinoma of the head and neck (SCCHN) cell line via this ligand-targeted, liposome complex was also able to revert the radiation resistant phenotype of these cells in vitro. Here we optimized the Tf/liposome/DNA ratio of the complex (LipT) for maximum tumor cell targeting, even in the presence of serum. The efficient reestablishment of wtp53 function in these SCCHN tumor cells in vitro, via the LipT complex, restored the apoptotic pathway, resulting in a significant increase in radiation-induced apoptosis that was directly proportional to the level of exogenous wtp53 in the tumor cells. More significantly, intravenous administration of LipT-p53 markedly sensitized established SCCHN nude mouse xenograft tumors to radiotherapy. The combination of systemic LipT-p53 gene therapy and radiation resulted in complete tumor regression and inhibition of their recurrence even 6 months after the end of all treatment. These results indicate that this tumor-specific, ligand-liposome delivery system for p53 gene therapy, when used in concert with conventional radiotherapy, can provide a new and more effective means of cancer treatment.     

Correction of a genetic defect in multipotent germline stem cells using a human artificial chromosome

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including regulatory elements. Multipotent germline stem (mGS) cells have a great potential for gene therapy because they can be generated from an individual's testes, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we herein report the functional restoration of a genetic deficiency in mouse p53-/- mGS cells, using a HAC with a genomic human p53 gene introduced via microcell-mediated chromosome transfer. The p53 phenotypes of gene regulation and radiation sensitivity were complemented by introducing the p53-HAC and the cells differentiated into several different tissue types in vivo and in vitro. Therefore, the combination of using mGS cells with HACs provides a new tool for gene and cell therapies. The next step is to demonstrate functional restoration using animal models for future gene therapy.     

稳定表达反义ATM／PI3K细胞系U937—ASPI3K的建立

为进一步研究DNA损伤杀灭肿瘤细胞的机制，为增强肿瘤治疗疗效提供一种有价值的细胞模型，构建了高效表达ATM基因编码羧基端100kD功能片段的反义cDNA，通过转染和筛选建立稳定表达反义ATM/PI3K cDNA的细胞系U937-ASPI3K。结果显示，构建的ATM/PI3K cDNA经测序证明反向定位于表达载体pZeoSV2( )中。经转染和筛选得到稳定表达pZeoSV( )-ATM/PI3K的细胞系U937-ASPI3K和对照细胞系U937-pZeoSV(-)，前ATM蛋白表达极度受抑制，后以及淋巴瘤细胞系U937ATM蛋白的高表达未受影响。本实验为阐明细胞周期关卡（checkpoint)在DNA损伤信号传导通路中的作用机制，探索肿瘤治疗新方法提供了一种实用的细胞模型。