GROWTH INHIBITION OF HUMAN LARYNGEAL CANCER CELL WITH THE ADENOVIRUS-MEDIATED p53 GENE

Objective: In most laryngeal cancers, the function of p53 gene is down regulated. To explore the potential use of p53 in gene therapy of laryngeal cancer, by introducing wild-type p53 into laryngeal cancer cell line via a recombinant adenoviral vector, Ad5CMV-p53 and analyzing its effects on cell and tumor growth. Methods: A human laryngeal cancer, cell line Hep-2 was used. Recombinant cytomegalovirus-promoted adenoviruses containing human wild-type p53 cDNA was transiently introduced into Hep-2 line. The growth suppression of the Hep-2 cells and established s.c. squamous, carcinoma model was examined. The p53 protein expression was detected using immunohistochemical analysis. Results: The transduction efficiencies of Hep-2 cell line were 100% at a multiplicity of 100 or greater. The p53 protein expression peaked on day 2 after infection and lasted far 5 days. In vitro growth assays revealed cell death following Ad5CMV-p53 infected. In vivo studies, Ad5CMV-p53 inhibited the tumorigenicity of Hep-2 cell, and in nude mice with established s.c. squamous, carcinoma, nodules showed that tumor volumes were significantly reduced in mice that received peritumoral infiltration of Ad5CMV-p53. Conclusion: Adenovirus-mediated antitumor therapy carrying the p53 gene is an efficient method to inhibit laryngeal cancer growth. Transfection of laryngeal cancer cells with the wild-type p53 gene via Ad5CMV-p53 is a potential novel approach to the therapy of laryngeal cancer.

rAd-p53联合放化疗治疗晚期头颈部鳞状细胞癌

p53基因是迄今为止发现与人类肿瘤相关性最高的基因，在头颈部癌中，p53基因在喉癌、上颌窦癌、唾液腺癌、鼻咽癌等中均可见到阳性表达，而以复制缺陷型重组腺病毒为载体的p53基因替代疗法作为一种肿瘤治疗的新方法在头颈部鳞状细胞癌（鳞癌）中取得了很好的疗效，且临床应用安全。自2006年3月至2006年12月，我科应用重组人p53腺病毒（recombinant adenovirus—p53，rAd—p53，商品名为今又生）注射液对14例局部晚期或复发性头颈部鳞癌联合放化疗进行治疗，获得了较好的近期疗效，报道如下。

The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines

AIM:There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status. METHODS:Human pancreatic cancer cell lines Capan-1^(p53mut), Capan-2^(p53wt),FAMPAC^(p53mut),PANC1^(p53mut),and rat pancreatic cancer cell lines AS^(p53wt) and DSL6A^(p53null) were used for in vitro studies.Following infection with different ratios of Ad- p53-particles (MOI) in combination with 5-FU,proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining).In addition,DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size,apoptosis (TUNEL) and survival were determined. RESULTS:Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53.In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU.Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU. CONCLUSION:Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function.These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.

RADIATION-INDUCED APOPTOSIS OF TWO NASOPHARANGEALCARCINOMA CELL LINES

Objective: To study apoptosis induced by radiation in two nasopharyngeal carcinoma (NPC) cell lines, CNE and CNE-2. Methods: Hoechst 33342 staining, immuno-histochemical staining, RT-PCR, DNA dot blotting and Southern blotting were used to identify apoptosis. Results: A single dose of X-irradiation resulted in apoptosis, the apoptotic index (AI) was time- and dose-dependent. Different apoptotic responses existed in the two cell lines. Immunohistochemical staining showed that bcl-2 protein was strongly positive in CNE but negative in CNE-2. However, RT-PCR revealed p53 mRNA in CNE-2 but not in CNE. P53 and bcl-2 genes were both present in the two cell lines as shown by DNA blotting, but the 2.8 kb fragment of the p53 gene was much lower than the 5.6 kb fragment on CNE which was clearly shown in Southern hybridization, suggestive of partial deletion of p53 gene in CNE. Conclusion: Apoptotic response to radiation is different in two NPC cell lines. CNE is more radioresistant than CNE-2. Overexpression of bcl-2 protein and partial deletion of p53 gene may explain their difference in radiosensitivity.

喉癌p53基因表达与放疗效果的关系

应用抗p53基因单克隆抗体，以免疫组织化学DACOCSASystem法染色技术，对73例喉鳞癌组织标本进行检测。结果显示，p53基因表达分为强度、中度表达及阴性表达。基因表达与放疗效果间有明显相关性，随着表达增强疗效相应提高。因此认为，可以把p53基因表达作为判定喉癌放疗预后及指导临床治疗的可靠指标。

人端粒酶逆转录酶启动子在裸鼠中增强人喉癌对基因放射治疗的敏感性

目的探索人端粒酶逆转录酶启动子（hTERTp）介导自杀基因辣根过氧化酶（HRP）-吲哚乙酸（IAA）系统联合射线，对相同来源而放射敏感性不同的人喉癌移植瘤模型的治疗作用。方法建奇相同来源而放射敏感性不同的人喉癌（Hep-2和Hep-2R细胞）移植瘤模型，并分为联合治疗组（A组和AR组）、基因治疗组（B组和BR组）、单纯放射组（C组和CR组）和对照组（D组和DR组）。瘤内注射脂质体包裹的质粒phTERTp—HRP，腹腔内注射IAA并联合放疗30Gy，观察对移植瘤生长的抑制作用。应用原位末端转移酶标记技术（TUNEL）检测肿瘤细胞的凋亡情况；应用AP法检测瘤内HRP蛋白的表达。结果裸鼠移植瘤生长以联合治疗组最慢，以对照组最快。A组的肿瘤抑制率为54．8％，B组为10．0％，C组为31．9％，AR组为52．7％，BR组为24．8％，CR组为17．0％。A组和AR组可见大量肿瘤细胞坏死和凋亡，凋亡指数分别为16．6％±1．3％和17．6％±1．3％，高于其他组（P〈0．05）。B组的HRP蛋白表达率（21．9％±5．7％）低于BR组和（33．3％±8．9％），辐射诱导后，A组和AR组的HRP蛋白表达率分别增加2．1和1．6倍（P〈0．05）。结论在不同放射敏感性的喉癌移植瘤模型中，hTERTp能被射线诱导，并根据瘤内端粒酶活性增强HRP基因的表达。hTERTp—HRP—IAA系统通过诱导肿瘤细胞凋亡和引起细胞坏死，抑制裸鼠移植瘤生长，并与射线协同作用，起到放射增敏作用。