Research at molecular and gene level on enhancement mechanisms combined with irradiation and hyperthermia in culture cell lines

辐射和热疗联合培养细胞系的增强机制的分子和基因水平研究

• 批准号: 15591277
• 负责人: KAWASAKI Shoji
• 金额: $ 2.18万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591277/
• 关键词: culture cell; heat shock protein; heating; western bloting; methylation; histone; KNK437; apoptosis; histone; HSP27; 温熱; 細胞分裂遅延; HSP; ヒストン; メチレーション; アセチレーション; cdc-2遺伝子; エピゼノイック; 扁平上皮癌; 核蛋白質

1 Relationship between radiation/or heating induced cell cycle disturbance and gene expression of 14-3-3a, cdc2 and 25 were studied in human cancer cells lines. When cells were heated for 20 to 40 min. at 45 degree, G2-block(G2 cells accumulation) were induced. At that time, decrease of cdc2 and increase of cdc25-P, was observed. When cells were released from G2-block, it was observed increase of cdc2 and decrease of cdc25-P.2 We established the method of synchronization of culture human cancer cells. Fractions of S phase cells in population reached about 90 percent 13 hours after stimulation of serum. It was suggested that this method is useful for experiments to determine gene expression after cells were exposed to several conditions.3 Using a pEYFP-Nuc vector, which contains nucleic acid sequences of a nuclear localization signal, we established a Jurkat-YN cell line that expressed enhanced yellow fluorescent protein (EYFP) in the nucleus. The nuclear forms visualized by EYFP were a … More lmost equal in quality to those visualized by SYTO59, a nucleic acid stain for living cells. Three-dimensional deformities in the nuclear form were observed during apoptosis before chromatin condensation became apparent, indicating these deformities are characteristic morphological changes of the early stage of apoptosis.4 We report here an analysis of the mechanisms of the inhibition by KNK437 in 2 human oral squamous cell carcinma cell lines whose heat sensitivities are different (HSC4; heat-resistant and KB; heat-sensitive) with a view to identifying which if any HSPs are targeted for KNK437. Combined administration of KNK437 to the medium after heat shock specifically inhibited the induction of HSPs including HSP27, HSP40, HSP70 and HSP90 in time dependence. The hyperthermic treatment combined with heating and KNK437 produced supra-reductive effects (i.e. HSP27 and HSP90 in HSC4, HSP27, HSP40 and HSP90 in KB). Careful examination of the ability of KNK437 to inhibit the induction of various HSPs may therefore be valuable in future attempts to improve the efficacy of oral cancer hyperthermic therapy.5 Heat-induced changes in histone H3 methylation especially for H3-Lys4 and H3-Lys9 methylation in combination with KNK-437 have been studied in two human oral cancer cell lines such as HSC4 and KB,. Heating of HSC4 cells at 45C for 20 min and KB cells for 3 min gradually increased H3-Lys4 and H3-Lys9 methylation. Treatment of both cells with KNK-437 before or after heat-treatment inhibited methylation of H3-Lys4. Our results indicate a possibility of alteration in histone structure by heat treatment as a result of change in the methylation of H3 histone. Use of KNK-437, an inhibitor of HSPs gene expression, inhibited methylation of H3 Lys4. From these results, there exist some mechanism to regulate the expressions of genes in DNA by heat treatment. Less

1研究了辐射/加热诱导的人癌细胞系细胞周期紊乱与14-3-3a、cdc 2和25基因表达的关系。当细胞在45 ℃下加热20至40分钟时，诱导G2阻滞（G2细胞积累）。此时，观察到cdc 2减少和cdc 25-P增加。当细胞从G2期阻滞释放时，观察到cdc 2的增加和cdc 25-P的减少。2我们建立了同步化培养人癌细胞的方法。血清刺激后13小时，S期细胞占细胞总数的90%左右。这表明，这种方法是有用的实验，以确定基因表达后，细胞暴露于几种条件。3使用pEYFP-Nuc载体，其中包含一个核定位信号的核酸序列，我们建立了一个Jurkat-YN细胞系，在核中表达增强型黄色荧光蛋白（EYFP）。EYFP显示的细胞核形态为： ...更多信息 l质量与SYTO 59（活细胞的核酸染色剂）显示的质量相同。在染色质浓缩变得明显之前，在细胞凋亡过程中观察到核形态的三维变形，表明这些变形是细胞凋亡早期阶段的特征性形态变化。4我们在这里报告了KNK 437对2种人口腔鳞状细胞癌细胞系的抑制机制的分析，其热敏感性不同（HSC 4;耐热和KB;热敏），以便鉴定哪些HSP（如果有的话）是KNK 437的靶向。KNK 437对热休克后的培养液中HSP 27、HSP 40、HSP 70和HSP 90的诱导有明显的抑制作用，且呈时间依赖性。与加热和KNK 437组合的热处理产生超还原效应（即HSC 4中的HSP 27和HSP 90，KB中的HSP 27、HSP 40和HSP 90）。因此，仔细检查KNK 437抑制各种HSP诱导的能力可能对未来提高口腔癌热疗疗效的尝试很有价值。5已在两种人口腔癌细胞系（如HSC 4和KB）中研究了热诱导的组蛋白H3甲基化变化，特别是H3-Lys 4和H3-Lys 9甲基化与KNK-437联合。在45 ℃加热HSC 4细胞20分钟和KB细胞3分钟逐渐增加H3-Lys 4和H3-Lys 9甲基化。在热处理之前或之后用KNK-437处理两种细胞抑制H3-Lys 4的甲基化。我们的结果表明，由于H3组蛋白甲基化的变化，热处理可能会改变组蛋白结构。HSPs基因表达抑制剂KNK-437的使用抑制了H3 Lys 4的甲基化。从这些结果来看，热处理对DNA中基因表达的调控存在一定的机制。少

项目成果

Kawasaki et al.: "pEYFP-Nuc vector is a useful tool for three-dimensional and time-lapse observation of nuclear morphology of Jurkat cells during apoptosis"Int J Mol Med.. 13(2). 235-242 (2004)

Kawasaki 等人：“pEYFP-Nuc 载体是对 Jurkat 细胞凋亡过程中核形态进行三维和延时观察的有用工具”Int J Mol Med.. 13(2)。

Effects of histone deacetylase inhibitor FR901228 on the expression level of telomerase reverse transcriptase in oral cancer

• DOI: 10.1007/s00280-004-0976-x
• 发表时间: 2005-07-01
• 期刊: CANCER CHEMOTHERAPY AND PHARMACOLOGY
• 影响因子: 3
• 作者: Murakami, J;Asaumi, J;Kishi, K
• 通讯作者: Kishi, K

Kawasaki S. et al.: "Effects of p53 gene therapy in radiotherapy or thermotherapy of human head and neck squamous cell carcinoma cell lines"Oncol Rep.. 10(3). 671-677 (2003)

Kawasaki S.等人：“p53基因治疗对人头颈鳞状细胞癌细胞系放射治疗或热疗的影响”Oncol Rep.. 10(3)。

Manganese superoxide dismutase overexpression changes plating efficiency bidirectionally according to change in redox for SaOS2 human osteosarcoma cell line.

• DOI: 10.3892/ijo.26.4.853
• 发表时间: 2005-04
• 期刊: International journal of oncology
• 影响因子: 5.2
• 作者: M. Komatsu;M. Kuroda;Yadi Wang;D. S. St. Clair;M. Urano;S. Akaki;J. Asaumi;S. Kawasaki;Y. Hiraki;S. Kanazawa

Effect of KNK437 on induction of Heat shock proteins with heating in human oral cancer cell lines

KNK437 对人口腔癌细胞系加热诱导热休克蛋白的影响

• 发表时间: 2006
• 期刊: JPN J Hyperthermic Oncology, 2006 22(2)
• 作者: Shin-ya Nakagawa;Keiji Matsuda;Tomoya Nakano.;Ganesh C.;Jagetia;Jun-ichi Asaumi;Shoji Kawasaki
• 通讯作者: Shoji Kawasaki