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 在人类癌细胞系中研究了辐射/或加热诱导的细胞周期紊乱与 14-3-3a、CDC2 和 25 基因表达之间的关系。当细胞在 45 度下加热 20 至 40 分钟时，G2 阻断（。诱导G2细胞积累，此时观察到cdc2减少和cdc25-P增加。当细胞从G2-块释放时，观察到cdc2增加。和CDC25-P.2的减少我们建立了同步培养人类癌细胞的方法，在血清刺激后13小时，群体中S期细胞的分数达到约90%，这表明该方法对于确定实验是有用的。细胞暴露于多种条件后基因表达。 3 使用包含核定位信号核酸序列的 pEYFP-Nuc 载体，我们建立了 Jurkat-YN 细胞系，该细胞系在细胞中表达增强型黄色荧光蛋白 (EYFP) EYFP 可视化的核形态与 SYTO59 可视化的核形态几乎相同，SYTO59 是一种活细胞核酸染色剂。这些畸形是细胞凋亡早期阶段的特征性形态学变化。4 我们在此报告了 KNK437 在 2 种人口腔鳞状细胞癌细胞系中的抑制机制的分析，这些细胞系热敏感性不同（HSC4；耐热；KB；热敏感），以鉴定哪些 HSP 是 KNK437 的靶标。 HSP40、HSP70 和 HSP90 具有时间依赖性。热疗结合加热和 KNK437 产生超还原效应。 （即，HSC4 中的 HSP27 和 HSP90，KB 中的 HSP27、HSP40 和 HSP90）因此，仔细检查 KNK437 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计划有价值。5 热-诱导组蛋白 H3 甲基化的变化，尤其是 H3-Lys4 和 H3-Lys9 甲基化与KNK-437 已在两种人类口腔癌细胞系（例如 HSC4 和 KB）中进行了研究，将 HSC4 细胞在 45°C 加热 20 分钟，将 KB 细胞加热 3 分钟，逐渐增加两种细胞的 H3-Lys4 和 H3-Lys9 甲基化。在热处理之前或之后使用 KNK-437 抑制 H3-Lys4 的甲基化。我们的结果表明热处理可能会改变组蛋白结构。由于H3组蛋白甲基化的改变，使用HSPs基因表达抑制剂KNK-437抑制了H3 Lys4的甲基化。从这些结果来看，存在一些通过热处理调节DNA中基因表达的机制。 。较少的

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

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

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)。

Kawasaki S. et al.: "65 H89, a protein kinase inhibitor, promotes the development of thermorolerance in"Jap.J.of Hyperthermia Oncol. 19(4). 187-194 (2003)

Kawasaki S. 等人：“65 H89，一种蛋白激酶抑制剂，促进耐热性的发展”Jap.J.of Hyperthermia Oncol。