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

1研究了辐射/加热诱导的细胞周期紊乱与人癌细胞14-3-3 3a、cdc2和25基因表达的关系。45度加热20 ~ 40 min，诱导G2细胞阻滞（G2细胞积累）。同时观察到cdc2降低，cdc25-P升高。释放G2-block后，cdc2升高，cdc25- p2降低建立了同步培养人癌细胞的方法。血清刺激13小时后，群体中S期细胞的分离率达到90%左右。结果表明，该方法可用于测定细胞在不同条件下的基因表达利用含有细胞核定位信号核酸序列的pEYFP-Nuc载体，我们建立了在细胞核中表达增强黄色荧光蛋白（EYFP）的Jurkat-YN细胞系。EYFP显示的细胞核形态与SYTO59（活细胞核酸染色）显示的细胞核形态在质量上几乎相等。在细胞凋亡过程中，在染色质凝结之前，细胞核形态出现了三维变形，表明这些变形是细胞凋亡早期的特征性形态学变化我们在此报道了KNK437对2种热敏性不同（HSC4，耐热和KB，热敏）的人口腔鳞状细胞癌细胞系的抑制机制分析，以确定哪些HSPs是KNK437的靶标。热休克后联合给药KNK437对HSP27、HSP40、HSP70和HSP90的诱导有明显的时间依赖性。高温处理联合加热和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抑制各种热敏感蛋白诱导的能力，可能对未来尝试提高口腔癌热疗的疗效有价值热诱导组蛋白H3甲基化的变化，特别是H3- lys4和H3- lys9甲基化与KNK-437的结合，已经在两种人类口腔癌细胞系如HSC4和KB中进行了研究。HSC4细胞在45℃下加热20分钟，KB细胞加热3分钟，H3-Lys4和H3-Lys9的甲基化逐渐增加。在热处理前后用KNK-437处理两种细胞均可抑制H3-Lys4的甲基化。我们的研究结果表明，由于H3组蛋白甲基化的改变，热处理可能会改变组蛋白结构。使用HSPs基因表达抑制剂KNK-437，可以抑制H3 Lys4的甲基化。由此可见，热处理对DNA中基因表达的调控存在一定的机制。少

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