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

1研究人癌细胞系辐射/加热诱导的细胞周期紊乱与14-3-3a、cdc2、25基因表达的关系。当细胞加热20~40min时。在45度时，诱导了G2期阻滞(G2细胞积聚)。CDc2降低，CDC25-P升高。当细胞从G2期阻滞中释放时，cdc2增加，cdc25-p2减少。血清刺激13h后，群体中S期细胞比例达到90%左右。3利用含有核定位信号的核酸序列的pEYFP-Nuc载体，建立了在细胞核内表达增强型黄色荧光蛋白(EYFP)的Jurkat-YN细胞系。EYFP所显示的核形态为…在质量上更接近于SYTO59，一种活细胞的核酸染色。在染色质凝聚变得明显之前，在细胞凋亡过程中观察到了核形态的三维变形，表明这些变形是早期凋亡的典型形态变化。4我们报告了KNK437对2种不同热敏性(HSC4；耐热性和KB；热敏性)人口腔鳞状细胞癌细胞株的抑制机制，以确定如果有HSPs针对KNK437的话。热休克后联合应用KNK437可明显抑制HSP27、HSP40、HSP70和HSP90的诱导，并呈时间依赖性。高温联合加热和KNK437可产生超还原效应(即HSC4中的HSP27和HSP90，KB中的HSP27、HSP40和HSP90)。因此，仔细检测KNK437抑制各种热休克蛋白诱导的能力，对于未来尝试提高口腔癌热疗的疗效可能是有价值的。5在两种人类口腔癌细胞系，如HSC4和KB，研究了热诱导的组蛋白H3甲基化的变化，特别是H3-Lys4和H3-Lys9甲基化与KNK-437的结合。HSC4细胞在45℃加热20min，KB细胞加热3min，H3-Lys4和H3-Lys9甲基化逐渐增加。在热处理前后用KNK-437处理这两种细胞均可抑制H3-Lys4的甲基化。我们的结果表明，由于H3组蛋白甲基化的改变，热处理可能会改变组蛋白的结构。使用HSPs基因表达的抑制剂KNK-437可以抑制H3Lys4的甲基化。由此可见，热处理对DNA中基因表达的调控可能存在一定的机制。较少

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