Molecular Chaperone Inducers Facilitate the Functional Restoration of Temperature-sensitive Mutant p53 Protein

D. Kawashima, Minoru Soga, Rika Takeuchi, H. Matsumoto, K. Ohtsuka
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引用次数: 2

Abstract

The tumor suppressor gene p53 encodes a transcription factor and is known to be the most frequently mutated gene (approximately 50%) in human cancer. The functional restoration of mutant p53 protein is considered to be one type of anticancer treatment ; and some chemical compounds, including CP-31398, PRIMA-1 (p53 reactivation and induction of massive apoptosis), and glycerol, have been shown to restore its function. We here investigated whether molecular chaperone inducers such as carbenoxolone (CBX), paeoniflorin (PF), and sodium salicylate (SA) could restore the functional defect of a temperature-sensitive mutant p53 protein (V143A). Functional restoration of p53 was detected by the induction of wild-type p53 activated fragment 1 (WAF1) and mouse double minute 2 (MDM2), both of which are gene products transactivated by an active p53. When H1299/tsp53 cells were cultured continuously at a nonpermissive temperature (37°C), no apparent expression of WAF1 and MDM2 was observed. Upon the temperature shift-down from 37°C to a permissive temperature (32°C), WAF1 and MDM2 gradually accumulated in the cells at 6 to 12 h later, probably owing to the gradual appearance of wild-type p53. When the cells were treated with molecular chaperone inducers at 37°C and then the temperature was shifted down, WAF1 and MDM2 appeared much earlier at 3 to 6 h, and also in much higher amounts than those in the control cells. Inhibition of molecular chaperone induction by quercetin or heat shock factor 1 (HSF1) siRNA diminished the facilitative effect of molecular chaperone inducers. Also, long-term overexpression (48 h) of molecular chaperones by CBX led to the accumulation of wild-type p53 even at 37°C. These results suggested that moderately overexpressed molecular chaperones could facilitate the correct folding and functional restoration of mutant p53 protein.
分子伴侣诱导剂促进温度敏感突变p53蛋白的功能恢复
肿瘤抑制基因p53编码一种转录因子,是人类癌症中最常见的突变基因(约50%)。突变型p53蛋白的功能恢复被认为是一种抗癌治疗方法;一些化合物,包括CP-31398, PRIMA-1 (p53的再激活和诱导大量凋亡)和甘油,已被证明可以恢复其功能。我们在这里研究了分子伴侣诱诱剂如卡贝诺洛酮(CBX)、芍药苷(PF)和水杨酸钠(SA)是否可以恢复温度敏感突变体p53蛋白(V143A)的功能缺陷。通过诱导野生型p53激活片段1 (WAF1)和小鼠双分钟2 (MDM2)来检测p53的功能恢复,这两种基因都是被活性p53反激活的基因产物。H1299/tsp53细胞在非允许温度(37℃)下连续培养,未观察到WAF1和MDM2的明显表达。当温度从37℃降至允许温度(32℃)时,WAF1和MDM2在6 ~ 12 h后逐渐在细胞中积累,这可能是野生型p53逐渐出现的原因。37℃分子伴侣诱导剂处理细胞后,温度降低后,在3 ~ 6 h时,WAF1和MDM2的出现时间明显早于对照细胞,出现量也明显高于对照细胞。槲皮素或热休克因子1 (HSF1) siRNA对分子伴侣诱导的抑制作用减弱了分子伴侣诱导剂的促进作用。此外,CBX长期过表达分子伴侣(48 h)导致野生型p53即使在37°C下也会积累。这些结果表明,适度过表达的分子伴侣可以促进突变p53蛋白的正确折叠和功能恢复。
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