Beere H M, Morimoto R I, Hickman J A
Cancer Research Campaign Molecular and Cellular Pharmacology Group, School of Biological Sciences, University of Manchester, United Kingdom.
Cancer Res. 1993 Jul 1;53(13):3034-9.
HL-60 cells were treated with the differentiating agent N-methylformamide and early changes in gene expression and protein content were investigated. Analysis of protein synthesis had previously shown an early (< 12 h) fall in the synthesis of M(r) 70,000 heat shock proteins (F. M. Richards, A. Watson, and J. A. Hickman. Cancer Res., 48: 6715-6720, 1988). The changes have now been characterized in detail and their kinetics compared to those of the expression of the c-myc protein. Immunoanalysis, using antibodies to either the stress-inducible heat shock protein hsp70 (4G4) or a pan-M(r) 70,000 heat shock protein antibody (3A3), showed that there was a striking reduction in the levels of the constitutive heat shock protein hsc70 when cells were incubated continuously with 170 mM N-methylformamide. A reduction in the level of hsc70 RNA was observed within 3 h and continued thereafter. In contrast, transcription of the hsc70 gene was induced within 1-2 h, after which the rate returned to basal level. There were no significant changes in the rate of transcription of the stress-inducible heat shock proteins hsp70 or hsp90. When N-methylformamide was removed from the cells, prior to commitment to differentiation, the levels of hsc70 were reestablished, whereas after 36 h of treatment there was no recovery. Western blotting with an antibody to the c-myc protein showed this to fall to virtually undetectable levels by 3 h under the same conditions. The results suggest that the loss of hsc70, which may perform a protein chaperoning role, was mediated at both transcriptional and posttranscriptional levels of regulation and was an early event closely associated with the commitment of HL-60 cells to differentiation. The fall in hsc70 was not associated with alterations in the cell cycle, nor were the kinetics of the change suggestive of a relationship with the decrease in content of c-myc protein.
用分化剂N-甲基甲酰胺处理HL-60细胞，并研究基因表达和蛋白质含量的早期变化。先前对蛋白质合成的分析表明，早期（<12小时）分子量为70,000的热休克蛋白的合成下降（F.M.理查兹、A.沃森和J.A.希克曼。《癌症研究》，48:6715 - 6720，1988）。现在已详细表征了这些变化，并将其动力学与c-myc蛋白表达的动力学进行了比较。使用针对应激诱导型热休克蛋白hsp70（4G4）或泛分子量70,000热休克蛋白抗体（3A3）的抗体进行免疫分析表明，当细胞与170 mM N-甲基甲酰胺连续孵育时，组成型热休克蛋白hsc70的水平显著降低。在3小时内观察到hsc70 RNA水平下降，并在此后持续下降。相反，hsc70基因的转录在1 - 2小时内被诱导，之后速率恢复到基础水平。应激诱导型热休克蛋白hsp70或hsp90的转录速率没有显著变化。当在细胞承诺分化之前去除N-甲基甲酰胺时，hsc70的水平得以恢复，而在处理36小时后则没有恢复。用针对c-myc蛋白的抗体进行蛋白质印迹分析表明，在相同条件下，到3小时时c-myc蛋白几乎降至不可检测的水平。结果表明，可能发挥蛋白质伴侣作用的hsc70的丧失是在转录和转录后调控水平介导的，并且是与HL-60细胞承诺分化密切相关的早期事件。hsc70的下降与细胞周期的改变无关，其变化动力学也不表明与c-myc蛋白含量的降低存在关系。
