Investigations of mechanisms of drug-induced changes in gene expression: N-methylformamide-induced changes in synthesis of the M(r) 72,000 constitutive heat shock protein during commitment of HL-60 cells to granulocyte differentiation.

Abstract

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.