TY - JOUR
T1 - Investigations of Mechanisms of Drug-induced Changes in Gene Expression
T2 - N-Methylformamide-induced Changes in Synthesis of the Mr 72,000 Constitutive Heat Shock Protein during Commitment of HL-60 Cells to Granulocyte Differentiation
AU - Beere, Helen M.
AU - Morimoto, Richard I.
AU - Hickman, John A.
PY - 1993/7
Y1 - 1993/7
N2 - 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 Mr 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-Mr 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 dim 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.
AB - 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 Mr 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-Mr 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 dim 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.
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M3 - Article
C2 - 8319210
AN - SCOPUS:0027304211
SN - 0008-5472
VL - 53
SP - 3034
EP - 3039
JO - Cancer Research
JF - Cancer Research
IS - 13
ER -