Hoechst 33342-induced apoptosis is associated with intracellular accumulation of E2F-1 protein in BC3H-1 myocytes and HL-60 cells.
BACKGROUND
Hoechst 33342 induces apoptosis, inhibits topoisomerase I, and disrupts TATA box-binding protein/TATA box element binding in BC3H-1 myocytes and HL-60 cells. In contrast, Hoechst 33258 does not have any of these actions.


OBJECTIVE
To determine if Hoechst 33342 or Hoechst 33258 treatment of BC3H-1 myocytes or HL-60 cells is associated with the intracellular accumulation of the nuclear transcription factor E2F-1, known to induce apoptosis.


METHODS
The gel mobility shift assay was used to study the effect of the 2 compounds on the binding capacity of nuclear proteins extracted from the 2 cell lines to a 30-base pair double-stranded oligonucleotide that contained an E2F-1-binding element. The DNA sequence of the protein-binding region was determined by the protection footprinting method and the Maxam-Gilbert guanosine plus adenosine chemical sequencing reaction.


RESULTS
Nuclear extracts from each cell line treated with 26.7 micromol/L Hoechst 33342 or Hoechst 33258 for 3 to 24 hours were incubated with [32P]-labeled 30-base pair oligonucleotide (5'GGCGCGGAGACTTGGAGAAATTTGGCGCGG3'). Three protein and DNA bands were altered by Hoechst 33342, but not by Hoechst 33258: band I, increased, then decreased in both cell lines; band II (2 adjacent bands) markedly decreased in both cell lines; band III markedly increased only in HL-60 cells. Footprinting and sequencing demonstrated that the nuclear protein-binding sequence was TTTGGCGC, an E2F-1 binding site. Hoechst 33342 treatment increased the concentration of E2F-1 protein after a 3-hour incubation in both cell lines.


CONCLUSIONS
Hoechst 33342-induced apoptosis is associated with intracellular accumulation of E2F-1 protein, another step in this specific apoptotic pathway.
Zhang
X
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Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Mich 48073-6769, USA.
Kiechle
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001483:Base Sequence; D001562:Benzimidazoles / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics; D006690:Bisbenzimidazole / Q000494:pharmacology; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D056465:Retinoblastoma-Binding Protein 1; D059004:Topoisomerase I Inhibitors; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*
C533431:Arid4a protein, mouse; D001562:Benzimidazoles; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D009838:Oligodeoxyribonucleotides; D056465:Retinoblastoma-Binding Protein 1; D059004:Topoisomerase I Inhibitors; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D004264:DNA Topoisomerases, Type I; D006690:Bisbenzimidazole; C017807:bisbenzimide ethoxide trihydrochloride