Hoechst 33342 alters luciferase gene expression in transfected BC3H-1 myocytes.
BACKGROUND
Hoechst 33342 and Hoechst 33258 bind to the minor groove of DNA. Hoechst 33342 induces apoptosis in a variety of cell types by a mechanism that is associated with disruption of the formation of the TATA box-binding protein/DNA complex.


OBJECTIVE
To further investigate the role of Hoechst 33342 in gene regulation using BC3H-1 myocytes transfected with 4 different pGL3 luciferase reporter vectors constructed with or without the SV40 promoter and/or enhancer regions or with 2 synthetic Renilla luciferase vectors (phRL-null and phRL-TK).


METHODS
Luciferase messenger RNA content was measured by reverse transcriptase-polymerase chain reaction, and luciferase activity was measured by luminometry. The ability of transcription factors in nuclei prepared from BC3H-1 myocytes to bind to a [32P]-labeled 24-base pair oligonucleotide containing the TATA box-binding element was determined by a gel mobility shift assay.


RESULTS
In vivo, 4.4 and 8.9 microM of Hoechst 33342 (sublethal doses) increased luciferase enzyme activity in cells transfected with each of the 4 pGL3 luciferase reporter vectors and both of the Renilla luciferase vectors. Hoechst 33258 had no effect on luciferase enzyme activity. In vitro, Hoechst 33342 increased transcription factor binding to the 24-mer oligonucleotide containing the TATA box-binding element, which would be favorable to increased RNA polymerase II efficiency.


CONCLUSIONS
Hoechst 33342 stimulates luciferase activity by a pathway that is independent of the integrity of the promoters in the luciferase gene expression vectors used (pGL3 basic, pGL3 control, pGL3 enhancer, and pGL3 promoter vectors, phRL-null, or phRL-TK).
Zhang
Xinbo
X
Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Mich 48073-6769, USA. fkiechle@beaumont.edu
Kiechle
Frederick L
FL
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics; D006690:Bisbenzimidazole / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D005456:Fluorescent Dyes / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016385:TATA Box / Q000235:genetics; D035181:TATA-Box Binding Protein / Q000378:metabolism; D013997:Time Factors; D019473:Transcription Factors, TFII / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; D019473:Transcription Factors, TFII; D008156:Luciferases; D006690:Bisbenzimidazole; C017807:bisbenzimide ethoxide trihydrochloride