Hoechst 33342 alters luciferase gene expression in transfected BC3H-1 myocytes

Archives of Pathology & Laboratory Medicine, Sep 2003 by Zhang, Xinbo, Kiechle, Frederick L

* 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 [^sup 32^P]-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 [mu]M 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.

Conclusion.-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).

Hoechst 33342 or 2'[4-ethoxyphenyl]-5-[4-methyl-piperazinyl]-2,5'-bi-1H-benzimidazole and Hoechst 33258 or 2'-[4-hydroxyphenyl]-5-[4-methyl-piperazinyl]-2,5'-bi-1H-benzimidazole are cell-permeable adenine-thymine-binding fluorescent dyes1-3 that are used to stain DNA for evaluating the cell cycle and apoptosis and for quantifying viable cells by flow cytometry.4,5 The only structural difference between the 2 dyes is that Hoechst 33258 has a hydroxy group, whereas Hoechst 33342 has an ethoxy group. This structural difference confers greater lipophilic properties and greater cell membrane permeability to Hoechst 33342.1,6 Nonspecific fluorescence has been reported to be caused by the dye binding to glycogen7 or sodium dodecyl sulfate.8 Both dyes bind in the minor groove of B-DNA covering at least 6 base pairs (bp), and a minimum of 4 AT sequences are required for tight drug-DNA electrostatic interaction.2,3,9 Hoechst 33258 binds with a higher affinity for the bent sequence of the ligated decamers (CAAAATTTTG)^sub n^ by 3 to 4 times compared to the straight sequence of (CTTTTAAAAG)^sub n^.10 The nonplanar N-methylpiperazine prefers the wider minor groove conferred by the G[middot]C base pair for binding compared to the narrower contiguous A[middot]T base pair.2,3

Both dyes have been reported to be either nontoxic11,12 or cytotoxic.13,14 Hoechst 33342 protects DNA and cells from ionizing radiation by an undefined mechanism.15 Hoechst 33342, but not Hoechst 33258, has been shown to induce apoptosis or programmed cell death in a variety of benign and malignant cell culture lines, usually after a 3-hour lag phase.13,16-28 Hoechst 33342-induced apoptosis is associated with characteristic morphologic changes, including cell shrinkage, rounding, and nuclear chromatin condensation,13,16-18 oligonucleosomal DNA fragmentation into multiples of 180 bp determined by agarose gel electrophoresis,16-18 and annexin V binding to phosphatidylserine translocated to the outer leaflet of the plasma membrane detected by flow cytometry.19 The concentration of Hoechst 33342 required to initiate apoptosis is dependent on the cell type and media used. For example, 17.8 [mu]M of Hoechst 33342 was required to initiate apoptosis in BC3H-1 myocytes grown in Dulbecco modified Eagle media compared to the 26.7 [mu]M required for cells grown in RPMI-1640 media.16 In both types of media, lower concentrations of Hoechst 33342 (4.4 or 8.9 [mu]M) failed to initiate the apoptotic pathway.16 Therefore, there are 2 concentrations of Hoechst 33342 defined for each cell line: a sublethal concentration, which does not induce apoptosis, and a lethal concentration, which does induce apoptosis. These findings are consistent with the observation that apoptotic events exhibit a biphasic relationship that results in a U-shaped or inverted U-shaped dose response.29

Hoechst 33342 disrupts the 3 primary enzymatic reactions initiated in the minor groove of DNA, including DNA polymerase,26,30 RNA polymerase II,13,16,21,22,31 and topoisomerase I.16,18-20,22,28 For example, Hoechst 33342-induced apoptosis decreases the TATA box-binding protein (TBP)/TATA box promoter complex formation in a variety of cell lines treated with a lethal dose (26.7 [mu]M) of Hoechst 33342 for 24 hours.18,21,22 The TBP is a general transcription factor required for the initiation of transcription in the minor groove of DNA by RNA polymerase II.32 After the binding of the TBP to its TATA box promoter region, additional transcription factors (TFIID, TFIIB, TFIIE, etc) are added to the complex before new RNA synthesis begins. The presence of these protein/DNA complexes is readily detected by the electrophoretic mobility shift assay, which provides a means of quantifying DNA/protein complex formation.33 The disrupted complexes were detected by this method after cells had been incubated in vivo with a lethal dose (26.7 [mu]M) of Hoechst 33342 for 24 hours.18,21,22 The assay was not performed with sublethal doses of this dye.