New insight into the molecular mechanisms of the biological effects of DNA minor groove binders

Our plain-English summary of New insight into the molecular mechanisms of the biological effects of DNA minor groove binders, by X. Zhang, S.C. Zhang, D. Sun, et al (PloS One, 2011).

The study

• Authors: X. Zhang, S.C. Zhang, D. Sun, J. Hu, A. Wali, H. Pass, F. Fernandez-Madrid, M.R. Harbut, N. Tang
• Journal: PloS One
• Published: 2011;6(10):e25822
• Cited by: 21 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Bisbenzimides, or Hoechst 33258 (H258), and its derivative Hoechst 33342 (H342) are archetypal molecules for designing minor groove binders, and widely used as tools for staining DNA and analyzing side population cells. They are supravital DNA minor groove binders with AT selectivity. H342 and H258 share similar biological effects based on the similarity of their chemical structures, but also have their unique biological effects. For example, H342, but not H258, is a potent apoptotic inducer and both H342 and H258 can induce transgene overexpression in in vitro studies. However, the molecular mechanisms by which Hoechst dyes induce apoptosis and enhance transgene overexpression are unclear.

Methodology/principal Findings: To determine the molecular mechanisms underlying different biological effects between H342 and H258, microarray technique coupled with bioinformatics analyses and multiple other techniques has been utilized to detect differential global gene expression profiles, Hoechst dye-specific gene expression signatures, and changes in cell morphology and levels of apoptosis-associated proteins in malignant mesothelioma cells. H342-induced apoptosis occurs in a dose-dependent fashion and is associated with morphological changes, caspase-3 activation, cytochrome c mitochondrial translocation, and cleavage of apoptosis-associated proteins. The antagonistic effect of H258 on H342-induced apoptosis indicates a pharmacokinetic basis for the two dyes' different biological effects. Differential global gene expression profiles induced by H258 and H342 are accompanied by unique gene expression signatures determined by DNA microarray and bioinformatics software, indicating a genetic basis for their different biological effects.

Conclusions/significance: A unique gene expression signature associated with H342-induced apoptosis provides a new avenue to predict and classify the therapeutic class of minor groove binders in the drug development process. Further analysis of H258-upregulated genes of transcription regulation may identify the genes that enhance transgene overexpression in gene therapy and promote recombinant protein products in biopharmaceutical companies.

Data Deposition: The microarray data reported in this article have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no.GSE28616).

In plain English

This laboratory study used human malignant pleural mesothelioma cells grown in dishes. It did not study patients or asbestos exposure. The authors treated seven mesothelioma cell lines with the DNA dye Hoechst 33342 (H342). Most other experiments, including those with the related dye Hoechst 33258 (H258), used one line, H2373. H342 killed cells in all seven lines, more at higher doses. In H2373 cells it produced signs of apoptosis (programmed cell death), such as caspase 3 activation and release of cytochrome c from mitochondria. In H2373 cells, H258 alone did not induce cell death after 6 or 24 hours at the concentrations tested. Given together with H342, it reduced H342-induced cell death by 33% to 57%; one hour of H258 pretreatment did not protect the cells. A software analysis indicated that the two dyes counteracted each other. In H2373 cells after 3 hours, H258 turned up 99 genes and turned down 582, while H342 turned up 191 and turned down 490, in different patterns. The authors concluded that H342 is a potent apoptotic inducer in mesothelioma cell lines and that such DNA minor groove binders may hold promise for treatment. They noted that cleaved caspase 3 was not detected.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: laboratory (in vitro) experiment on human mesothelioma cell lines, using cell viability tests, protein measurements, gene microarrays and bioinformatics analysis
• Who was studied: Seven human malignant pleural mesothelioma cell lines derived from patients (H2373, H2452, H2596, H2461, H2591, H-meso, H2714). H342 viability was tested in all seven; the H258, combination, protein and gene experiments used H2373.
• Exposure: Treatment of cells with the DNA dyes Hoechst 33342 and Hoechst 33258, alone, in combination, or with one hour of H258 pretreatment before H342. Concentrations are printed as up to 36 mM (H342), and the unit symbol may be damaged; see text problems. Gene profiling was done after 3 hours of treatment. No asbestos exposure was studied.
• Main results:
  • H342 caused cell death in all seven mesothelioma cell lines, increasing with dose
  • In H2373 cells, H342 treatment was accompanied by caspase 3 activation and movement of cytochrome c from mitochondria to the cytosol
  • In H2373 cells, H258 alone did not induce cell death after 6 and 24 hours at the concentrations tested
  • In H2373 cells given H258 together with H342, H258 reduced H342-induced apoptosis by 33% to 57%, depending on dose
  • In H2373 cells, one hour of H258 pretreatment before H342 did not protect the cells from H342-induced apoptosis
  • Combination indices were above 1.0, which the authors interpreted as antagonism between the two dyes (Table 1 values ranged from 1.186 to 2.356)
  • In H2373 cells after 3 hours, H258 increased 99 genes and decreased 582 genes; H342 increased 191 genes and decreased 490 genes
  • The authors judged that turning down transcription-regulation genes was the gene signature specific to H342
  • The H342-downregulated and H258-upregulated transcription-regulation gene sets overlapped by 7 genes. Of 10 enriched pathways for the H342 genes, 9 overlapped with the H258 pathways.
• The authors' conclusion: The authors concluded that H342 is a potent apoptotic inducer in mesothelioma cell lines and that DNA minor groove binders may hold promise as a new treatment approach for malignant pleural mesothelioma
• Limitations the authors state:
  • Cleaved (active) caspase 3 was not detected by immunoblotting
  • The authors state that the mechanism of H342-induced apoptosis remains unclear
  • Further experiments are needed to determine how the dyes cross the cell membrane

Topics

Medical terms in this study, explained in our Lexicon: Apoptosis, Malignant Mesothelioma, Translocation.

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