The principles of large screening strategies, which are developed by industrial companies, have been recently adopted by researchers in the fields of molecular biology and oncology as invaluable tools for translational medicine. The declining costs of laboratory robotic machines have allowed high-throughput screening to become more available to academic centres with limited resources. Here, we describe how a robotic conventional liquid handling system could be used on a daily basis in laboratories to obtain consistent and reproducible results. Our approach allowed us to quickly screen a panel of more than 20 tumorigenic and non-tumorigenic cell lines for their responses to hydroxyurea, which is a DNA-damaging anticancer therapeutic drug. The format of 384-well microplates was used for manual cell seeding, and the effect of hydroxyurea was screened at multiple concentrations. The fluorescence-based CyQuant assay was employed as the readout method to analyse the cellular DNA content. The effectiveness of our approach was demonstrated in the experimental results.
- MeSH
- automatizace MeSH
- buněčné kultury metody MeSH
- hydroxymočovina farmakologie MeSH
- karcinogeneze účinky léků patologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- poškození DNA * MeSH
- protinádorové látky farmakologie MeSH
- regresní analýza MeSH
- screeningové testy protinádorových léčiv * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Platinum diamine complexes are able to crosslink the guanines of d(GC)(2) dinucleotides within double-stranded DNA. The interstrand crosslink thus formed causes a bend of the double helix toward the minor groove and the helical sense changes locally to left-handed, resulting in a considerable unwinding. The bend and unwinding angles have been shown to depend on the platinum ligands. Here, we have used molecular dynamics simulations to investigate the DNA 20-mer d(C(1)T(2)C(3)T(4)C(5)C(6)T(7)T(8)G*(9)C(10)T(11)C(12)T(13)C(14)C(15)T(16)T(17)C(18)T(19)C(20))-d(G(21)A(22)G(23)A(24)A(25)G(26)G(27)A(28)G(29)A(30)G*(31)C(32)A(33)A(34)G(35)G(36)A(37)G(38)A(39)G(40)) with the G* guanines crosslinked by cis-Pt(NH(3))(2)(2+), Pt(R,R-DACH)(2+), or Pt(S,S-DACH)(2+). Previous investigations on cisplatin interstrand adducts indicated that the structure is similar in solid state and in solution; thus, we used the reported X-ray structure of a cisplatin adduct as a starting model. Replacing in the MD-relaxed model for the DNA duplex crosslinked with cis-Pt(NH(3))(2)(2+) the two NH(3) platinum ligands by R,R-DACH or S,S-DACH led to clashes between the DACH residue and the deoxyribose of C(12). Confrontation of MD-derived models with gel shift measurements suggested that these clashes are avoided differently in the adducts of Pt(R,R-DACH)(2+)versus Pt(S,S-DACH)(2+). The R,R-isomer avoids the clash by untwisting the T(11)/A(30)-C(12)/G(29) step, thus increasing the global unwinding. In contrast, the S,S-isomer modifies the shift and slide parameters of this step, which dislocates the helical axis and enhances the bend angle. The clash that leads to the differentiation of the structures as a function of the diamine ligand is related to a hydrogen bond between the platinum complex and the T(11) base and could be characteristic of interstrand crosslinks at d(pyG*Cpy)-d(puG*Cpu) sequences.
The global modification of mammalian and plasmid DNAs by the novel platinum compounds cis-[PtCl(2)(isopropylamine)(1-methylimidazole)] and trans-[PtCl(2)(isopropylamine)(1-methylimidazole)] and the reactivity of these compounds with reduced glutathione (GSH) were investigated in cell-free media using various biochemical and biophysical methods. Earlier cytotoxicity studies had revealed that the replacement of the NH(3) groups in cisplatin by the azole and isopropylamine ligands lowers the activity of cisplatin in both sensitive and resistant cell lines. The results of the present work show that this replacement does not considerably affect the DNA modifications by this drug, recognition of these modifications by HMGB1 protein, their repair, and reactivity of the platinum complex with GSH. These results were interpreted to mean that the reduced activity of this analog of cisplatin in tumor cell lines is due to factors that do not operate at the level of the target DNA. In contrast, earlier studies had shown that the replacement of the NH(3) groups in the clinically ineffective trans isomer (transplatin) by the azole and isopropylamine ligands results in a radical enhancement of its activity in tumor cell lines. Importantly, this replacement also markedly alters the DNA binding mode of transplatin, which is distinctly different from that of cisplatin, but does not affect reactivity with GSH. Hence, the results of the present work are consistent with the view and support the hypothesis systematically tested by us and others that platinum drugs that bind to DNA in a fundamentally different manner from that of conventional cisplatin may have altered pharmacological properties.
- MeSH
- bezbuněčný systém MeSH
- cirkulární dichroismus MeSH
- DNA chemie účinky léků MeSH
- financování organizované MeSH
- glutathion chemie účinky léků MeSH
- kultivační média chemie MeSH
- lidé MeSH
- organoplatinové sloučeniny farmakologie chemie MeSH
- protinádorové látky farmakologie chemie MeSH
- spektrofotometrie ultrafialová MeSH
- stereoizomerie MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
