Primer extension activity of RT HIV-1 using the 17mer/44mer primer/template duplex

<p><b>Copyright information:</b></p><p>Taken from "Mechanism of the formation of DNA–protein cross-links by antitumor cisplatin"</p><p></p><p>Nucleic Acids Research 2007;35(6):1812-1821.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874601.</p><p>© 2007 The Author(s)</p> The experiments were conducted for the times indicated in the figure (5–90 min) using undamaged templates (lanes 2–5), the template containing single, site-specific 1,2-GG intrastrand CL of cisplatin (lanes 6–9) and the template containing single DPCL formed by the transformation of the template containing site-specific 1,2-GG intrastrand CL of cisplatin incubated with histone H1 (lanes 10–13). Lane 1, 17-mer primer. The pause sites opposite the platinated guanines and the nucleotide preceding the platinated guanines (thymine residue on the 3′ side of the CL) are marked 34, 33, 32, respectively. The nucleotide sequences of the templates and the primers are shown beneath the gels. See the text for other details

DPCL formation of unmodified and platinated oligodeoxyribonucleotide duplexes containing single, site-specific platinum adduct with KF assessed by SDS/PAA gel electrophoresis

<p><b>Copyright information:</b></p><p>Taken from "Mechanism of the formation of DNA–protein cross-links by antitumor cisplatin"</p><p></p><p>Nucleic Acids Research 2007;35(6):1812-1821.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874601.</p><p>© 2007 The Author(s)</p> Lanes: 1, 4, 7, 10, 13, the duplexes incubated with the protein for 4 h; 2, 5, 8, 11, 14 for 8 h; 3, 6, 9, 12, 15 for 24 h. Lanes: 1–3, the duplex TGT (20) containing monofunctional adduct of cisplatin; 4–6, the duplex TGT (20) containing monofunctional adduct of transplatin; 7–9, the duplex TGGT (20) containing 1,2-GG intrastrand CL of cisplatin; 10–12, the duplex TGTGT (20) containing 1,3-GTG intrastrand CL of cisplatin; 13–15, the duplex TGCT (20) containing interstrand CL of cisplatin. See the text for other details

Valuable Insight into the Anticancer Activity of the Platinum-Histone Deacetylase Inhibitor Conjugate, <i>cis</i>-[Pt(NH₃)₂malSAHA_–2H)]

<i>cis</i>-[Pt^II(NH₃)₂(malSAHA_–2H)], a cisplatin adduct conjugated to a potent histone deacetylase inhibitor (HDACi), suberoylanilide hydroxamic acid (SAHA), was previously developed as a potential anticancer agent. This Pt–HDACi conjugate was demonstrated to have comparable cytotoxicity to cisplatin against A2780 ovarian cancer cells but significantly reduced cytotoxicity against a representative normal cell line, NHDF. Thus, with a view to (i) understanding more deeply the effects that may play an important role in the biological (pharmacological) properties of this new conjugate against cancer cells and (ii) developing the next generation of Pt–HDACi conjugates, the cytotoxicity, DNA binding, cellular accumulation and HDAC inhibitory activity of <i>cis</i>-[Pt^II(NH₃)₂(malSAHA_–2H)] were investigated and are reported herein. <i>cis</i>-[Pt^II(NH₃)₂(malSAHA_–2H)] was found to have marginally lower cytotoxicity against a panel of cancer cell lines as compared to cisplatin and SAHA. <i>cis</i>-[Pt^II(NH₃)₂(malSAHA_–2H)] was also found to accumulate better in cancer cells but bind DNA less readily as compared to cisplatin. DNA binding experiments indicated that c<i>is</i>-[Pt^II(NH₃)₂(malSAHA_–2H)] bound DNA more effectively in cellulo as compared to in cell-free media. Activation of the Pt–HDACi conjugate was therefore investigated. The binding of c<i>is</i>-[Pt^II(NH₃)₂(malSAHA_–2H)] to DNA was found to be enhanced by the presence of thiol-containing molecules such as glutathione and thiourea, and activation occurred in cytosolic but not nuclear extract of human cancer cells. The activity of <i>cis</i>-[Pt­(NH₃)₂(malSAHA_–2H)] as a HDAC inhibitor was also examined; the conjugate exhibited no inhibition of HDAC activity in CH1 cells. In light of these results, novel Pt–HDACi conjugates are currently being developed, with particular emphasis, through subtle structural modifications, on enhancing the rate of DNA binding and enhancing HDAC inhibitory activity

Combined Theoretical and Computational Study of Interstrand DNA Guanine–Guanine Cross-Linking by <i>trans</i>-[Pt(pyridine)₂] Derived from the Photoactivated Prodrug <i>trans,trans,trans</i>-[Pt(N₃)₂(OH)₂(pyridine)₂]

Molecular modeling and extensive experimental studies are used to study DNA distortions induced by binding platinum­(II)-containing fragments derived from cisplatin and a new class of photoactive platinum anticancer drugs. The major photoproduct of the novel platinum­(IV) prodrug <i>trans,trans,trans</i>-[Pt­(N₃)₂(OH)₂(py)₂] (<b>1</b>) contains the <i>trans</i>-{Pt­(py)₂}²⁺ moiety. Using a tailored DNA sequence, experimental studies establish the possibility of interstrand binding of <i>trans</i>-{Pt­(py)₂}²⁺ (<b>P</b>) to guanine N7 positions on each DNA strand. Ligand field molecular mechanics (LFMM) parameters for Pt–guanine interactions are then derived and validated against a range of experimental structures from the Cambridge Structural Database, published quantum mechanics (QM)/molecular mechanics (MM) structures of model Pt–DNA systems and additional density-functional theory (DFT) studies. Ligand field molecular dynamics (LFMD) simulation protocols are developed and validated using experimentally characterized bifunctional DNA adducts involving both an intra- and an interstrand cross-link of cisplatin. We then turn to the interaction of <b>P</b> with the DNA duplex dodecamer, d­(5′-C₁C₂T₃C₄T₅C₆G₇T₈C₉T₁₀C₁₁C₁₂-3′)·d­(5′-G₁₃G₁₄A₁₅G₁₆A₁₇C₁₈G₁₉A₂₀G₂₁A₂₂G₂₃G₂₄-3′) which is known to form a monofunctional adduct with <i>cis</i>-{Pt­(NH₃)₂(py)}. <b>P</b> coordinated to G₇ and G₁₉ is simulated giving a predicted bend toward the minor groove. This is widened at one end of the platinated site and deepened at the opposite end, while the <b>P</b>–DNA complex exhibits a global bend of ∼67° and an unwinding of ∼20°. Such cross-links offer possibilities for specific protein–DNA interactions and suggest possible mechanisms to explain the high potency of this photoactivated complex

Novel Antitumor Cisplatin and Transplatin Derivatives Containing 1‑Methyl-7-Azaindole: Synthesis, Characterization, and Cellular Responses

The current work investigates the effect of new bifunctional and mononuclear Pt­(II) compounds, the cis- and trans-isomers of [PtCl₂(NH₃)­(L)] (L = 1-methyl-7-azaindole, compounds <b>1</b> and <b>2</b>, respectively), on growth and viability of human carcinoma cells as well as their putative mechanism(s) of cytotoxicity. The results show that substitution of 1-methyl-7-azaindole for ammine in cisplatin or transplatin results in an increase of the toxic efficiency, selectivity for tumor cells in cisplatin-resistant cancer cells, and activation of the trans geometry. The differences in the cytotoxic activities of <b>1</b> and <b>2</b> were suggested to be due to their different DNA binding mode, different capability to induce cell cycle perturbations, and fundamentally different role of transcription factor p53 in their mechanism of action. Interestingly, both isomers make it possible to detect their cellular uptake and distribution in living cells by confocal microscopy without their modification with an optically active tag