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Triplex-DNA stabilization by hydralazine and the presence of anti-(triplex DNA) antibodies in patients treated with hydralazine.

ABSTRACT: Hydralazine is an antihypertensive drug that elicits andti-nuclear antibodies in patients as an adverse effect. We investigated the ability of hydralazine to promote/stabilize the triplex DNA form of poly(dA).2poly(dT). Under conditions of low ionic strength, the polynucleotide melted as a double helix with a melting temperature (Tm) of 55.3 degrees C. Hydralazine destabilized this duplex form by reducing its Tm to 52.5 degrees C. Spermidine (2.5 microM), a natural polyamine, provoked the triplex form of poly(dA)-.2poly(dT) with two melting transitions, Tm1 of 42.8 degrees C corresponding to triplex-->duplex+single-stranded DNA and Tm2 of 65.4 degrees C, corresponding to duplex melting. Triplex DNA thus formed in the presence of spermidine was further stabilized by hydralazine (250 microM) with a Tm1 of 53.6 degrees C. A similar stabilization effect of hydralazine was found on triplex DNA formed in the presence of 5 mM Mg2+. CD spectra revealed conformational perturbations of DNA in the presence of spermidine and hydralazine. These results support the hypothesis that hydralazine is capable of stabilizing unusual forms of DNA. In contrast with the weak immunogenicity of DNA in its right-handed B-DNA conformation, these unusual forms are immunogenic and have the potential to elicit anti-DNA antibodies. To test this possibility, we analysed sera from a panel of 25 hydralazine-treated patients for anti-(triplex DNA) antibodies using an ELISA. Our results showed that 72% of sera from hydralazine-treated patients contained antibodies reacting toward the triplex DNA. In contrast, there was no significant binding of normal human sera to triplex DNA. Taken together our data indicate that hydralazine and related drugs might exert their action by interacting with DNA and stabilizing higher-order structures such as the triplex DNA.

SUBMITTER: Thomas TJ

PROVIDER: S-EPMC1136136 | biostudies-other | 1995 Oct

REPOSITORIES: biostudies-other

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Project description:Importance:Longitudinal circulating tumor DNA (ctDNA) has been shown to predict response and survival in patients with metastatic melanoma treated with anti-programmed cell death 1 (PD-1) antibodies. Pseudoprogression, defined as radiologic finding of disease progression prior to response, has been a challenge to clinicians. Objective:To establish whether ctDNA at baseline and up to week 12 of treatment can differentiate between the radiologic findings of pseudoprogression and true progression in patients with metastatic melanoma. Design, Setting, and Participants:This explorative biomarker study examined circulating BRAF and NRAS mutations in a cohort of 125 patients with melanoma receiving PD-1 antibodies alone or in combination with ipilimumab between July 3, 2014, and May 24, 2016. Pseudoprogression was defined retrospectively as radiologic progression not confirmed as progressive disease at the next radiologic assessment. Plasma samples of ctDNA at baseline and while receiving treatment were taken for analysis prospectively over the first 12 weeks of treatment. Favorable ctDNA profile (undetectable ctDNA at baseline or detectable ctDNA at baseline followed by >10-fold decrease) and unfavorable ctDNA profile (detectable ctDNA at baseline that remained stable or increased) were correlated with response and prognosis. Main Outcomes and Measures:Early differentiation of pseudoprogression from true progression using longitudinal ctDNA profile. Results:According to guidelines by Response Evaluation Criteria in Solid Tumors (RECIST), progressive disease occurred in 29 of the 125 patients (23.2%). Of the 29 patients, 17 (59%) were 65 years or younger, 18 (62%) were men, 9 (31%) had pseudoprogression, and 20 (69%) had true progression. Of the 9 patients (7%) with confirmed pseudoprogression, all patients had a favorable ctDNA profile. At a median follow-up of 110 weeks, 7 of 9 patients (78%) were alive. All but 2 patients with true progression had an unfavorable ctDNA profile. Sensitivity of ctDNA for predicting pseudoprogression was 90% (95% CI, 68%-99%) and specificity was 100% (95% CI, 60%-100%). The 1-year survival for patients with RECIST-defined progressive disease and favorable ctDNA was 82% vs 39% for unfavorable ctDNA (hazard ratio [HR], 4.8; 95% CI, 1.6-14.3; P?=?.02). Overall survival was longer in patients with a partial response (54 of 125 patients [43%]) compared with patients with progressive disease and a favorable ctDNA profile (11 of 125 patients [9%]; HR, 0.09; 95% CI, 0.01-0.80; P?<?.01). Conclusions and Relevance:The results demonstrate that ctDNA profiles can accurately differentiate pseudoprogression from true progression of disease in patients with melanoma treated with PD-1 antibodies. Results of this blood test performed at regular intervals during systemic treatment reflect tumor biology and have potential as a powerful biomarker to predict long-term response and survival.

Project description:Natural polyamines, i.e. putrescine, spermidine and spermine, are excellent promoters of triplex DNA. Using melting temperature (Tm) measurements and CD spectroscopy, we found that structural alterations on spermidine backbone, including methylation, or acetylation at the N1-, N4- and/or N8-positions had a profound influence on the stability and conformation of poly(dA).2poly(dT) triplex. The conformation of the polynucleotide complex underwent sequential changes from B-DNA to triplex DNA as the concentration of spermidine increased from 0 to 50 microM in a buffer containing 10 mM sodium cacodylate and 1 mM EDTA (pH 7.2). At 60 microM spermidine, the CD spectrum of triplex DNA was comparable with that of psi-DNA, with a strong positive band centred around 260 nm. A negative band was also found at 295 nm. At higher concentrations of spermidine, however, the intensity of the positive band progressively decreased and the peak intensity was found at a 1:0.3 molar ratio of DNA phosphate:spermidine. Temperature-dependent CD analysis showed that the psi-DNA structure melted to single-stranded DNA at temperatures above the Tm determined from the absorbance versus temperature profile. Comparable effects were exerted on the conformation of triplex DNA by Co(NH3)6(3+), an inorganic trivalent cation. Substitution of the N4-hydrogen of spermidine by a cyclohexyl ring or the fusion of the N4-nitrogen in a cyclic ring system, as in piperidine, enhanced the ability of spermidine analogues to stabilize triplex and psi-DNA forms over a wider concentration range compared with spermidine. These data demonstrate a differential effect of trivalent cations in stabilizing triplex DNA and provoking unusual conformations such as psi-DNA. Synthetic homologues of spermidine that stabilize triplex DNA over a wider range of concentrations than that stabilized by spermidine itself might have potential therapeutic applications in the development of an anti-gene strategy against several diseases, including cancer and AIDS.

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Triplex-DNA stabilization by hydralazine and the presence of anti-(triplex DNA) antibodies in patients treated with hydralazine.

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