Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides.

The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex

Evidences for a catalytic activity of the DNA double helix in the reaction between DNA, platinum(II), and intercalators.

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DNA double helix promotes a linkage isomerization reaction in trans-diamminedichloroplatinum(II)-modified DNA.

In the reaction between trans-diamminedichloroplatinum(II) and a single-stranded pyrimidin-rich oligodeoxyribonucleotide (22-mer) containing the central sequence TGAGT, the 1,3-trans-[Pt(NH3)2[d(GAG)]] cross-link is formed. The 1,3-intrastrand cross-link is inert within the single-stranded oligonucleotide. In contrast, it rearranges to an interstrand cross-link when the platinated oligonucleotide is paired with its complementary deoxyribo- or ribonucleotide strand. The half-life of the 1,3-intrastrand cross-link, approximately 6 h at 37 degrees C, is independent of the nature and concentration of the salt (NaCl or NaClO4). It is not dramatically affected when the intervening adenine residue between the chelated guanine residues is replaced by a cytosine or a thymine residue or when the T.A base pair adjacent to the 5' or 3' side of the adduct is replaced by a C.G base pair. On the other hand, a mismatch on the 3' or 5' side of the adduct prevents the rearrangement. We propose that the linkage isomerization reaction results from a direct nucleophilic attack of the cytosine residue complementary to the platinated 5' guanine residue on the platinum residue. Among others, the potential use of the DNA.RNA-promoted reaction is discussed in the context of the antisense strategy to irreversibly cross-link the antisense oligonucleotides to their targets

Trans-Diamminedichloroplatinum (II) Is Not an Antitumor Drug: Why?

International audienceThe clinical activity of the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) is thought to be related to its ability to form bifunctional lesions in DNA. trans-Diamminedichloroplatinum(II) (trans-DDP), the stereoisomer of cis-DDP, is clinically ineffective, although it forms bifunctional cross-links in the in vitro reaction with DNA1–4. A question still unsolved is why only cis-DDP is an antitumor drug

Intrastrand cross-links are not formed in the reaction between transplatin and native DNA: relation with the clinical inefficiency of transplatin.

The reaction between trans-diamminedichloroplatinum(II) and single-stranded oligonucleotides containing the sequence d(GXG) (X being an adenine, cytosine or thymine residue) yields trans-[Pt(NH3)2[(GXG)-GN7,GN7]] intrastrand cross-links. These cross-links do not prevent the pairing of the platinated oligonucleotides with their complementary strands but they decrease the thermal stability of the duplexes. The thermal stability is not much affected by the chemical nature of the X residue and its complementary base. By gel electrophoresis, it is shown that the trans- [Pt(NH3)2[d(GTG)-GN7,GN7]] cross-link bends the DNA double helix (26 degrees) and unwinds it (45 degrees). The pairing of the platinated oligonucleotides with their complementary strands promotes the rearrangement of the 1,3-intrastrand cross-links into interstrand cross-links. At a given temperature, the nature of the X residue, its complementary base and of the base pairs adjacent to the adducts do not dramatically affect the rate of the reaction. To know whether trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links do not rearrange in some sequences, the location of these adducts was searched in double-stranded DNA after reaction with trans-diamminedichloroplatinum(II) by means of the 3'-5' exonuclease activity of T4 DNA polymerase. At low level of platination, trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links were not detected. Monofunctional adducts and interstrand cross-links were mainly formed. These results are discussed in relation with the clinical inefficiency of trans-diamminedichloroplatinum(II)

102 IDENTIFICATION OF CONTRASTED PHENOTYPES IN THE BOVINE FROM REPEATED IN VIVO AND IN VITRO EMBRYO PRODUCTION FOLLOWING SUPEROVULATION

International audienceThis study was initiated to evaluate maternal influence on in vivo and in vitro bovine embryo production and identify animals with contrasted phenotypes for reproductive parameters. Nine Montbéliard cows raised on the same farm and with various genetic origins were included in the study. In vivo-derived embryos were collected nonsurgically from superovulated cows on day 7 after AI (34 collections). Immature oocytes were collected by ovum pickup from the same (superovulated) cows (36 sessions) then matured, fertilized (day 0) with the same bull, and cultured in vitro until day 7 on Vero cell monolayers in B2 medium. Grade 1 to 3 in vivo and grade 1 and 2 in vitro produced embryos deemed viable according to IETS criteria. The mean numbers of blastocysts and viable blastocysts per session per cow were, respectively, 8.3 ± 5.5 and 4.8 ± 3.6 in the in vivo system and 2.5 ± 2.6 and 1.8 ± 2.2 in the in vitro system. Individual cow data of in vivo and in vitro embryo production were analyzed by ANOVA (GLM program in SAS; SAS Institute Inc., Cary, NC, USA). Results are presented in Table 1: mean ± SD. Quantity and quality of produced embryos varied significantly among females, and production in vivo and in vitro was not systematically related. Contrasted phenotypes were identified according to their viable blastocyst rates in both systems (in vivo: no viable/recovered; in vitro: no viable/inseminated). Two females presented a relatively high percentage of viable blastocysts in both systems (over 30% in vitro and over 70% in vivo, Table 1). On the contrary, 2 females showed low percentages of blastocysts in the 2 systems (50%), but in vitro development rates were low. Only one female (C3) presented the inverse situation. Oocytes collected from animals with contrasted phenotypes will be analysed for gene expression to identify marker genes associated with oocyte developmental competence