Similar studies of dna platinated with trans ddp have

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Similar studies of DNA platinated with trans-DDP have been carried out. In these, oligonucleotides containing the 1,3-trans-{Pt(NH 3 hd(GpNpG)} intra- strand crosslink were examined. The electrophoresis gels of polymerized 15- mers and 22-mers containing this adduct showed cooperative bending. This re- sult indicates that bends at the sites of platination by trans-DDP are not phase sensitive, and has been interpreted to imply the formation of a "hinge joint" at these positions. 92 95 The directed bends and local unwinding of DNA produced by cisplatin could be an important structural element that triggers a response by cellular proteins. This subject is discussed in greater detail in Section V.D.7.d. d. Biological Consequences of Platinum-DNA Binding (i). Inhibition of replication Binding of cis-DDP to DNA inhibits replica- tion both in vivo and in vitro, as shown by a variety of assays. Inhibition of replication of SV40 viral DNA in African green monkey cells as a function of the concentration of added cis-DDP is shown in Figure 9.17. When SV40 virus infects monkey cells, it does not integrate its DNA into the genome of the host.
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547 100 e "E 80 0 (,) ~ ~ c 60 0 cis ~ .2 Ci. ~ 40 « z 0 a 20 ,.. > Cf) 0 25 50 75 100 125 platinum (flM) o 125 100 (B) (C) 100 80 e "E 0 (,) ~ ~ 75 ~ 60 c 6 0 E ~ :::J .2 c Ci. .~ 50 ~ 40 Ci. « z 0 a ,.. > 25 Cf) 20 DIN X 10 4 DIN X 10 4 Figure 9.17 SV40 DNA replication in CV-l cells as a function of platinum concentration in the medium (panel A) or DIN (panel C). In panel B, DIN is plotted as a function of platinum concentration in the medium. SV40-infected cells were treated with cis-DDP (e) or trans-DDP (0) at the indi- cated concentrations for 40 h. SV40 DNA replication relative to control (untreated) cells was measured by incorporation of [3H]thymidine, added after the first 24 h of platinum treatment, and Pt in isolated SV40 chromosomes was measured by AAS. The data shown are from a repre- sentative experiment. Experiments were carried out in quadruplicate. Reproduced by permission from Reference 85.
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548 9 I METALS IN MEDICINE Instead, it forms its own chromosomes in the cell nucleus. These so-called mini- chromosomes consist of = 20 nucleosomes, fundamental chromosome building blocks. SV40 has its own life cycle, using virally encoded and cellular proteins to replicate and, ultimately, reassemble virus particles before lysing the cell and departing to infect neighboring cells. In the experiment shown in Figure 9.17, the SV40-infected cells were treated with cisplatin. After 24 h, [3H]thymidine was added and, after 24 more hours, the cells were harvested, and SV40 DNA was isolated; the amount of DNA synthesis was recorded by comparing incorporated radiolabel with results from control experiments where no platinum was present. The data show that, when 25 pM platinum was present, SV40 DNA replication was reduced to about 5 percent of control. Quantitation reveals that, at =2 platinum atoms bound per thousand nucleotides (drug-per-nucleotide, or (DlNh, = 0.002), synthesis is only 10 percent that of control.
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