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S J Lippard

Publications and source records attributed to S J Lippard.

At least 127 records · Page 7Linked to original sources
PubMed1985

In vivo effects of cis- and trans-diamminedichloroplatinum(II) on SV40 chromosomes: differential repair, DNA-protein cross-linking, and inhibition of replication.

The mechanism of action of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, was investigated by using the approximately 5200 base pair (bp) chromosome of simian virus 40 (SV40) as an in vivo chromatin model. Comparative studies were also carried out with the clinically ineffective isomer trans-DDP. Although 14 times more trans- than cis-DDP in the culture medium is required to inhibit SV40 DNA replication in SV40-infected green monkey CV-1 cells, the two isomers are equally effective at inhibiting replication when equimolar amounts are bound to SV40 DNA in vivo. Since both isomers are transported into CV-1 cells at similar rates, differential uptake cannot account for the greater ability of cis-DDP to inhibit SV40 DNA replication. Rather, this result is explained by the finding that cis-DDP-DNA adducts accumulate continuously over the incubation period, whereas trans-DDP binding to DNA reaches a maximum at 6 h and thereafter decreases dramatically. We suggest that the different accumulation behavior of cis-DDP and trans-DDP on DNA is due to their differential repair in CV-1 cells. A variety of non-histone proteins, including SV40 capsid proteins but virtually no histones, are cross-linked to SV40 DNA in vivo by either cis- or trans-DDP. More DNA-protein cross-links are formed by trans-DDP than by cis-DDP at equivalent amounts of DNA-bound platinum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

X-ray structure of the major adduct of the anticancer drug cisplatin with DNA: cis-[Pt(NH3)2(d(pGpG))].

Crystals of the adduct of the anticancer drug cis-diamminedichloroplatinum(II), cis-DDP, with d(pGpG), its putative target on DNA in the cancer cell, have been obtained and used in an x-ray crystallographic study to elucidate the molecular structure to atomic resolution. Each of the four crystallographically independent cis-[Pt(NH3)2(d(pGpG))] molecules is comprised of a square-planar platinum atom bonded to two ammonia ligands and two N(7) atoms of guanosine nucleosides from the same chain. Base stacking of the two adjacent guanine rings is completely disrupted by coordination to the cis-(Pt(NH3)2)2+ unit. Comparison of the backbone and deoxyribose ring torsion angles with those found by previous (nuclear magnetic resonance spectroscopy) studies of this adduct in solution demonstrates that the solid state geometry is substantially the same as that in solution. The relevance of these results to the molecular mechanism of action of cis-DDP is discussed.

Cisplatin↗
PubMed1985

Sequence-dependent termination of in vitro DNA synthesis by cis- and trans-diamminedichloroplatinum (II).

Inhibition of DNA replication by the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) has been proposed to be responsible for its cytotoxicity. Treatment of primed phage M13 mp8 viral DNA templates with the drug followed by second-strand synthesis using large fragment DNA polymerase I reveals that cis-DDP forms an adduct with DNA that inhibits DNA synthesis in vitro. This inhibition occurs at all (dG)n (n greater than or equal to 2) sequences in the template strand, confirming that these regions are the major cis-DDP binding sites on DNA. trans-Diamminedichloroplatinum (II), which is inactive as a drug, also forms adducts that inhibit DNA synthesis. Although considerably lower specificity is observed with the trans isomer, there appears to be a preference for d(GpNpG) sequences, where N is any intervening nucleotide. The monofunctional adduct formed between chlorodiethylenetriamineplatinum(II) chloride and DNA does not inhibit DNA synthesis in this system.

Base Sequence↗
PubMed1983

Alteration and activation of sequence-specific cleavage of DNA by bleomycin in the presence of the antitumor drug cis-diamminedichloroplatinum(II).

The antitumor drug cis-diamminedichloroplatinum(II) dramatically alters the sequence-specific cleavage of the bleomycin A2-iron(II)-O2 system. Preferred bleomycin cleavage sites adjacent to oligo(dG) regions on two restriction fragments of plasmid pBR322 DNA were masked by pretreatment with cis-diamminedichloroplatinum(II). trans-Diamminedichloroplatinum(II), which is inactive as an antitumor drug, showed similar but not identical behavior. The DNA-cleaving activity of bleomycin was substantially modified by cis-diamminedichloroplatinum(II), and a number of specific new cutting sites in guanine-rich parts of the sequence were activated by both isomers of the platinum complex. The results further emphasize the possibility that the synergism found when cis-diamminedichloroplatinum(II) and bleomycin are used in combination chemotherapy may be due to interactions at the level of DNA-drug binding.

Animals↗
PubMed1982

New chemistry of an old molecule: cis-[Pt(NH3)2Cl2].

The discovery that cis-diamminedichloroplatinum(II) (cis-DDP) has clinically useful antitumor properties and can form platinum blues spawned an extensive investigation of its chemistry in water. Several new molecules have been synthesized, some rather old ones have been characterized for the first time, and clues have begun to emerge about the chemical interaction of cis-DDP with its likely biological target, DNA.

Chemical Phenomena↗
PubMed1982

Binding of [(dien)PtCl] Cl to poly(dG-dC)-poly(dG-dC) facilitates the B goes to Z conformational transition.

Chlorodiethylenetriamineplatinum(II) chloride, [(dien)PtCl]Cl, bound to less than or equal to 10% of the nucleotide bases of poly(dG-dC) . poly(dG-dC) reduces the amount of ethanol necessary to bring about the B goes to Z conformational transition in proportion to the amount of platinum complex bound as monitored by CD spectroscopy. The transition may be effected by 25% ethanol with 9.3% of the bases modified polymer an ethanol with 5.4% of the bases modified. With an unmodified polymer an ethanol concentration of 55-60% is necessary to bring about the transition. The assignment of the Z conformation was supported by 31P NMR spectroscopy. This covalent modification of the DNA is reversed by treatment with cyanide ion after which the normal amount of ethanol is necessary to achieve the transition. The platinum complex shows no enhanced binding to DNA in the Z versus the B conformation. Between 20 and 33% (saturation binding) modification, [(dien)PtCl]Cl binds cooperatively to the heterocopolymer as judged by CD spectroscopy. At this high level of modification it is no longer possible to induce the Z DNA structure with ethanol. When [(dien)PtCl]Cl is bound to preformed (with ethanol) Z DNA at saturating levels the CD spectrum is altered but reverts to the spectrum of highly modified DNA upon removal of ethanol. The antitumor drug cis-diaminedichloroplatinum(II), cis-DDP, binds to poly(dG-dC) . poly(dG-dC) and alters the CD spectrum. It does not facilitate the B goes to Z conformational change, however, and actually prevents it from happening even at very high ethanol concentrations.

Chemical Phenomena↗
PubMed1982

A pH-dependent superoxide dismutase activity for zinc-free bovine erythrocuprein. Reexamination of the role of zinc in the holoprotein.

The zinc-free derivative of bovine erythrocuprein, Cu2E2BE, was prepared and its superoxide dismutase activity was measured and compared with that of the holoprotein, Cu2Zn2BE. The dismutase activity of these proteins was measured by quantitating their inhibition of the superoxide-mediated autooxidation of 6-hydroxydopamine, dihydroxyfumaric acid, pyrogallol, and epinephrine. It was found that the superoxide dismutase activity of the zinc-free protein is pH dependent, ranging between 82 +/- 5% (relative to Cu2Zn2BE) at pH 5.8, and 25 +/- 10% at pH 10.2. The overlapping range of assays and buffers verified that these measurements are independent of the method of assay, buffer, and ionic strength (in the range of mu = 0.10 to 0.20). The variation in activity with pH is probably due, at least in part, to the migration of Cu(II) at high pH as described previously [J. S. Valentine, M. W. Pantoliano, P. J. McDonnell, A. R. Burger, and S. J. Lippard, Proc. Natl. Acad. Sci. USA 76, 4245 (1979)], since Cu(II) bound at the zinc binding site has been shown to have little or no dismutase activity. The observation of high activity (82%) for the zinc-free protein at pH 5.8, where Cu(II) is predominantly in the native Cu binding site, and less susceptible to removal by ethylenediaminetetraacetic acid, demonstrates that the presence of Zn(II) in Cu2Zn2BE does not greatly enhance the inherent dismutase activity of Cu(II) in the holoprotein.

Amino Acid Sequence↗
PubMed1982

Ethidium bromide changes the nuclease-sensitive DNA binding sites of the antitumor drug cis-diamminedichloroplatinum(II).

Exonuclease III has been shown previously to reveal the binding sites of the antitumor drug cis-diamminedichloroplatinum(II) on DNA. Pretreatment of the same DNA with the intercalator ethidium bromide causes new platinum binding sites to be detected by the exonuclease III method. In particular, a 5'd(G6-D-G2)3' sequence in a 165-base-pair restriction fragment of plasmid pBR322 becomes a preferred site for exonuclease III-detectable cis-diamminedichloroplatinum(II)binding. This switching of nuclease-sensitive platinum binding to new sites by the influence of another drug, ethidium bromide, offers an explantation at the molecular level for the phenomenon of synergism in combination chemotherapy.

Base Sequence↗
PubMed1982

Antibodies elicited against cis-diamminedichloroplatinum(II)-modified DNA are specific for cis-diamminedichloroplatinum(II)-DNA adducts formed in vivo and in vitro.

Rabbit antiserum elicited against calf thymus DNA modified to 4.4% (Pt drug/nucleotide ratio = 0.044) with the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) contains antibodies specific for the Pt-modified DNA immunogen as well as for Pt-DNA adducts formed in both cultured mouse leukemia L1210 cells and in L1210 cells from the ascites fluid of tumor-bearing mice exposed to cis-DDP. Pt-modified DNA was electrostatically complexed to methylated bovine serum albumin and injected into rabbits. Early bleedings of the derived antiserum were used to establish a competitive enzyme-linked immunosorbent assay (ELISA), which demonstrated specificity for the Pt-modified DNA but not for DNA or the Pt drug alone. In the ELISA, 50% inhibition occurred at a concentration of 0.5 nM Pt (on DNA) as determined by atomic absorption spectroscopy. This value corresponds to a lower limit of detectability of one adduct in 10(7) nucleotides, with 50 micrograms of sample DNA added per microtiter well. DNA isolated from cultured mouse L1210 cells exposed to increasing doses of the Pt drug was found by ELISA to contain from 0.2 to 10.0 fmol of Pt adduct per microgram of DNA. These levels remained stable for up to 4 hr after a 1-hr drug treatment, during which time DNA interstrand crosslinks developed. Thus, the antiserum appears not to be specific for DNA interstrand crosslinks. DNAs from L1210 cells exposed to trans-diamminedichloroplatinum(II) and L-phenylalanine mustard were not recognized in the ELISA. DNA prepared from the ascites cells of mice bearing the L1210 tumor 5 hr after injection of cis-DDP was found to contain about 2 fmol of Pt per microgram of DNA. This work establishes that cis-DDP-DNA adducts prepared in vitro are relevant to the in vivo binding of the Pt drug to its biological target, DNA, and opens new avenues for studying the mechanism of action of the Pt anticancer drugs.

Animals↗
PubMed1981

Anion binding to the four-copper form of bovine erythrocyte superoxide dismutase: Mechanistic implications.

The binding of thiocyanate, azide, and other anions to superoxide dismutase (SOD) containing copper in both the copper and zinc sites of the native enzyme (Cu2Cu2SOD) has been studied. Electron spin resonance spectroscopy was used to show that binding of SCN- to Cu2Cu2SOD breaks the imidazolate bridge between the two copper centers. Thiocyanate displaces the bridging histidine from the Cu2+ ion in the copper site and also replaces the aspartic acid ligand from the Cu2+ ion in zinc site. This conclusion is supported by studies with Ag2Cu2SOD, where silver is in the native copper site and copper is in the native zinc site. At low concentrations, N3- ion displaces axially coordinated water from Cu2Cu2SOD. At higher concentrations, N3- also breaks the imidazolate bridge. Parallel behavior was observed for SCN- and N3- binding to a model compound for Cu2Cu2SOD, showing that the difference between the two anions is a consequence of their copper binding properties and is not due to secondary interactions with the protein active site. The SCN- complex of Cu2Cu2SOD is catalytically active, indicating that the intact imidazolate bridge is not essential to the mechanism of superoxide dismutase action. Phosphate ion breaks the histidine bridge in Cu2Cu2SOD, whereas cyanate, formate, and fluoride ions do not. At about pH 11, hydroxide ion promotes the irreversible formation of a copper complex of deprotonated peptide nitrogen atoms for Cu2Cu2SOD but not for Cu2Zn2SOD in the same pH range.

Animals↗
PubMed1980

Nuclear magnetic resonance study of the exchangeable histidine protons in bovine and wheat germ superoxide dismutases.

Nuclear magnetic resonance studies at 220 MHz of the exchangeable histidine NH protons in bovine erythrocyte superoxide dismutase (BESOD) [EC 1.15.1.1] and the two isoenzymes of wheat germ superoxide dismutase (WGSODI and WGSODII) have been carried out. NMR spectral similarities reveal substantial structural homology of WGSOD with BESOD. Comparison of the spectra of the apoproteins and copper-free, native, and reduced proteins suggests that zinc has a structural role in WGSOD similar to that previously reported for BESOD [Lippard, S. J., Burger, A. R., Ugurbil, K., Pantoliano, M. W., & Valentine, J. S. (1977) Biochemistry 16, 1136]. Four resonances are assigned to conserved histidine residues, three of which are coordinated to the zinc atom while the fourth is nonligating.

Animals↗
PubMed1979

Binding of platinum(II) intercalation reagents to deoxyribnonucleic acid. Dependence on base-pair composition, nature of the intercalator, and ionic strength.

The DNA binding of three platinum(II) intercalation reagents has been studied and found to depend upon base composition, the nature of the intercalator, and the ionic strength of the solvent medium. In 0.2 M NaCl, binding data for calf thymus DNA show the association constants to be approximately 10(4) M-1. The binding constants decrease in the order [(o-phen)Pt(en)]2+ greater than or equal to [(terpy)Pt(HET)]+ greater than [(bipy)Pt(en)]2+. The number of available intercalation sites for the doubly charged intercalators is only 70% of the number expected from the nearest-neighbor exclusion model. Binding of [(o-phen)Pt(en)]2+ and [(terpy)Pt(HET)]+ to various DNAs depends linearly on G.C content. Both reagents exhibit essentially the same degree of G.C specificity. Intercalative binding is a function of ionic strength. Increasing the salt concentration minimizes the importance of metallointercalator charge, and extrapolation to 1 M salt reveals the intercalative abilities, as reflected in binding constants, to be equivalent for [(terpy)Pt(HET)]+ and [o-phen)Pt(en)]2+ and about 1 order of magnitude less than that of ethidium.

Animals↗
PubMed1979

Visualization of polymercurimethane-labeled for bacteriophage in the scanning transmission electron microscope.

Each of the 2700 coat proteins of fd bacteriophage was labeled with tetrakis(acetoxymercuri)methane (TAMM) or aquoglycylmethionineplatinum(II). The TAMM-labeled specimens reveal striking bright spots in the scanning transmission electron microscope which arise from clustering. Measurements of mass show increases consistent with the addition of four mercury atoms or one platinum atom, respectively, to each coat protein.

Coliphages↗
PubMed1979

Cooperative binding of a platinum metallointercalation reagent to poly(A).poly(U).

The cationic complex (2-hydroxyethanethiolato)(2,2',2''-terpyridine)platinum(II), [(terpy)Pt(HET)]+, binds cooperatively to poly(A).poly(U) by intercalation. The melting temperature of poly(A).poly(U) in low-salt buffer is increased by 6 degrees C in the presence of [(terpy)Pt(HET)]+, indicating stabilization of the duplex structure by the bound platinum reagent. Viscosity measurements provide evidence for comparable lengthening of the polynucleotide in the presence of [(terpy)Pt(HET)]+ and the intercalating dye, ethidium bromide. Scatchard plots of the binding of [(terpy)Pt(HET)]+ to poly(A).poly(U) and poly(I).poly(C), determined through ultracentrifugation pelleting methods, show large positive curvature, reflecting the strong cooperativity associated with the platinum complex-RNA interaction. The characteristics of the binding isotherms are interpreted in terms of a model where cooperative pair units of [(terpy)Pt(HET)]+ intercalate into the double-stranded polymer. At saturation, two platinum molecules are bound for every three base pairs. This stoichiometry may be compared with the nearest-neighbor-exclusion binding observed previously in the interaction of [(terpy)Pt(HET)]+ and the ethidium cation with DNA, in which one intercalator occupies every other interbase-pair site at saturation. The striking differences observed in the interaction of [(terpy)Pt(HET)]+ with DNA and RNA suggest that drug recognition is sensitive to the constraints imposed by nucleic acid secondary structure.

Chemical Phenomena↗
PubMed1979

Binding of cis- and trans-dichlorodiammineplatinum(II) to DNA: evidence for unwinding and shortening of the double helix.

The antitumor drug cis-dichlorodiammineplatinum(II) (cis-DDP) and the inactive trans isomer bind and produce cooperative changes in closed and nicked circular duplex DNA's. Covalent binding of both platinum complexes to the closed circular DNA alters the degree of supercoiling, presumably by disrupting and unwinding the double helix. Electron micrographs show the platinated DNA's to be shortened by up to 50 percent of their original length. At similar ratios of bound platinum per nucleotide, the electrophoretic mobilities of the DNA's in gels containing the dye ethidium bromide are the same for both isomers. The only detectable difference in the binding of the two platinum isomers is an increase in the electrophoretic mobility in nondye gels of closed circular DNA having small amounts of bound cis-DDP that is not apparent for the trans complex.

Cisplatin↗