Methylation of chloroplatinate by methylcobalamin.

Incubation of microM levels of K2PtCl6 and methylcobalamin (MeB-12) results in the complete conversion of MeB-12 to aquoB-12. Demethylation is optimal at pH 2.0 and is accelerated by the addition of K2PtCl4. The reaction is stoichiometric between MeB-12 and K2PtCl6 (1:1). Incubation of 40 microM K2PtCl6 with either 40 microM [Me-14C]MeB-12 or [Me-3H]MeB-12 followed by lyophilization converts 70% of the label to a stable form which is associated with Pt upon subsequent paper chromatography and electrophoresis. There is no preferential loss of 3H relative to 14C in the reaction product. When 50 mumoles each of [Me-14C]MeB-12 and K2PtCl6 were reacted and subjected to column chromatography, a labeled UV-absorbing product was separated with a 14C/Pt ratio of 0.9-1.2. The 14C-Pt product has absorption maxima at 260 nm and 208 nm with a minimum at 240 nm (A240 nm/A260 nm = 0.5). Proton NMR spectroscopy confirmed the presence of an H-C-Pt covalent bonding pattern (J for 1H, 195Pt = 78.2 Hz; tau for 194Pt-Me + 196Pt-Me = 6.956).     

Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

The effects of platinum complexes on seven enzymes.

The effects of K2PtCl4, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 on the activities of glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, fructose-1,6-bisphosphate aldolase, catalase, tyrosinase, and peroxidase have been investigated. All of the enzymes which are thought to have essential sulfhydryl groups (glyceraldehyde-3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase) were significantly inhibited by K2PtCl4. The other four enzymes studied are not known to have essential sulfhydryl groups, and were not significantly affected by the Pt compounds under the conditions employed. Glyceraldehyde-3-phosphate dehydrogenase was the only enzyme inhibited by all three Pt compounds tested, with K2PtCl4 being the most effective and cis-Pt(NH3)2Cl2 the least effective inhibitor. Semilogarithmic plots of residual activity versus inhibition time indicated that the inhibition reactions were not simple first-order processes, except for the inhibition of glucose-6-phosphate dehydrogenase by K2PtCl4 which appeared to be first-order with respect to enzyme concentration.     

A circular dichroism study of DNA.platinum complexes. Differentiation between monofunctional, cis-bidentate and trans-bidentate platinum fixation on a series of DNAs

The circular dichroism (CD) spectra of a series of DNA . platinum complexes are presented. The following platinum compounds, [Pt(dien)Cl]Cl, cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, trans-Pt-(NH3)2Cl2, K[Pt(NH3)Cl3] and K2[PtCl4] were complexed with the DNA extracted from bacteria Micrococcus lysodeikticus (72% dG + dC), Escherichia coli (50% dG + dC), Clostridium perfringens (32% dG + dC) and salmon sperm (41% dG + dC). Strong differences were found between the different DNA . Pt complexes. Three types of spectra clearly demonstrate the different platinum binding modes on DNA. In the first type, the platinum compound, i.e. [Pt(dien)Cl]Cl, is fixed to DNA with only one bond (monofunctional complex formation) and no significant change of the CD positive band of DNA is found. The main feature of the second type is a continuous intensity decrease of the positive band as observed for trans-Pt(NH3)2Cl2 (trans-bidentate complex formation). The third type concerns the cis-bidentate platinum fixation obtained with cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, K[Pt(NH3)Cl3] and K2[PtCl4]. The CD spectra are in this case characterized by an increase in the positive Cotton effect which is dG + dC-dependent up to an rb value around 0.10 (where rb = number of platinum atoms bound per nucleotide), followed by a decrease until DNA saturation with platinum is reached. A linear decrease in the amplitude of the negative band is detected in all the complexes except in the case of the monofunctional DNA . Pt complexes. For the cis-bidentate and trans-bidentate platinum fixation, a continuous bathochromic shift occurs.     

Tracer Studies to Locate the Site of Platinum Ions Within Filamentous and Inhibited Cells of Escherichia coli

The distribution of platinum ions within Escherichia coli after the induction of filaments with cis-Pt(NH(3))(2)Cl(4), and after growth inhibition by PtCl(6) (2-), has been determined with radioactive metal compounds ((191)Pt, with a half-life of approximately 3 days) by the simple chemical procedure of Roberts et al. In the filamentous cells, the platinum metal is associated with metabolic intermediates, nucleic acids, and cytoplasmic proteins; whereas, in inhibited cells, the platinum is combined only with the cytoplasmic protein. Similar experiments with gram-positive cells of Bacillus cereus and Staphyloccus aureus, which show no filamentous growth in the presence of cis-Pt(NH(3))(2)Cl(4), reveal that the metal complex does penetrate the cell wall and subsequently becomes bound predominantly by metabolic intermediates.     

The new antitumor compound, cis-[Pt(NH3)2(4-methylpyridine)Cl]Cl, does not form N7,N7-d(GpG) chelates with DNA. An unexpected preference for platinum binding at the 5'G in d(GpG)

The reaction of the antitumor active agent cis-[Pt(NH3)2(4-mepy)Cl]Cl (4-mepy stands for 4-methylpyridine) with d(GpG) has been investigated by 1H magnetic resonance spectroscopy. Initially, two mononuclear complexes cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(1)] 1 and cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(2)] 2 are formed in an unexpected ratio 65:35, as determined by 1H NMR and enzymatic digestion techniques. Both products react further with a second equivalent of cis-[Pt(NH3)2(4-mepy)Cl]Cl forming the dinuclear platinum complex [cis-Pt(NH3)2(4-mepy)]2[mu-d(GpG)- N7(1),N7(2)] 3. With [Pt(dien)Cl]Cl and [Pt(NH3)3Cl]Cl similar complexes are formed. No evidence was found for the formation of chelates cis-Pt(NH3)(4-mepy) [d(GpG)-N7(1),N7(2)], which would be formed upon ammonia release from the mononuclear complexes 1 and 2. Even addition of strong nucleophiles, like sodium diethyldithiocarbamate, thiourea, cysteine, or methionine, before or after reaction, do not induce the formation of a chelate. Under all conditions the N-donor ligands remain coordinated to Pt in 1,2 and 3. In addition, the results of bacterial survival and mutagenesis experiments with E. coli strains show that the in vivo formation of bifunctional adducts in DNA, comparable to those induced by cis-Pt(NH3)2Cl2, by treatment of cells with cis-[Pt(NH3)2(4-mepy)Cl]Cl is unlikely. Also, a mechanism of binding and intercalation is not supported by experimental data. All experiments suggest that the mechanism of action of this new class of antitumor agents must be different from that of cis-Pt(NH3)2Cl2.     

Reactions of cisplatin hydrolytes with methionine, cysteine, and plasma ultrafiltrate studied by a combination of HPLC and NMR techniques

The reactions of cis-[PtCl(NH3)2(H2O)]+ with L-methionine have been studied by 1D 195Pt and 15N NMR, and by 2D[1H, 15N] NMR. When the platinum complex is in excess, the initial product, cis-[PtCl(NH3)2(Hmet-S)]+ undergoes slow ring closure to [Pt(NH3)2(Hmet-N,S)]2+. Slow ammine loss then occurs to give the isomer of [PtCl(NH3)(Hmet-N,S)]+ with chloride trans to sulfur. When methionine is in excess, a reaction sequence is proposed in which trans-[PtCl(NH3)(Hmet-S)2]+ isomerises to the cis-isomer, with subsequent ring closure reactions leading to cis-[Pt(Hmet-N,S)2]2+. Near pH 7, methionine is unreactive toward cis-[PtCl(OH)(NH3)2]. By contrast, L-cysteine reacts readily with cis-[PtCl(OH)(NH3)2] at pH 7, but there were many reaction products, including bridged species. Cis-[PtCl(OH)(NH3)2] reacts with reduced thiols in ultrafiltered plasma but these are oxidized if the plasma is not fresh or appropriately stored. With very low concentrations of the platinum complexes (35.5 microM), HPLC experiments (UV detection at 305 nm) indicate that the thiolate (probably cysteine) reactions become simpler as bridging becomes less important.     

Chiral differentiation of DNA adducts formed by enantiomeric analogues of antitumor cisplatin is sequence-dependent

1,2-GG intrastrand cross-links formed in DNA by the enantiomeric complexes [PtCl(2)(R,R-2,3-diaminobutane (DAB))] and [PtCl(2)(S,S-DAB)] were studied by biophysical methods. Molecular modeling revealed that structure of the cross-links formed at the TGGT sequence was affected by repulsion between the 5'-directed methyl group of the DAB ligand and the methyl group of the 5'-thymine of the TGGT fragment. Molecular dynamics simulations of the solvated platinated duplexes and our recent structural data indicated that the adduct of [PtCl(2)(R,R-DAB)] alleviated this repulsion by unwinding the TpG step, whereas the adduct of [PtCl(2)(S,S-DAB)] avoided the unfavorable methyl-methyl interaction by decreasing the kink angle. Electrophoretic retardation measurements on DNA duplexes containing 1,2-GG intrastrand cross-links of Pt(R,R-DAB)(2+) or Pt(S,S-DAB)(2+) at a CGGA site showed that in this sequence both enantiomers distorted the double helix to the identical extent similar to that found previously for the same sequence containing the cross-links of the parent antitumor cis-Pt(NH(3))(2)(2+) (cisplatin). In addition, the adducts showed similar affinities toward the high-mobility-group box 1 proteins. Hence, whereas the structural perturbation induced in DNA by 1,2-GG intrastrand cross-links of cisplatin does not depend largely on the bases flanking the cross-links, the perturbation related to GG cross-linking by bulkier platinum diamine derivatives does.     

Effect of pKM101 on cell killing and specificity of mutation induction by cis-diaminedichloroplatinum(II) in Escherichia coli K-12.

Cell killing and mutation induction in the lacI gene of Escherichia coli by cis-Pt(NH3)2Cl2 were studied in cells with different repair capacities, with and without pKM101. The presence of the plasmid pKM101 made repair-proficient cells more susceptible to killing by cis-Pt(NH3)2Cl2 and strongly enhanced mutation induction by that compound. Both effects were shown to be dependent upon excision repair. Characterization of the induced mutations in the lacI gene after cis-Pt(NH3)2Cl2 treatment of E. coli cells, by the LacI system, revealed that the mutagenic specificity of the Pt compound was strongly influenced by the presence of the pKM101 plasmid. With pKM101, 23% of the induced amber and ochre mutations resulted from substitutions at AT base pairs, whereas these mutations were hardly induced in cells without pKM101. These results suggest that pKM101-induced repair differs from normal SOS repair.     

DNA-platinum interactions in vitro with trans- and cis-Pt(NH3)2Cl2.

In the present study the nature and the hydrolysis of DNA-Pt complexes with the platinum compounds, [Pt(dien)Cl]Cl, trans- and cis-Pt(NH3)2Cl2, using potentiometric chloride determinations, have been investigated. The trans-Pt(NH3)2Cl2 and the [Pt(dien)Cl]Cl react with the GC planes at the N7(G) sites, while the cis-Pt(NH3)2Cl2 compound reacts with the GC planes and forms a chelate by using the N7(G) and O6(G) sites. The complex is a specific 1:1 Pt:DNA adduct. The platinum atom in cis-Pt(NH3)2Cl2 liberates both chlorine atoms on chelation. A mechanism for the in vivo antitumor activity of the cis-Pt(NH3)2Cl2 is proposed and the structure activity relationship is discussed.     

Reaction of cis- and trans-[PtCl2(NH3)2] with reduced glutathione inside human red blood cells, studied by 1H and 15N-[1H] DEPT NMR

Reactions of cis- and trans-[PtCl2(NH3)2] with glutathione (GSH) inside intact red blood cells have been studied by 1H spin-echo nuclear magnetic resonance (NMR). Upon addition of trans-[PtCl2(NH3)2] to a suspension of red cells, there was a gradual decrease in the intensity of the resonances for free GSH, and new peaks were observed that were assignable to coordinated GSH protons in trans-[Pt(SG)Cl(NH3)2], trans-[Pt(SG)2(NH3)2], and possibly the S-bridged complex trans-[[NH3)2PtCl)2SG]+. Formation of trans-[Pt(SG)2(NH3)2] inside the cell was confirmed from the 1H NMR spectrum of hemolyzed cells, which were ultrafiltered to remove large protein molecules; the ABM multiplet of the coordinated GSH cys-beta CH2 protons was resolved using selective-decoupling experiments. Seventy percent of the total intracellular GSH was retained by the ultrafiltration membrane, suggesting that the mixed complex trans-[Pt(SG)(S-hemoglobin)(NH3)2] also is a major metabolite of trans-[PtCl2(NH3)2] inside red cells. The reaction of cis-[PtCl2(NH3)2] with intracellular GSH was slower; only 35% of the GSH had been complexed after a 4-hr incubation compared to 70% for the trans isomer. There was a gradual decrease in the intensity of the GSH 1H spin-echo NMR resonances, but no new peaks were resolved. This was interpreted as formation of high-molecular weight Pt:GSH and mixed GS-Pt-S(hemoglobin) polymers. By using a 15N-[1H] DEPT pulse sequence, we were able to study the reaction of cis-[PtCl2(15NH3)2] with red cells at concentrations as low as 1 mM. 15NH3 ligands were released, and no resonances assignable to Pt-15NH3 species were observed after a 12-hr incubation.     

Immunochemical quantitation of adducts induced in DNA by cis-diamminedichloroplatinum (II) and analysis of adduct-related DNA-unwinding

Antibodies elicited against the haptens cis-Pt(NH3)2dGuodGMP and its ribo-analog, both covalently coupled to bovine serum albumin, recognize adducts of cis-diamminedichloroplatinum(II) (cis-DDP) in DNA. Antibody-binding to cis-DDP-DNA strongly depends on the accessibility of the adducts to the antibodies. In double-stranded cis-DDP-DNA with low Pt: nucleotide ratios (rb's), this accessibility is enhanced by unwinding of the cis-DDP-DNA, e.g. by heat-denaturation. An unwinding effect is also induced by the cis-DDP treatment itself. A260nm readings of cis-DDP-DNA samples indicate an increased denaturation of the DNA at increasing Pt-contents. The data obtained after heat-denaturation of the same samples show a growing capability to renaturation when the rb-values increase from 0 to 0.04; at 0.04 less than rb less than 0.18 the renaturation effect gradually disappears. In the competitive enzyme-linked immunosorbent assay (ELISA), the cis-DDP-adducts in heat-denatured DNA are detected in the pmol range; in DNA-digests, however, they are recognized in fmol amounts. For the individual Pt-containing (oligo)nucleotides the amounts causing 50% inhibition in the ELISA were established for the two anti-sera; they were 13.3 +/- 3.8 (fmol +/- S.D.) and 5.4 +/- 1.8 for cis-Pt(NH3)2d(GMP)2; 15.5 +/- 5.4 and 4.0 +/- 1.5 for cis-Pt(NH3)2d(pGpG); (2.6 +/- 1.1) X 10(3) and (2.0 +/- 1.0) X 10(3) for cis-Pt(NH3)2d(pApG); (5.6 +/- 1.9) X 10(3) and (2.9 +/- 0.4) X 10(3) for Pt(NH3)3dGMP. Pt-adducts in a trans-DDP-DNA digest are recognized in pmol amounts and dGMP in nmol quantitatives. Finally, the usefulness of these antibodies for the detection and quantitation of individual cis-DDP-adducts in cis-DDP-DNA digests was demonstrated.     

Mechanism of the reversal reaction of platinated DNA with thiourea studied by platinated 5'-GMP

The reversal reactions of cis-[Pt(NH3)2(5'-GMP)2] 2-(1) and trans-[Pt(NH3)2(5'-GMP)2] 2-(2) with thiourea were examined by reversed phase HPLC and monothioureido intermediate cis-[Pt(NH3)2(5'-GMP) (tu)] (4) was detected. This result suggested that Pt-[5'-GMP-N(7)] bond was more labile than Pt-NH3 bond and the release of ammonia from cis-Pt(II)-DNA base complexes is a result of trans-labilizing effect of sulfur containing molecule displaced with DNA base.     

Preparation of platinum(IV) complexes with dipeptide and diimine. X-ray crystal structure and 195Pt NMR spectra

We prepared platinum(IV) complexes containing dipeptide and diimine or diamine, the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complex, where -N,N,O means dipeptide coordinated as a tridentate chelate, dipeptide=glycylglycine (NH(2)CH(2)CON(-)CH(2)COO(-), digly, where two protons of dipeptide are detached when the dipeptide coordinates to metal ion as a tridentate chelate), glycyl-L-alanine (NH(2)CH(2)CON(-)CHCH(3)COO(-), gly-L-ala), L-alanylglycine (NH(2)CH CH(3)CON(-)CH(2)COO(-), L-alagly), or L-alanyl-L-alanine (NH(2)CHCH(3)CON(-)CHCH(3)COO(-), dil-ala), and diimine or diamine=bipyridine (bpy), ethylenediamine (en), N-methylethylenediamine (N-Me-en), or N,N'-dimethylethylenediamine (N,N'-diMe-en). In the complexes containing gly-L-ala or dil-ala, two separate peaks of the (195)Pt NMR spectra of the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complexes appeared in, but in the complexes containing digly or L-alagly, one peak which contained two overlapped signals appeared. One of the two complexes containing gly-L-ala and bpy, [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3), crystallized and was analyzed. This complex has the monoclinic space group P2(1)2(1)2(1) with unit cell dimensions of a=9.7906(3)A, b=11.1847(2)A, c=16.6796(2)A, Z=4. The crystal data revealed that this [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex has the near- (Cl, CH(3)) configuration of two possible isomers. Based on elemental analysis, the other complex must have the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) configuration. The (195)Pt NMR chemical shifts of the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex and the far- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex are 0 ppm and -19 ppm, respectively (0 ppm for the Na(2)[PtCl(6)] signal). The additive property of the (195)Pt NMR chemical shift is discussed. The (195)Pt NMR chemical shifts of [PtCl(dipeptide-N,N,O)(bpy)]Cl appeared at a higher field when the H attached to the dipeptide carbon atom was replaced with a methyl group. On the other hand, the (195)Pt NMR chemicals shifts of [PtCl(dipeptide-N,N,O)(diamine)] appeared at a lower field when the H attached to the diamine nitrogen atom was replaced with a methyl group, in the order of [PtCl(digly-N,N,O)(en)]Cl, [PtCl(digly-N,N,O)(N-Me-en)]Cl, and [PtCl(digly-N,N,O)(N,N'-diMe-en)]Cl.     

Ouabain-resistant human lymphoblastoid lines altered in the (Na+ + K+)-dependent ATPase membrane transport system.

Diploid human lymphoblastoid cells with altered response to ouabain inhibition of the (Na+ + K+)-dependent ATPase transport system, manifest both in whole cells and in purified plasma membrane vesicles, were selected for their resistance to 0.1 muM ouabain. Ouabain-resistant (OUA(R)) cells with normal growth at 50 times this dose were recovered at a frequency 1 X 10(-6). This frequency was increased 9-fold after exposure to ethyl methane sulphonate but was decreased by the frameshift mutagen ICR-191, under conditions where both increased the frequency of 8-azaguanine-resistant colonies. The ouabain resistance phenotype was stable after 200 population doublings in the absence of ouabain. OUA(R) clones show showed 30-50% of the wild type amount of 3H-ouabain bound per cell, with the same dissociation constant for ouabain, 0.1 muM at 0.5 mM K+, as observed in wild-type cells. Both the initial rate of uptake of 86Rb+ in OUA(R) cells and the (Na+ + K+)-dependent ATPase activity of OUA(R) plasma membranes showed decreased sensitivity to ouabain inhibition. However, growth and transport properties of OUA(R) cells in the absence of ouabain were unchanged compared with wild type cells.     

Influence of the position of substituents in the cytotoxic activity of trans platinum complexes with hydroxymethyl pyridines

The synthesis and chemical characterization of two trans platinum complexes, (1) trans-[PtCl(2)NH(3)(2-hydroxymethylpyridine)] and (2) trans-[PtCl(2)NH(3)(3-hydroxymethylpyridine)], are described. The structures and chemical behaviour of these compounds have been compared to those of their isomer (3) trans-[PtCl(2)NH(3)(4-hydroxymethylpyridine)] previously studied. X-ray structures of all of them were solved and some interesting differences were found. The values of the dihedral angle (85 degrees , 57 degrees and 42 degrees for 1, 2 and 3, respectively) demonstrate how important is the position of substituent from a structural point of view. Studies of circular dichroism (CD), electrophoretic mobility (EM) in agarose gel and atomic force microscopy (AFM) showed differences in the modifications caused by the three complexes on DNA. Studies of antiproliferative activity of complexes 1 and 2 against cell tumour lines (HL-60) and apoptosis assays have also been carried out, showing that 1 as well as 2 are far less active than the previously described complex 3 (IC(50)=19; 19 and 3 microM, respectively). This fact probes that slight modifications on the drug's design may generate significant differences in the final antitumour activity by modifying the DNA-drug adducts, performance of resistance mechanisms and all the factors that play a fundamental role in Pt complexes' cytotoxicity.     

An in vivo highly antitumor-active tetrazolato-bridged dinuclear platinum(II) complex largely circumvents in vitro cisplatin resistance: two linkage isomers yield the same product upon reaction with 9-ethylguanine but exhibit different cytotoxic profiles

Cytotoxicity assays of azolato-bridged dinuclear Pt(II) complexes, [{cis-Pt(NH(3))(2)}(2)(μ-OH)(μ-azolato)](2+), where the azolato was pyrazolato (1), 1,2,3-triazolato-N1,N2 (2), tetrazolato-N1,N2 (3), or tetrazolato-N2,N3 (4), were performed in cisplatin-sensitive and -resistant human non-small-cell lung cancer cell lines (PC-9 and PC-14). These complexes largely circumvented the cisplatin resistance in both cell lines, with resistance factors for 1-4 in the range of 0.5-0.8 and 0.9-2.0 for PC-9 and PC-14 cells, respectively. Complex 4 exhibited approximately 10 times the cytotoxicity of 3. When 3 and 4 were reacted with 2 molar equiv. of 9-ethylguanine (9EtG), they yielded an identical product, [{cis-Pt(NH(3))(2)(9EtG-N7)}(2)(μ-tetrazolato-N1,N3)](3+), that had N1,N3 platinum coordination through a Pt(II) migration process on the tetrazolate ring. The second-order rate kinetics of these isomers were almost the same as each other and faster than those of 1 and 2. The cytotoxicity of azolato-bridged complexes, except for 3, increases as their kinetic rates in the 9EtG reaction increase.     

Dissociation of double-stranded poly(I) . poly(C) by cis-diammine-dichloro-Pt(II).

The covalent binding of cis-Pt(NH3)2Cl2 on the double stranded poly(I) . poly(C) induced an irreversible dissociation of the two strands. This dissociation was evidenced mainly by poly(I)-Agarose affinity chromatography which allowed to recover free strands of cis-Pt(NH3)2Cl2-poly(I) from a cis-Pt(NH3)2Cl2-poly(I) . poly(C) complex, by density equilibrium centrifugation where free poly(C) could be isolated, and by acid titrations of the metal-poly(I) . poly(C) complexes. The separation of the two strands of the polyribonucleotide upon cis-Pt(NH3)2Cl2 fixation was shown not to exceed 90--95%. A dissociation curve of the polynucleotide double helix as a function of the amount of bound cis-Pt(NH3)2Cl2 was determined and was shown to be of a characteristic cooperative effect. The fixation of the paltinum compound to poly(I) . poly(C) seemed also to be cooperative.     

Evolution of a recombinant (gucoamylase-producing) strain of Fusarium venenatum A3/5 in chemostat culture

Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited chemostat cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 chemostat. The highly branched mutants detected in this chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 chemostat complemented representative strains previously isolated from glucose-limited chemostat cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 chemostat culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates.     

The reaction of Pt-antitumor drugs with selected nucleophiles. II. Preparation and characterization of coordination compounds of Pt(II) and L-histidine

Various His-Pt(II) coordination compounds were prepared by reaction of K2PtCl4 or cis-[Pt(NH3)2Cl2](cis-DDP) with His and analyzed by 1H and 13C NMR spectroscopy, electrophoresis, and ion-exchange chromatography. His may be coordinated to Pt by the imidazol iminogroup and/or the alpha-aminogroup; the carboxygroup remains always free. Both bidentate as well as monodentate ligands were identified. Cis-DDP reacts with His to give a mixture of compounds where all these possibilities are present: cis-diamine-(histidine-N,N-)Pt(II) and three different types of cis-diammine-bis(histidine). HCl trans cleavage of compounds with bidentate His ligands leads to a mixture of two compounds having His ligated to Pt by an amino or imin group. The methods applied are suitable for analyzing reactions of His with cis-DDP under model conditions similar to physiological conditions.