DNA repair: enzymatic mechanisms and relevance to drug response.

A number of chemotherapeutic agents, such as platinum drugs, nitrogen mustards, and chloroethylnitrosoureas, act by forming bifunctional DNA adducts. It is likely that abortive attempts to replicate and/or repair the damaged DNA cause chromosome aberrations and breakage, leading to cell death. Any substantial increase in cellular capacity to repair damaged DNA may result in resistance to chemotherapeutic agents. In this review, we examine the types of DNA adducts formed by the major classes of chemotherapeutic agents, the enzymatic pathways that play a role in the repair of those adducts, the evidence that DNA repair is enhanced in drug-resistant cell lines and tumors, and strategies for utilizing selective inhibition of DNA repair to overcome resistance.

Role of carrier ligand in platinum resistance of human carcinoma cell lines.

We have examined the effects of the cis-diammine and 1,2-diaminocyclohexane (dach) carrier ligands on cytotoxicity, platinum accumulation and efflux, platinum incorporation into DNA, cytotoxicity of Pt-DNA adducts, and repair of Pt-DNA adducts in the human ovarian carcinoma A2780 cell line, the human colon carcinoma HCT8 cell line, and their cis-diamminedichloroplatinum(II) (cisplatin)-resistant derivatives, A2780/DDP and HCT8/DDP. The A2780/DDP cell line was 7.7-fold resistant to cisplatin, and the HCT8/DDP cell line was 1.6-fold resistant to cisplatin compared to their parental cell lines. Both were considered as examples of acquired cisplatin resistance. The HCT8/S cell line was 4.6-fold resistant to cisplatin compared with the A2780/S cell line and was considered an example of intrinsic resistance. Decreased accumulation of cisplatin made a significant contribution to acquired cisplatin resistance in the A2780/DDP cell line, probably contributed to intrinsic resistance in the HCT8/S cell line, but made little or no contribution to acquired resistance in the HCT8/DDP cell line. Decreased cytotoxicity of Pt-DNA adducts made a major contribution to both acquired and intrinsic cisplatin resistance in all three cell lines. Increased repair activity made a significant contribution to the decreased cytotoxicity of Pt-DNA adducts in the HCT8/S cell line, a weak contribution in the A2780/DDP cell line, and no contribution in the HCT8/DDP cell line. Glutathione levels were elevated in all the cell lines with acquired and intrinsic resistance, but the increased glutathione levels were not associated with decreased incorporation of platinum into DNA. These data suggest that both decreased accumulation and increased repair contribute to cisplatin resistance to different degrees in these human carcinoma cell lines. In addition, mechanism(s) other than repair may contribute to the decreased cytotoxicity of cis-diammine-Pt-DNA adducts. Of the cells with acquired cisplatin resistance, the HCT8/DDP cell line showed no resistance to tetrachloro(trans-DL)1,2-diaminocyclohexaneplatinum(IV) (ormaplatin, formerly known as tetraplatin), while the A2780/DDP cell line was just as resistant to ormaplatin as to cisplatin. The intrinsically cisplatin-resistant HCT8/S cell line showed only partial cross-resistance to ormaplatin. The effects of the dach carrier ligand on both acquired and intrinsic resistance in these cell lines appeared to occur primarily at the level of cytotoxicity of dach-Pt adducts, but the differences in the cytotoxicity of cis-diammine-Pt and dach-Pt adducts could not be explained by differences in repair of those adducts.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid reduction of tetrachloro(D,L-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in RPMI 1640 tissue culture medium.

Tetrachloro(D,L-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) is a new platinum analogue which is less nephrotoxic than cisplatin and is effective in some cell lines which have become resistant to cisplatin. Since platinum(IV) compounds are thought to require reduction to their platinum(II) analogues for activity, the biotransformations of tetraplatin and its platinum(II) analogue, dichloro(D,L-trans)1,2-diaminocyclohexaneplatinum(II) [PtCl2(trans-dach)], were studied. For L1210 cells cultured in RPMI 1640 medium, the time course for inhibition of DNA synthesis and cytotoxicity was virtually identical for both drugs. The time course for binding to fetal calf serum in the tissue culture medium was also the same for both drugs. In the complete tissue culture medium, tetraplatin was reduced to PtCl2(trans-dach) with a half-life of 5 to 15 min, depending on the level of protein sulfhydryl in the medium. The major reducing agent in the medium was the protein sulfhydryl, with glutathione and glucose making minor contributions. No other component of the medium reacted with tetraplatin. The rapid reduction of tetraplatin to PtCl2(trans-dach) was followed by much slower substitution reactions involving the chloro ligands of PtCl2(trans-dach). The major transformation products which accumulated in RPMI 1640 medium were identical for both drugs. These data suggest that tetraplatin should be considered a prodrug which is very rapidly converted to PtCl2(trans-dach) with subsequent biotransformations as expected for the platinum(II) analogue. These data also indicate that in tissue culture most of the tetraplatin is reduced extracellularly.

In vitro biotransformations of tetrachloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in rat plasma.

The in vitro biotransformation of tetrachloro(d,l-trans)-1,2,-diaminocyclohexaneplantinum(IV) (tetraplatin) in the plasma of Fischer 344 rats were studied by the two-column high-performance liquid chromatography technique described previously (Mauldin et al., Cancer Res., 48: 5136-5144, 1988). The reduction of tetraplatin to dichloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(II) [PtCl2(dach)] was extremely rapid. From experiments with diluted plasma, it was possible to estimate a t1/2 for tetraplatin of approximately 3 s at 37 degrees C in undiluted plasma. By titrating with N-ethylmaleimide, it was possible to show that sulfhydryl groups were responsible for 70-80% of the total reducing potential of plasma. The rapid reduction of tetraplatin to PtCl2(dach) was followed by slower substitution reactions involving the chloro ligands of PtCl2(dach). The t1/2 for PtCl2(dach) in plasma at 37 degrees C was 1.5 h. The monoaquamonochloro complex was an important biotransformation product at early times, reaching 10 to 12% of the total platinum present from 15 min to 2 h, when it was gradually replaced with more stable biotransformation products. Three major stable biotransformation products accumulated in the plasma. One of these biotransformation products was identified as the Pt(methionine)(dach) complex. The other two were tentatively identified as the Pt(cysteine)(dach) or Pt(ornithine)(dach) complex and the Pt(urea)(dach) or Pt(citrato)(dach) complex on the basis of coelution in two different high-performance liquid chromatography separation systems. These biotransformation products could play a role in tetraplatin effectiveness and/or toxicity.

Effects of the bidentate malonate ligand on the utilization and cytotoxicity of platinum compounds in the L1210 cell line.

Previous studies have investigated the cytotoxicity of platinum(II) compounds with bidentate leaving ligands, but little is known about their uptake or mechanism of action inside the cell. We have compared the uptake and intracellular effects of cis-1,2-diaminocyclohexanemalonatoplatinum(II) [Pt(mal)(cis-DACH)] and cis-1,2-diaminocyclohexanedichloroplatinum(II) [PtCl2(cis-DACH)] in the L1210 cell line. In the 3-h colony formation assay, PtCl2(cis-DACH) is almost 10 times more cytotoxic than Pt(mal)(cis-DACH). However, when the time of incubation with platinum drug is increased from 3 to 24 h the cytotoxicity of PtCl2(cis-DACH) changes relatively little while the cytotoxicity of Pt(mal)(cis-DACH) increases over 10-fold. As a consequence, Pt(mal)(cis-DACH) is only slightly less cytotoxic than the dichloro compound by 24 h. These data can readily be explained by differences in the time course of uptake. Pt(mal)(cis-DACH) is initially taken by the cell at a much slower rate than PtCl2(cis-DACH), but the uptake continues linearly for at least 24 h with the malonate compound, while uptake for the dichloro compound begins to plateau between 5 and 8 h. The earlier plateauing of PtCl2(cis-DACH) uptake is most likely to be due to a quicker approach to equilibrium between intracellular and extracellular concentrations of unchanged drug which is caused by a more rapid inactivation of PtCl2(cis-DACH) in the media coupled with the more rapid uptake of PtCl2(cis-DACH) by the cells. When compared at equal cytotoxicity, the uptake of platinum into the intracellular pool of low molecular weight platinum compounds is significantly greater for Pt(mal)(cis-DACH) even though the rate of platinum incorporation into DNA is essentially the same for both compounds at early times. These data suggest that a smaller percentage of the low molecular weight platinum is in a reactive form inside the cell for Pt(mal)(cis-DACH). While the inhibition of cell survival exactly parallels incorporation of platinum into DNA for both compounds, there is a significant lag before the onset of inhibition of DNA synthesis for Pt(mal)(cis-DACH). This observation suggests that the conversion of monoadducts to diadducts may be slower for platinum compounds with bidentate leaving ligands.

Effect of the chemoprotective agent WR-2721 on disposition and biotransformations of ormaplatin in the Fischer 344 rat bearing a fibrosarcoma.

The effects of the phosphorothioate agent, WR-2721, have been investigated with respect to the biotransformations of ormaplatin in the Fischer 344 rat bearing a transplanted fibrosarcoma. A number of different paradigms of dosing route and schedule for the administration of the two agents have been investigated. In the first group of experiments, WR-2721 (200 mg/kg, i.p.) was administered 30 min before ormaplatin (12.5 mg/kg, i.p.), and then peritoneal fluid, plasma, and tissues were harvested at 30 min after the ormaplatin administration. Our results suggest that a significant interaction between WR-2721 and ormaplatin is occurring in the peritoneal cavity. The interaction was evident in terms of both effects on distribution and disposition of total platinum and in alterations of the profiles of biotransformation products formed in the various tissues and fluids. Plasma protein binding of ormaplatin was decreased by 50% in the presence of WR-2721. Total platinum in the spleen was decreased by 66% and in the liver by 50%. There were no trends among the findings that would indicate any selectivity between tumor and nontumor tissue with respect to the effects of WR-2721 on the parameters measured. Subsequent investigations examined the effects of dosing the WR-2721 by the i.v. route while continuing with the i.p. administration of the ormaplatin. WR-2721 was administered either 30 or 5 min before the ormaplatin, and the plasma and tissues were harvested at 15, 30, or 60 min after ormaplatin administration. The reverse-phase HPLC peak, which behaved chromatographically as a Pt(dach)(WR-1065) standard, was less prominent after the i.v. administration of WR-2721 than it was after i.p. administration under any of the paradigms tested. There was again no evidence for selectivity between tumor and nontumor tissue in the findings from any of the paradigms. It is concluded that if WR-2721 is capable of selectively protecting nontumor tissue from the toxicities of platinum-based chemotherapy, it is doing so by some mechanism other than its selective uptake into normal tissue and subsequent nonspecific inactivation of any reactive cytosolic platinum species formed. Other possible mechanisms are briefly discussed.

An unexpected biotransformation pathway for tetrachloro-(d,l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in the L1210 cell line.

Tetrachloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) has been considered a prodrug which would be converted rapidly to dichloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(II) [PtCl2(dach)] under physiological conditions. However, the biotransformations of tetraplatin have not been studied in detail. We have followed the intracellular biotransformations of tetraplatin and PtCl2(dach) in the L1210 cell line by a two-step high performance liquid chromatography separation procedure described previously (Mauldin et al., Cancer Res., 48: 5136-5144, 1988). At early times the intracellular biotransformation pathways appeared to be very different in tetraplatin- and PtCl2(dach)-treated cells. The tetraplatin present in the medium initially was taken up preferentially by the L1210 cells. However, no intracellular tetraplatin and very little intracellular PtCl2(dach) were found in the tetraplatin-treated cells. Instead, two previously unidentified biotransformation products predominated at early times. The same biotransformation products were present in cells incubated in Hank's balanced salt solution, so they most likely did not arise from extracellular reactions. The unidentified biotransformation products present in tetraplatin-treated cells at early times appeared to be at the platinum(II) level of oxidation. Model reactions suggested that these compounds could have been formed by platinum(II)-assisted platinum(IV) substitution reactions, followed by reduction of the platinum(IV) complex to the platinum(II) level. Thus, there appear to exist unique features of tetraplatin metabolism which are observed only when tetraplatin is taken up directly by the cell without prior reduction. These reaction products did not react with DNA and presumably represent an inactivation pathway.

Intracellular biotransformation of platinum compounds with the 1,2-diaminocyclohexane carrier ligand in the L1210 cell line.

We have previously reported the development of a two-column high performance liquid chromatography system for separation of platinum(II) complexes with the 1,2-diaminocyclohexane (DACH) carrier ligand (Mauldin et al., Anal. Biochem., 157: 129, 1986). Here we report the application of this technique to the study of the intracellular biotransformations of (DL)-trans-1,2-diaminocyclohexanemalonatoplatinum(II) [PtCl2(trans-DACH)] and (DL)-trans-1,2-diaminocyclohexanemalonatoplatinum(II) [Pt(mal)(trans-DACH)] in the L1210 cell line. The two-column high performance liquid chromatography system allowed separation and identification of both parent drugs and intracellular biotransformation products containing glutathione, methionine, cysteine, arginine, lysine, aspartate or glutamate, and serine or threonine. With the exception of the platinum-glutathione complex, the relative abundance of each biotransformation product was independent of drug concentration. The relative abundance of the platinum-glutathione biotransformation product increased with increasing platinum concentration, suggesting that platinum drugs cause an increase in intracellular glutathione levels in a dose-dependent manner. This hypothesis was verified by direct measurement of intracellular glutathione levels. In continuous uptake experiments, the intracellular levels of the parent compounds peaked between 2 and 5 h and declined to negligible levels by 24 h. In pulse-chase experiments, the chemical t1/2 for PtCl2(trans-DACH) and Pt(mal)(trans-DACH) inside the cell at 37 degrees C was determined to be 12-15 and 21-28 min, respectively. This is far shorter than previously determined rates for the displacement of either ligand in vitro. The platinum-amino acid complexes accumulated gradually throughout the 24-h incubation. The free trans-DACH carrier ligand also accumulated to a level approaching 20% of filterable counts during the 24-h incubation, probably due to trans-labilization of the carrier ligand by sulfur-containing nucleophiles. A combination of reverse phase high performance liquid chromatography and a DNA binding assay was used to identify and quantitate the reactive biotransformation products. As expected from previous studies (Mauldin et al., Cancer Res., 46: 2876, 1986), the PtCl2(trans-DACH)-treated cells had approximately 3 times more reactive platinum biotransformation product at early times, but the levels of reactive biotransformation product fell much more rapidly than in Pt(mal)(trans-DACH)-treated cells. In the PtCl2(trans-DACH)-treated cells, the major reactive biotransformation product was the aquachloro species at all time points tested. In Pt(mal)(trans-DACH)-treated

Efficient nucleotide excision repair of cisplatin, oxaliplatin, and Bis-aceto-ammine-dichloro-cyclohexylamine-platinum(IV) (JM216) platinum intrastrand DNA diadducts.

Tumors exhibit a spectrum of cellular responses to chemotherapy ranging from extreme sensitivity to resistance, either intrinsic or acquired. These variable responses are both patient and tumor specific. For platinum DNA-damaging agents, drug resistance depends on the carrier ligand of the platinum complex and is due to a combination of mechanisms including DNA repair. Nucleotide excision repair is the only known mechanism by which bulky adducts, including those generated by platinum chemotherapeutic agents, are removed from DNA in human cells. In this report, we show that the types of DNA lesions generated by three platinum drugs, cisplatin, oxaliplatin, and (Bis-aceto-ammine-dichloro-cyclohexylamine-platinum(IV) (JM216), are repaired in vitro with similar kinetics by the mammalian nucleotide excision repair pathway.

Role of carrier ligand in platinum resistance in L1210 cells.

We have examined the effect of carrier ligands on platinum accumulation, incorporation of platinum into DNA, cytotoxicity of Pt-DNA adducts, and repair of Pt-DNA adducts in three L1210 cell lines: L1210/0, which is sensitive to most types of platinum compounds; L1210/DDP, which is resistant to platinum compounds with the ethylenediamine (en) carrier ligand but sensitive to those with the diaminocyclohexane (dach) ligand; and L1210/DACH, which is resistant to dach-Pt compounds but sensitive to en-Pt compounds. There was a selective decrease in accumulation of dach-Pt in the L1210/DACH line and of en-Pt in the L1210/DDP line. Intracellular dach-Pt was incorporated into DNA to a lesser extent than en-Pt in both resistant cell lines. Cytotoxicity of en-Pt adducts was less than that of dach-Pt adducts in the L1210/DDP line, while the reverse was true in the L1210/DACH line. Increased repair was seen in both resistant cell lines; a carrier ligand effect was seen only in the L1210/DDP line, which showed a greater initial rate of repair for en-Pt than dach-Pt adducts. These data suggest that carrier ligand effects seen in resistant cell lines may be due, in part, to differences in accumulation of platinum, repair of Pt-DNA adducts, and tolerance of Pt-DNA adducts.

The role of hMLH1, hMSH3, and hMSH6 defects in cisplatin and oxaliplatin resistance: correlation with replicative bypass of platinum-DNA adducts.

Defects in mismatch repair are associated with cisplatin resistance, and several mechanisms have been proposed to explain this correlation. It is hypothesized that futile cycles of translesion synthesis past cisplatin-DNA adducts followed by removal of the newly synthesized DNA by an active mismatch repair system may lead to cell death. Thus, resistance to platinum-DNA adducts could arise through loss of the mismatch repair pathway. However, no direct link between mismatch repair status and replicative bypass ability has been reported. In this study, cytotoxicity and steady-state chain elongation assays indicate that hMLH1 or hMSH6 defects result in 1.5-4.8-fold increased cisplatin resistance and 2.5-6-fold increased replicative bypass of cisplatin adducts. Oxaliplatin adducts are not recognized by the mismatch repair complex, and no significant differences in bypass of oxaliplatin adducts in mismatch repair-proficient and -defective cells were found. Defects in hMSH3 did not alter sensitivity to, or replicative bypass of, either cisplatin or oxaliplatin adducts. These observations support the hypothesis that mismatch repair defects in hMutL alpha and hMutS alpha, but not in hMutS beta, contribute to increased net replicative bypass of cisplatin adducts and therefore to drug resistance by preventing futile cycles of translesion synthesis and mismatch correction.

Enhanced replicative bypass of platinum-DNA adducts in cisplatin-resistant human ovarian carcinoma cell lines.

We have examined the relationship between cis-diamminedichloroplatinum(II) (cisplatin) resistance and replicative bypass in the human ovarian carcinoma cell lines 2008, A2780, and their respective cisplatin-resistant derivatives C13* and A2780/DDP. Replicative bypass is defined as the ability of a replication complex to proceed past a DNA adduct known to block or stall the complex during synthesis. Previous studies in our laboratory have shown a 3-4-fold increase in the replicative bypass of platinum-DNA adducts in platinum-resistant murine leukemia cell lines [G. R. Gibbons et al, Carcinogenesis (Lond.), 12: 2253-2257, 1991]. To test for this effect in the human lines, we used a steady-state replication assay which measures the inhibition of DNA chain elongation (based on the incorporation of [3H]thymidine into nascent DNA strands) as a function of the number of platinum-DNA adducts present on the DNA following cisplatin treatment. With this technique we demonstrated a 4.5-fold increase in the replicative bypass ability of the C13* line compared to the 2008 line and a 2.3-fold increase in the bypass ability of the A2780/DDP line compared to the A2780 line. To confirm these results, we performed a pulse-chase replication assay on the 2008 and C13* lines. This assay differs from the first in that DNA chain elongation is measured in a time-dependent manner. With the pulse-chase assay we observed a 4.8-fold increase in the replicative bypass ability of the C13* line compared to the 2008 line. We then examined the specificity of this enhanced bypass by repeating the steady-state assay with the 2008 and C13* lines using as damaging agents 1,2-diaminocyclohexanedichloroplatinum(II), UV radiation (producing pyrimidine dimers), and benzo(a)pyrene-7,8-diol-9,10-epoxide. In both cell lines, 1,2-diaminocyclohexanedichloroplatinum(II)-DNA adducts caused a greater inhibition of DNA chain elongation than cisplatin-DNA adducts. The level of enhanced bypass of 1,2-diaminocyclohexanedichloroplatinum(II)-DNA adducts in the resistant line was 2.1-fold (approximately 2-fold less than the level of enhanced bypass observed with cisplatin-DNA adducts). There was no evidence of enhanced bypass in the resistant line when cells were treated with UV light or benzo(a)pyrene-7,8-diol-9,10-epoxide. These results indicate that the bypass response in the C13* line has some degree of specificity for cisplatin adducts. The specificity of bypass in these cell lines coincided well with the specificity of resistance to each agent.(ABSTRACT TRUNCATED AT 400 WORDS)

Escherichia coli mutants deficient in RNA accumulation at high temperature.

A selection technique is described which has permitted isolation of 10 mutants that continue to form protein, but are deficient in the accumulation of rRNA at 42 degrees C. DNA-RNA hybridization experiments have demonstrated that most of these mutants are specifically defective in their ability to synthesize rRNA at the restrictive temperature. The bulk of the pulse-labeled RNA formed in such mutants at 42 degrees C appears to be mRNA, with normal instability as measured in the presence of rifampicin. The remaining mutants appear to synthesize normal levels of rRNA, but that rRNA does not accumulate in a stable form. Since all of these mutants continue to form protein, and most do not accumulate significant levels of ppGpp at 42 degrees C, it appears likely that the shut-off of rRNA synthesis at 42 degrees C does not act through a lesion in the rel locus. Thus, these mutants may reveal another element(s) required to promote ribosomal RNA formation.

Guanine nucleotide metabolism in a mutant strain of Escherichia coli with a temperature sensitive lesion in rRNA synthesis.

We have described a mutant of Escherichia coli (designated 2S142) which shows specific inhibition of rRNA synthesis at 42 degrees C. ppGpp levels increase at the restrictive temperature, as expected. However, when the cells are returned to 30 degrees C, rRNA synthesis resumes before ppGpp levels have returned to normal. Furthermore, when ppGpp levels are decreased by the addition of tetracycline or choramphenicol, rRNA synthesis does not resume at 42 degrees C. Also, a derivative of 2S142 with a temperature-sensitive G factor (which cannot synthesize either protein or ppGpp at 42 degrees C) shows identical kinetics of rRNA shut-off at 42 degrees C as 2S142. Thus, the elevated ppGpp levels in this mutant do not appear to be directly responsible for the cessation of rRNA synthesis at 42 degrees C.

Selective inactivation of rabbit reticulocyte initiation factor eIF-3 by helenalin.

Helenalin, a sesquiterpene lactone which reacts primarily with exposed sulfhydryl groups, was shown to be an effective inhibitor of protein synthesis in rabbit reticulocyte lysates. Optimal inhibition required a 30 min preincubation in the absence of any added thiol compound. Beta-Mercaptoethanol was more effective than reduced glutathione in protecting enzyme sulfhydryl groups from inactivation by helenalin. Using partially fractionated systems, it was possible to show that helenalin had no effect on the elongation reactions or on the formation of the ternary initiation complex. However, the conversion of the ternary complex to the 48 S initiation complex was strongly inhibited. In this assay, only the initiation factor(s) were sensitive to helenalin. Using an assay system which requires all the initiation factors for optimal activity it was possible to show that the 0-40% ammonium sulfate cut of initiation factors (containing eIF-3 and eIF-4B) was sensitive to helenalin, while the 40-50% ammonium sulfate cut (containing eIF-2 and eIF-5) was not. Both ammonium sulfate cuts were equally sensitive to inhibition by the sulfhydryl reagent N-ethylmaleimide. Three purified rabbit reticulocyte initiation factors were then tested in the same assay system. Only eIF-3 showed appreciable sensitivity to helenalin, while eIF-2, eIF-3 and eIF-4B were all sensitive to inactivation by N-ethylmaleimide. These data suggest that helenalin may possess a relatively high degree of specificity as a sulfhydryl reagent.

Investigation of sesquiterpene lactones as protein synthesis inhibitors of P-388 lymphocytic leukemia cells.

The mode of action of helenalin and bis(helenalinyl) malonate as protein synthesis inhibitors of P-388 lymphocytic leukemia cells was investigated. The initial characterizations were carried out in crude lysates of the P-388 cells. In the lysate, there was a 4 min lag after the addition of drug before inhibition of protein synthesis occurred. Both drugs allowed run-off of preformed polysomes, but did significantly inhibit the formation of the 80 S initiation complex suggesting a preferential inhibition of one or more initiation reactions. The effect of these drugs on inhibition of both elongation and initiation reactions was further investigated using more fractionated systems prepared from P-388 cells. Poly(U)-directed polyphenylalanine synthesis was marginally inhibited by both drugs, but the degree of inhibition was not sufficient to explain the inhibition observed in either the lysate or in whole cell preparations of P-388. The formation of the ternary initiation complex was not significantly inhibited by either drug, but the conversion of this complex to the 48 and 80 S initiation complexes was inhibited. The inhibition of 48 S initiation complex formation by both drugs was sufficient to explain their inhibition of protein synthesis in whole cells.

Inhibition of inosine monophosphate dehydrogenase by sesquiterpene lactones.

Inosine monophosphate (IMP) dehydrogenase had previously been determined to be a likely target enzyme for the sesquiterpene lactones, a class of potential anti-neoplastic drugs. IMP dehydrogenase was purified approx. 770-fold from the P-388 lymphocytic leukemia tumor cell line. The Km values for the substrates, IMP and NAD, were determined to be 12 microM and 25 microM, respectively. Xanthine monophosphate (XMP) was shown to be a competitive inhibitor with a Ki of 67 microM. Mycophenolic acid gave mixed-type inhibition with a Ki of 8 nM for the noncompetitive component and a Ki of 2 nM for the competitive component. Dissociation constants (Kd) and rate constants for inhibition of IMP dehydrogenase by nine different sesquiterpene lactones were determined. The highest Kd was seen with 2,3-dihydrohelenalin while the lowest Kd was observed with bis-helenalinyl malonate. Binding of the drugs by IMP dehydrogenase increased as the size of the drug increased. Also, changes in structure at position 6 had a relatively large effect on the Kd. There was no correlation with hydrophobicity, as determined by octanol/water partition. The first-order rate constants for the reaction of the sesquiterpene lactones with IMP dehydrogenase (k1) and the second-order rate constants for the reaction of the sesquiterpene lactones with glutathione (k2) were also determined. The rate constants for most of the sesquiterpene lactones with the alpha-methylene-gamma-lactone moiety were similar and were approximately twice as great as the rate constants for those sesquiterpene lactones with only the alpha, beta-unsaturated cyclopentenone ring. Microlenin had approximately 5-times the reactivity of the other sesquiterpene lactones towards IMP dehydrogenase, but had approximately the same reactivity towards glutathione, suggesting that it was bound to the enzyme in a way which facilitated its reaction with one or more essential sulfhydryls. The same procedure was used for a series of N-substituted maleimide compounds with the N-substituent ranging in size from a methyl group to a benzyl group. The binding of the maleimide compounds was generally tighter than for the sesquiterpene lactones and there was an increase in binding with size.

Mechanism of eukaryotic protein synthesis inhibition by brusatol.

The mechanism by which brusatol inhibits protein synthesis in rabbit reticulocytes has been investigated. When added to reticulocyte lysates, brusatol inhibits endogenous protein synthesis only after a lag of 2-4 min at 30 degrees C. During this period 80 S ribosomes accumulate. Brusatol is equally effective in inhibiting endogenous protein synthesis in lysates and poly(U)-directed polyphenylalanine synthesis with runoff ribosomes. In fractionated reticulocyte systems, brusatol does not inhibit formation of the ternary, 40 S, and 80 S initiation complexes, but does inhibit the reaction of puromycin with initiation complexes containing [35S]Met-tRNAf. These data suggest that brusatol inhibits the peptidyl transferase elongation reaction of protein synthesis, but can do so only after one round of protein synthesis has been completed. Thus, the mechanism of action of brusatol in the rabbit reticulocyte system is very similar to the effects previously reported for bruceantin in a yeast system.

Oxaliplatin: mechanism of action and antineoplastic activity.

Oxaliplatin, a platinum-based chemotherapeutic agent with a 1,2-diaminocyclohexane (DACH) carrier ligand, has shown in vitro and in vivo efficacy against many tumor cell lines, including some that are resistant to cisplatin and carboplatin. The retention of the bulky DACH ring by activated oxaliplatin is thought to result in the formation of platinum-DNA adducts, which appear to be more effective at blocking DNA replication and are more cytotoxic than adducts formed from cisplatin. Studies by the National Cancer Institute (NCI) have suggested that oxaliplatin has a spectrum of activity different from that of either cisplatin or carboplatin, suggesting that it has different molecular targets and/or mechanisms of resistance. Oxaliplatin has been demonstrated to differ in some mechanisms associated with the development of cisplatin resistance. Compared with cisplatin-conditioned cells, deficiencies in mismatch repair (MMR) and increases in replicative bypass, which appear to contribute to cisplatin resistance, have not been shown to induce a similar resistance to oxaliplatin. A decreased likelihood of resistance development makes oxaliplatin a good candidate for first-line therapy. Studies also demonstrate additive and/or synergistic activity with a number of other compounds, however, suggesting the possible use of oxaliplatin in combination therapies.

In vivo and in vitro effects of helenalin on mouse hepatic microsomal cytochrome P450.

Helenalin, a natural plant product with significant antitumor activities, decreased male BDF1 mouse hepatic microsomal cytochrome P450 contents in vivo and in vitro. A single i.p. dose of 25 mg helenalin/kg body weight significantly (P less than 0.05) decreased microsomal cytochrome P450 contents and inhibited cytochrome P450-dependent mixed-function oxidase activities within 1-2 hr post-exposure. Helenalin (1.0 mM) decreased microsomal cytochrome P450 contents in vitro by 11% in the absence of NADPH and by 32% in the presence of NADPH. These in vitro and in vivo decreases in cytochrome P450 were accompanied by comparable decreases in total microsomal heme contents. Helenalin (1.0 mM) increased mouse hepatic microsomal oxygen consumption and NADPH utilization by 3.2 and 5.4 nmol/min/mg protein respectively. Helenalin (1.0 mM) significantly (P less than 0.05) increased microsomal lipid peroxidation in vitro, and this helenalin-induced increase in lipid peroxidation was inhibited completely by the addition of 0.05 mM EDTA. However, microsomal cytochrome P450 contents were equally affected by helenalin in the presence or absence of EDTA, suggesting that lipid peroxidation did not contribute to the helenalin-induced decrease in cytochrome P450. The addition of 0.05 mM hemin to microsomes treated in vitro with 1.0 mM helenalin resulted in a 58% recovery of cytochrome P450 contents. This ability of hemin to reconstitute cytochrome P450 in helenalin-treated microsomes suggests that helenalin produced a selective loss of heme from the cytochrome P450 holoprotein, and that the resulting cytochrome P450 apoprotein remained intact after helenalin treatment. The increased loss of microsomal cytochrome P450 produced by helenalin in the presence of NADPH suggests that a helenalin metabolite may be responsible for heme loss and the in vitro destruction of cytochrome P450.