Effects of alkylating agents on the DNA replication of cultured Yoshida sarcoma cells

Logarithmically growing Yoshida sarcoma cells were treated for 1 h with low (2 decades cell kill) or high (more than 6 decades cell kill) doses of alkylating agents. Pulse and chase labelled DNA from treated cells were studied by alkaline sucrose gradient centrifugation. Nitrogen mustard (HN-2), 4-hydroperoxycyclophosphamide (CY-OOH), melphalan (L-PAM) and chlorambucil (CA) had no effect on the elongation rate of newly replicated DNA, both at low and high doses, although per cell the rate of DNA synthesis declined as inferred from the rates of [³H] thymidine incorporation compared to the increase in numbers of S phase cells in the treated populations. It is concluded that these drugs act specifically on the initiation step of the DNA replication, leaving chain elongation undisturbed. At low doses the chemically related sulphur mustard (SM) had also no effect on the maturation of new DNA but at high doses a decreased elongation rate was observed. A transient inhibition of chain growth was observed following treatment with a low dose of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In contrast, the intercalating agent adriamycin showed a severe but delayed effect resulting in an almost complete block of the maturation.     

Effects of the bioreductive alkylating agent 2,3-bis(chloromethyl)-1,4-naphthoquinone on coupled mitochondria isolated from sarcoma 180 ascites cells.

The effect of CMNQ was studied on mitochondria isolated from S-180 ascites tumor cells. It was found that the primary metabolic event upon addition of CMNQ to S-180 mitochondria was a stimulation of oxygen uptake. The oxygen utilization rate was maximized at about 50 nmoles CMNQ/mg protein; at doses higher than this, inhibition of respiration was observed relative to the stimulation of respiration produced by CCCP. It was also up to 50 nmoles CMNQ/mg protein. S-180 ATPase activity is stimulated maximally by 125 nmoles CMNQ/mg protein; at doses higher than this, slight inhibition of the ATPase activity relative to the stimulation produced by CCCP is seen. In vivo treatment of CMNQ to tumor bearing animals leads to a significant reduction of in vitro S-180 cellular respiration rates. The data presented in this work coupled with previously published reports involving CMNQ support the proposal for a mitochondrial level of action for this bioreductive alkylating antineoplastic agent.     

A theoretical evaluation of the effect of netropsin binding on the reactivity of DNA towards alkylating agents.

The effect of netropsin binding on the electrostatic potential of DNA reactive sites is presented. Calculations are performed for atoms N7 and O6 of guanine, N3 and N7 of adenine of model, 25 base pair long, DNA-netropsin complexes. An important weakening of the potential is found spreading along all the oligonucleotide chain studied. The results are discussed in connection with the inhibitory effect of a related ligand, distamycin A, on DNA methylation.     

The effect of spermine binding on the reactivity of DNA towards carcinogenic alkylating agents

The effect of spermine binding on the electrostatic potential of DNA is evaluated. The calculations are performed for the essential reactive sites, atoms N7 and O6 of guanine, N3 and N7 of adenine, of the nucleic acid and for its surface envelope. An important weakening of the potential is found affecting all the important reactive sites in both grooves and spreading moreover along the polynucleotide chain far away from the site of binding of the ligand. These results are discussed in connection with the experimentally observed inhibitory effect of spermine binding on DNA methylation by carcinogenic agents.     

Study of the DNA primary structure effect on induction of mutations by alkylating agents

Analysis and comparison of mutation spectra is one of the major tasks of molecular biology, since mutation spectra often reveal important properties of various mutagens and proteins involved in the repair/replication systems. Mutability is known to vary significantly along the nucleotide sequence. Mutations are abundant at certain positions (mutation hotspots). In this work, we applied regression analysis based on the basic logic patterns to understand the role of the nucleotide sequence context in mutation induction. The spectra of mutations induced by various alkylating agents were studied. The nucleotide bases at positions -2, -1, +1 and +2 were shown to have the most significant effect in G:C-->A:T replacements.     

The effect of new non-cross resistant antitumour agents on the energy state of human erythrocytes

Multidrug resistance (MDR) of tumour cells is related to the overexpression of ATP-dependent pumps responsible for the active efflux of antitumour agents out of resistant cells. Benzoperimidine and anthrapyridone compounds exhibit comparable cytotoxic activity against sensitive and MDR tumour cells. They diffuse extremely rapidly across the plasma membrane and render the ATP-dependent efflux inefficient. Such uptake could disturb an energy metabolism of normal cells possessing an elevated level of ATP-dependent proteins, especially erythrocytes having a high level of the MRP1, MRP4 and MRP5 proteins. In this study the effect of five antitumour agents: benzoperimidine (BP1), anthrapyridones (CO1, CO7) and reference drugs used in the clinic: doxorubicin (DOX) and pirarubicin (PIRA), on the energetic state in human erythrocytes has been examined. These compounds have various types of structure and kinetics of cellular uptake (slow--DOX, CO7, moderate--PIRA, fast--BP1, CO1) resulting in their different ability to saturate ATP-dependent transporters. The energetic state of erythrocytes was examined by determination of purine nucleotide contents (ATP, ADP, AMP), NAD(+) and values of adenylate energy charge (AEC) using an HPLC method. It was found that the level of nucleotides as well as the AEC value of erythrocytes were not changed during 24 h of incubation with these agents independently of their structure and ability to saturate ATP-dependent pumps. This is a very promising result in view of their potential use in the clinic as antitumour drugs against multidrug resistant cancers.     

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents.

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.     

Increased urinary excretion of nucleic acid and nicotinamide derivatives by rats after treatment with alkylating agents.

Rats treated with di(2-chloroethyl)methylamine (HN2), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) excrete significantly larger amounts of deoxycytidine (dC) and thymidine in their urine 0-24 h after treatment. Ethyl methanesulphonate (EMS) and dimethylnitrosamine (DMN) gave negative results in this respect but all five alkylating agents increased the excretion of 1-methyl-nicotinamide (1-meNmd). In addition, a larger quantity of 7-methylguanine (7MG) and uric acid was excreted after DMN treatment. 1,4-Dimethanesulphonoxybutane (myleran), 2,2-dichlorovinyl dimethyl phosphate (dichlorvos), 5-fluorouracil (5FU), cytosine arabinoside (araC), 2-acetylaminofluorene (AAF) and 7-bromomethylbenz-[a]anthracene (7-BrMBA) gave negative results.