Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ALKYLATION”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources
PubMed2004

1,3- vs 1,5-intramolecular alkylation reactions in isophosphoramide and phosphoramide mustards.

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.

Alkylation↗
PubMedDate not supplied

Base sequence selectivity in the alkylation of DNA by 1,3-dialkyl-3-acyltriazenes.

The base sequence selectivity of DNA alkylation for a series of structurally related 1,3-dialkyl-3-acyltriazenes was examined with calf thymus DNA or polymers containing the sequences GGG, CGC, TGT, and AGA. The reaction products at the N7 and the O6 positions of guanine were identified, quantitated, and then correlated with the decomposition rates of the triazenes, 1-(2-chloroethyl)-3-methyl-3-carbethoxy- (CMC), 1-(2-chloroethyl)-3-methyl-3-acetyl- (CMA), 1-(2-hydroxyethyl)-3-methyl-3-carbethoxy- (HMC), 1-(2-hydroxyethyl)-3-methyl-3-acetyl- (HMA), and 1,3-dimethyl-3-acetyl- (DMA). The results of these studies revealed that DNA sequences with runs of purines were more reactive toward alkylation by all of the triazenes tested, irrespective of whether the alkylation was measured by N7, O6, or total guanine adducts. Within this generalization, the (hydroxyethyl)triazenes showed a preference for the AGA sequence, while the (chloroethyl)triazenes favored the GGG sequence. The structure of the 3-acyl group of the triazene also played a role in the extent of alkylation of a particular sequence of DNA. Both the (chloroethyl)- and the (hydroxyethyl)triazenes produced higher alkylation product yields for the 3-carbethoxytriazenes as compared with the 3-acetyl derivatives for most of the sequences examined. These overall patterns correlated well with the order of decomposition of the triazenes at 37 degrees C: HMC > DMA > HMA > CMC > CMA. This study has demonstrated how varying the structure of 1,3-dialkyl-3-acyltriazenes can modulate DNA alkylation, a finding which may be important in the design of new triazene antitumor agents.

Alkylation↗
PubMed1999

Activation of microsomal glutathione S-transferase and inhibition of cytochrome P450 1A1 activity as a model system for detecting protein alkylation by thiourea-containing compounds in rat liver microsomes.

The recent development of several promising new thiourea-containing drugs has renewed interest in the thiourea functionality as a potential toxicophore. Most adverse reactions of thiourea-containing compounds are attributed to the thionocarbonyl moiety. Oxidation of these thionocarbonyl compounds by flavin-containing monooxygenases (FMO) and cytochrome P450 isoenzymes (P450) to reactive sulfenic, sulfinic, or sulfonic acids leads to alkylation of essential macromolecules. To more rationally design thiourea-containing drugs, structure-toxicity relationships (STRs) must be derived. Since for the development of STRs a large number of thiourea-containing compounds must be investigated, it is important to develop rapid in vitro assays for alkylating potential. In this study, the utility of activation of microsomal glutathione S-transferase (mGST) and inactivation of P450 1A1 as markers of the alkylating potential of metabolites of thiourea-containing compounds was investigated. It was found that metabolites of thiourea-containing compounds inactivate P450 1A1 in a time-dependent manner, as evidenced by a decrease in 7-ethoxyresorufin O-dealkylation (EROD) activity. An extent of inactivation of P450 1A1 by 100 microM N-phenylthiourea (PTU) of 64% was found after 10 min. This inactivation was dependent on the presence of NADPH and the presence of the thionosulfur, since the carbonyl analogue of PTU was not found to inactivate P450 1A1, and was partially prevented by heat treatment of the microsomes which is known to selectively inactivate FMO enzymes. Inactivation of P450 1A1 could be reversed by treatment with dithiothreitol, indicating the formation of disulfide bonds. However, thiourea-containing compounds also inhibited the EROD activity of P450 1A1 in a competitive manner. This property complicates the usefulness of the EROD activity of P450 1A1 as a marker for the alkylating potential of thiourea-containing compounds. It was found that metabolites of thiourea-containing compounds could transiently activate the mGST. A maximal level of activation by 100 microM PTU of 162+/-16% was found after 10 min. Activation of mGST by 100 microM PTU was dependent on the presence of NADPH and the presence of the thionosulfur, since the carbonyl analogue of PTU was not found to activate mGST. Activation was completely prevented by heat treatment of the microsomes, indicating involvement of FMO in the bioactivation process. Finally, a series of structurally diverse thiourea-containing compounds were tested for their ability to activate mGST. It appeared that their potency in alkylating mGST was inversely related to their Vmax/Km value for the FMO enzyme. From this study, it is concluded that, whereas activation of mGST in rat liver microsomes may be a useful system with which to investigate the relationship between structure and alkylating potential of thiourea-containing compounds in vitro, inactivation of P450 1A1 is not.

Alkylation↗
PubMed2003

Thiol alkylation inhibits the mitogenic effects of platelet-derived growth factor and renders it proapoptotic via activation of STATs and p53 and induction of expression of caspase1 and p21(waf1/cip1).

Thiols provide the major intracellular redox milieu and can undergo reversible oxidation and reduction. To understand the role of thiols in redox signaling events, we have studied the effect of N-ethylmaleimide, a specific thiol alkylating agent, on platelet-derived growth factor-BB (PDGF-BB)-induced mitogenesis in vascular smooth muscle cells (VSMC). Thiol alkylation inhibited PDGF-BB-induced expression of the Fos and Jun family proteins and AP-1 activity in VSMC. Thiol alkylation also inhibited PDGF-BB-induced expression of cyclin A and growth in these cells. In contrast, thiol alkylation enhanced and sustained the effect of PDGF-BB on the activation of the Jak STAT pathway, and this event was correlated with inhibition of protein tyrosine phosphatase lB activity. Thiol alkylation via inducing the expression of p21(waf1/cip1) in a STAT1- and p53-dependent manner antagonized the downregulation of this cell cycle inhibitory molecule by PDGF-BB. The inhibition of AP-1 and activation of STATs, particularly STAT1, by thiol alkylation correlated with increased production of active caspase 1 and apoptosis in VSMC. Together, these findings suggest a role for thiols in mediating mitogenic and/or apoptotic signaling events in VSMC. These results also show that a sustained change in the intracellular thiol redox state can convert a mitogen into a death promoter.

Alkylation↗
PubMed2006

Monoalkyl sulfates as alkylating agents in water, alkylsulfatase rate enhancements, and the "energy-rich" nature of sulfate half-esters.

Alkyl sulfate monoesters are involved in cell signaling and structure. Alkyl sulfates are also present in many commercial detergents. Here, we show that monomethyl sulfate acts as an efficient alkylating agent in water, reacting spontaneously with oxygen nucleophiles >100-fold more rapidly than do alkylsulfonium ions, the usual methyl donors in living organisms. These reactions of methyl sulfate, which are much more rapid than its hydrolysis, are insensitive to the nature of the attacking nucleophile, with a Brønsted beta(nuc) value of -0.01. Experiments at elevated temperatures indicate a rate constant of 2 x 10(-11) s(-1) for the uncatalyzed hydrolysis of methyl sulfate at 25 degrees C (t(1/2) = 1,100 y), corresponding to a rate enhancement of approximately 10(11)-fold by a human alkylsulfatase. Equilibria of formation of methyl sulfate from methanol and sodium hydrogen sulfate indicate a group transfer potential (DeltaG'(pH7)) of -8.9 kcal/mol for sulfate ester hydrolysis. The magnitude of that value, involving release of the strong acid HSO(4)(-), helps to explain the need for harnessing the free energy of hydrolysis of two ATP molecules in activating sulfate for the biosynthesis of sulfate monoesters. The "energy-rich" nature of monoalkyl sulfate esters, coupled with their marked resistance to hydrolysis, renders them capable of acting as sulfating or alkylating agents under relatively mild conditions. These findings raise the possibility that, under appropriate circumstances, alkyl groups may undergo transfer from alkyl sulfate monoesters to biological target molecules.

Alkylating Agents↗
PubMed1998

Alkyl-dihydroxyacetonephosphate synthase. Fate in peroxisome biogenesis disorders and identification of the point mutation underlying a single enzyme deficiency.

Peroxisomes play an indispensible role in ether lipid biosynthesis as evidenced by the deficiency of ether phospholipids in fibroblasts and tissues from patients suffering from a number of peroxisomal disorders. Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme playing a key role in the biosynthesis of ether phospholipids, contains the peroxisomal targeting signal type 2 in a N-terminal cleavable presequence. Using a polyclonal antiserum raised against alkyl-dihydroxyacetonephosphate synthase, levels of this enzyme were examined in fibroblast cell lines from patients affected by peroxisomal disorders. Strongly reduced levels were found in fibroblasts of Zellweger syndrome and rhizomelic chondrodysplasia punctata patients, indicating that the enzyme is not stable in the cytoplasm as a result of defective import into peroxisomes. In a neonatal adrenoleukodystrophy patient with an isolated import deficiency of proteins carrying the peroxisomal targeting signal type 1, the precursor form of alkyl-dihydroxyacetonephosphate synthase was detected at a level comparable to that of the mature form in control fibroblasts, in line with an intraperoxisomal localization. A patient with an isolated deficiency in alkyl-dihydroxyacetonephosphate (DHAP) synthase activity had normal levels of this protein. Analysis at the cDNA level revealed a missense mutation leading to a R419H substitution in the enzyme of this patient. Expression of a recombinant protein carrying this mutation in Escherichia coli yielded an inactive enzyme, whereas a comparable control recombinant enzyme was active, providing further proof that this substitution is responsible for the inactivity of the enzyme and the phenotype. In line with this result is the observation that wild-type alkyl-DHAP synthase activity can be inactivated by the arginine-modifying agent phenylglyoxal. The enzyme is efficiently protected against this inactivation when the substrate palmitoyl-DHAP is present at a saturating concentration. The gene encoding human alkyl-dihydroxyacetonephosphate synthase was mapped on chromosome 2q31.

Alkyl and Aryl Transferases↗
PubMed2000

Low N-ethylmaleimide concentrations activate ryanodine receptors by a reversible interaction, not an alkylation of critical thiols.

Previous studies proposed that N-ethylmaleimide (NEM) alkylates 3 classes of thiols on skeletal muscle ryanodine receptors (RyRs) producing 3 phases of channel modification, as function of time and concentration. NEM (5 mm) decreased, increased, and then decreased the open probability (P(o)) of the channel by thiol alkylation, a reaction not reversed by reducing agents. We now show that low NEM concentrations (20-200 microm) elicit Ca(2+) release from sarcoplasmic reticulum (SR) vesicles, but contrary to expectations, the effect was fully reversed by reducing agents or by washing SR vesicles. In bilayers, NEM (0.2 mm) increased P(o) of RyRs within seconds when added to the cis (not trans) side, and dithiothreitol (DTT; 1 mm) decreased P(o) in seconds. High (5 mm) NEM concentrations elicited SR Ca(2+) release that was not reversed by DTT, as expected for an alkylation reaction. A non-sulfhydryl reagent structurally related to NEM, N-ethylsuccinimide (0.1-0.5 mm), also elicited SR Ca(2+) release that was not reversed by DTT (1 mm). Other alkylating agents elicited SR Ca(2+) release, which was fully (N-methylmaleimide) or partially (iodoacetic acid) reversed by DTT and inhibited by ruthenium red. Nitric oxide (NO) donors at concentrations that did not activate RyRs inhibited NEM-induced Ca(2+) release, most likely by an interaction of NO with NEM rather than an inactivation of RyRs by NO. Thus, at low concentrations, NEM does not act as a selective thiol reagent and activates RyRs without alkylating critical thiols indicating that the multiple phases of ryanodine binding are unrelated to RyR activity or to NEM alkylation of RyRs.

Alkylation↗
PubMed1991

Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents.

O6-Methylguanine-DNA methyltransferase (MGMT) is responsible for removal of O6-alkylguanine from DNA induced by alkylating mutagens/carcinogens. To analyze the involvement of O6-alkylguanine in the generation and MGMT in avoidance of various genotoxic effects of alkylating agents, we transfected Chinese hamster ovary (CHO) cells that lack MGMT activity with human MGMT cDNA cloned into a mammalian expression vector (pSV2MGMT). A high proportion (60-80%) of transfectants selected for a cotransfected neo gene survived treatment with high doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU). Parallel transfections with an expression vector containing the bacterial ada gene (pSV2ada) showed the human MGMT to be more effective than the ada expression vector in mediating alkylation resistance. Various clonal CHO cell lines have been established stably transfected with the human MGMT cDNA. The transfectants expressed human MGMT at levels ranging from 8600 to 210,000 molecules per cell. The high MGMT expressors became strongly resistant to the killing effects of MNNG, HeCNU, N-methyl-N-nitrosourea (MNU) and, to a significant lesser degree, methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). No killing resistance was observed to N-ethyl-N-nitrosourea (ENU), though the MGMT and ada transfectants showed reduction in mutation frequency induced by this agent. Protection from mutation induction by MGMT (and ada) expression was also demonstrated for MNNG. The transfectants were also protected from the sister chromatid exchange (SCE) inducing and, to a lesser degree, clastogenic effect of MNNG and MNU, and slightly to EMS and MMS. Again no protection was observed towards ENU. Correlations between MGMT activity and resistance to a given end point suggest that, for MNNG, O6-methylguanine is the preponderant toxic, mutagenic and SCE inducing lesion. About 90% of MNNG (and MNU) induced SCEs and nearly all of the MNNG-induced gene mutations seem to be due to this adduct. For alkylation-induced chromosomal aberrations, however, and for cell killing and SCEs induced by MMS, EMS and ENU, other lesions than O6-alkylguanine appear to be of major importance. The data strongly support the view that O6-methylguanine is a genotoxic lesion and MGMT a function decisively involved in avoidance of genotoxic effects in cells exposed to MNNG and related compounds. They indicate also that it is important to take into account the property and mode of action of any given alkylating agent in assessing the protective role of MGMT against alkylation-induced genotoxicity.

Alkylating Agents↗
PubMed1986

Potentiation of alkylation-induced sister chromatid exchange frequency by 3-aminobenzamide is mediated by intracellular loss of NAD+.

3-Aminobenzamide (3AB) is a competitive inhibitor of poly-(ADP-ribose) polymerase. It will interact synergistically with certain monofunctional alkylating agents to increase the frequency of sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. 3AB will also increase the baseline SCE frequency in exposed cells. The extent of interaction between 3AB and monofunctional alkylating agents varies depending on the alkylating agent used and appears to be due to the different amounts of membrane damage produced by the alkylating agents. In this study, exogenously added beta-NAD+ was found to reduce substantially SCE frequency in cells that had been treated with combinations of 3AB and methyl methanesulfonate (MMS) but not in cells treated with 3AB and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMS produces more cell membrane damage than MNNG at equitoxic doses. beta-NAD+ is the substrate for ADP-ribosylation and normally does not freely diffuse into cells. beta-NAD+ had no significant effect on SCE induction in intact cells or in cells treated with either 3AB or alkylating agent alone. In contrast to beta-NAD+, exogenously added alpha-NAD+, which is an inhibitor of poly(ADP-ribose) polymerase, increased SCE frequency in MMS-treated cells. Thus the interaction between 3AB and certain monofunctional alkylating agents in SCE formation is apparently due to cell membrane permeabilization and the loss of intracellular NAD+ which in turn probably results in a greater inhibition of ADP-ribosylation in the presence of 3AB.

Adenosine Triphosphate↗
PubMed2000

Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding.

Human O(6)-alkylguanine-DNA alkyltransferase (AGT), which directly reverses endogenous alkylation at the O(6)-position of guanine, confers resistance to alkylation chemotherapies and is therefore an active anticancer drug target. Crystal structures of active human AGT and its biologically and therapeutically relevant methylated and benzylated product complexes reveal an unexpected zinc-stabilized helical bridge joining a two-domain alpha/beta structure. An asparagine hinge couples the active site motif to a helix-turn-helix (HTH) motif implicated in DNA binding. The reactive cysteine environment, its position within a groove adjacent to the alkyl-binding cavity and mutational analyses characterize DNA-damage recognition and inhibitor specificity, support a structure-based dealkylation mechanism and suggest a molecular basis for destabilization of the alkylated protein. These results support damaged nucleotide flipping facilitated by an arginine finger within the HTH motif to stabilize the extrahelical O(6)-alkylguanine without the protein conformational change originally proposed from the empty Ada structure. Cysteine alkylation sterically shifts the HTH recognition helix to evidently mechanistically couple release of repaired DNA to an opening of the protein fold to promote the biological turnover of the alkylated protein.

Alkylation↗
PubMed1986

DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards.

Nitrogen mustards alkylate DNA primarily at the N7 position of guanine. Using an approach analogous to that of the Maxam-Gilbert procedure for DNA sequence analysis, we have examined the relative frequencies of alkylation for a number of nitrogen mustards at different guanine-N7 sites on a DNA fragment of known sequence. Most nitrogen mustards were found to have similar patterns of alkylation, with the sites of greatest alkylation being runs of contiguous guanines, and relatively weak alkylation at isolated guanines. Uracil mustard and quinacrine mustard, however, were found to have uniquely enhanced reaction with at least some 5'-PyGCC-3' and 5'-GT-3' sequences, respectively. In addition, quinacrine mustard showed a greater reaction at runs of contiguous guanines than did other nitrogen mustards, whereas uracil mustard showed little preference for these sequences. A comparison of the sequence-dependent variations of molecular electrostatic potential at the N7-position of guanine with the sequence dependent variations of alkylation intensity for mechlorethamine and L-phenylalanine mustard showed a good correlation in some regions of the DNA, but not others. It is concluded that electrostatic interactions may contribute strongly to the reaction rates of cationic compounds such as the reactive aziridinium species of nitrogen mustards, but that other sequence selectivities can be introduced in different nitrogen mustard derivatives.

Alkylation↗
PubMed1995

DNA sequence-specific adenine alkylation by the novel antitumor drug tallimustine (FCE 24517), a benzoyl nitrogen mustard derivative of distamycin.

FCE 24517, a novel distamycin derivative possessing potent antitumor activity, is under initial clinical investigation in Europe. In spite of the presence of a benzoyl nitrogen mustard group this compound fails to alkylate the N7 position of guanine, the major site of alkylation by conventional nitrogen mustards. Characterisation of DNA-drug adducts revealed only a very low level of adenine adduct formation. Using a modified Maxam-Gilbert sequencing method the consensus sequence for FCE 24517-adenine adduct formation was found to be 5'-TTTTGA-3'. A single base modification in the hexamer completely abolishes the alkylation of adenine. Using a Taq polymerase stop assay alkylations were confirmed at the A present in the hexamer TTTTGA and, in addition, in one out of three TTTTAA sequences present in the plasmid utilized. The sequence specificity of alkylation by FCE 24517 is therefore the most striking yet observed for an alkylating agent of small molecular weight.

Adenine↗
PubMed2005

N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts.

Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO2(CH2)2-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents.

Alkylating Agents↗
PubMed2003

Molecular design of hairpin pyrrole-imidazole polyamides possessing sequence specific DNA alkylating moiety.

By conjugating pyrrole (Py)/imidazole (Im) hairpin polyamides with CBI, a stable alkylating moiety, we have developed a new type of DNA-sequence-specific alkylating agent. The sequence specificity and alkylation efficiency of the CBI conjugates were analyzed with high-resolution denaturing gel electrophoresis using linear 450- and 1000-bp DNA. The results demonstrate that CBI conjugates selectively alkylate adenine in specific sequences, whereas the previous type of alkylating polyamide, which contains segment A of DU-86, alkylates both adenine and guanine.

Alkylation↗
PubMed1982

Injection defect in alkylated and depurinated T7 bacteriophage: analysis by DNA ejection.

Using DNA ejection in vitro as a model, we have studied the DNA injection defect caused by alkylation and depurination of T7 bacteriophage. Phage was alkylated with 0.02 M methyl methanesulfonate for 2 h at 37 degrees C; alkylated phage was then incubated 24 h at 30 degrees C to induce depurination. These samples were treated with formamide to cause DNA ejection without dissociation of the phage capsid. After ejection, the phage preparations were analyzed by electron microscopy. DNA lengths in capsid-DNA complexes were measured; relative numbers of full, empty, and partially empty phage heads were determined. To establish the direction of DNA ejection, E. coli RNA polymerase was bound to capsid-DNA complexes. The results showed that DNA was partially ejected from both alkylated and depurinated phages. In the alkylated sample, RNA polymerase was bound to the DNA end distal to the capsid; this showed that ejection started from the genetic left end. We interpret these results to show, in confirmation of earlier results obtained by marker rescue, that alkylation causes T7 phage to partially inject its DNA, starting from the genetic left end. For depurinated phage, our results suggest that partial DNA injection is responsible, in this case as well, for the already documented injection defect.

Alkylation↗
PubMed2006

A hypermutation phenotype and somatic MSH6 mutations in recurrent human malignant gliomas after alkylator chemotherapy.

Malignant gliomas have a very poor prognosis. The current standard of care for these cancers consists of extended adjuvant treatment with the alkylating agent temozolomide after surgical resection and radiotherapy. Although a statistically significant increase in survival has been reported with this regimen, nearly all gliomas recur and become insensitive to further treatment with this class of agents. We sequenced 500 kb of genomic DNA corresponding to the kinase domains of 518 protein kinases in each of nine gliomas. Large numbers of somatic mutations were observed in two gliomas recurrent after alkylating agent treatment. The pattern of mutations in these cases showed strong similarity to that induced by alkylating agents in experimental systems. Further investigation revealed inactivating somatic mutations of the mismatch repair gene MSH6 in each case. We propose that inactivating somatic mutations of MSH6 confer resistance to alkylating agents in gliomas in vivo and concurrently unleash accelerated mutagenesis in resistant clones as a consequence of continued exposure to alkylating agents in the presence of defective mismatch repair. The evidence therefore suggests that when MSH6 is inactivated in gliomas, alkylating agents convert from induction of tumor cell death to promotion of neoplastic progression. These observations highlight the potential of large scale sequencing for revealing and elucidating mutagenic processes operative in individual human cancers.

Aged↗
PubMed2004

Apurinic endonuclease activity in adult gliomas and time to tumor progression after alkylating agent-based chemotherapy and after radiotherapy.

PURPOSE: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair enzyme that cleaves DNA at cytotoxic abasic sites caused by alkylating agents and radiation. We have observed that human glioma cells deficient in Ap endo activity are hypersensitive to clinically used alkylators (Silber et al., Clin Cancer Res 2002;8:3008.). Here we examine the association of glioma Ap endo activity with clinical response after alkylating agent-based chemotherapy or after radiotherapy. EXPERIMENTAL DESIGN: Cox proportional hazards regression models were used to analyze the relationship of Ap endo activity with time to tumor progression (TTP). RESULTS: In a univariate model with Ap endo activity entered as a continuous variable, the hazard ratio (HR) for progression after alkylator therapy in 30 grade III gliomas increased by a factor of 1.061 for every 0.01 increase in activity (P = 0.013). Adjusting for age, gender, extent of resection, and prior treatment strengthened slightly the association (HR = 1.094; P = 0.003). Similarly, the HR for progression after radiotherapy in 44 grade II and III tumors increased by a factor of 1.069 (P = 0.008). Adjusting for the aforementioned variables had little effect on the association. In contrast, we observed no association between activity and TTP in grade IV gliomas after either alkylator therapy in 34 tumors or radiotherapy in 26 tumors. CONCLUSIONS: Our data suggest that Ap endo activity mediates resistance to alkylating agents and radiation and may be a useful predictor of progression after adjuvant therapy in a subset of gliomas.

Adult↗
PubMed1978

Patterns of resistance and therapeutic synergism among alkylating agents.

Alkylating anticancer drugs are varied in chemical structure, alkylating moieties, and likely mechanisms of cytotoxic activity for vital normal cells and sensitive tumor cells. This has been objectively documented by numerous examples illustrating: (1) different in vitro and in vivo reaction products; (2) greater than additive, additive, and less than additive cytotoxicity of drug combinations for vital normal cells in the mouse; (3) readily reproducible and often marked therapeutic synergism between a variety of 2-drug combinations of alkylating agents against a wide variety of histologic types of murine tumors, and (4) observed resistance and cross-resistance of a variety of murine tumors, selected for resistance to specific alkylating agents, compatible with recognized chemical and functional differences between these drugs. The most important observations on resistance and cross-resistance reported are: (a) L1210 cells selected for complete resistance to cyclophosphamide (CPA) retain full sensitivity to selected nitrosoureas (BCNU, CCNU, MeCCNU), chlorozotocin), dianhydrogalactitol, and cis-DDPt, while retaining marked but somewhat reduced sensitivity to L-PAM, piperazinedione, and thioTEPA. (B) L1210 cells selected for resistance to BCNU retain full sensitivity to CPA, L-PAM, and dianhydrogalactitol. They show complete cross-resistance to BIC and variable cross-resistance to other selected nitrosoureas and piperazinedione. (c) L1210/L-PAM has incomplete but marked resistance to L-PAM. It is similar to the parent drug-sensitive line (L1210/0) in response to BCNU, CCNU, MeCCNU, and BIC. It is variably (usually moderately) cross-resistant to CPA, chlorozotocin, dianhydrogalactitol, and thioTEPA, but is completely cross-resistant to cis-DDPt. These resistance and cross-resistance patterns, which are consistent with most other biological and chemical principles established with these alkylating agents, may be useful in selecting alkylating drug combinations for inclusion in chemotherapy protocols in man which, on the basis of diverse observations in animal tumor systems, appear to be clearly indicated.

Alkylating Agents↗