Induction of the common fragile site FRA3B does not affect FHIT expression.

A long-standing issue concerns the extent to which fragile sites predispose to cancer-associated chromosomal rearrangements. The FHIT gene at chromosome 3p14.2 spans the most common fragile site, FRA3B, in the human genome. Although the FHIT gene is altered in many human cancers, its status as a tumor suppressor gene has remained controversial, particularly since functional studies provided contradictory results. It had been suggested the FHIT alterations result from FRA3B induction promoted by the interference of carcinogens with DNA replication. Here we investigated the effect of FRA3B induction on FHIT expression. Common fragile sites were induced by treatment with aphidicolin and scored cytogenetically. FHIT transcription was analysed by RT--PCR and RNase protection analysis. Unexpectedly, FHIT transcription proceeded unchanged after fragile site induction. Aberrant FHIT transcripts lacking one or more exons were not observed. Moreover, Western blots revealed that the levels of FHIT prior to and following fragile site induction was unchanged, whereas p53 was found at elevated levels after induction. FRA3B induction thus has no direct effect on FHIT transcription and translation.

Suppression of ethylnitrosourea-induced schwannoma development involves elimination of neu/erbB-2 mutant premalignant cells in the resistant BDIV rat strain.

Contrary to the response of rats of the highly sensitive inbred strain BDIX, BDIV rats are resistant to the induction of malignant schwannomas by exposure to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). In BDIX rats, a point mutation at nucleotide 2012 in the transmembrane region of the neu/erbB-2 gene has proved to be a very early marker of initiated Schwann precursor cells with an elevated risk of malignant transformation, and is diagnostic of the resulting schwannomas. To gain insight into the cellular and molecular mechanisms responsible for the resistance of the BDIV strain, comparative quantitative neu mutation analyses combined with histomorphological studies were performed on the trigeminal nerves of EtNU-treated BDIV and BDIX rats as well as on their (BDIX x BDIV) F1 progeny. It was found that neu-mutant Schwann cells are initially present at comparable frequency in the trigeminal nerves of both resistant and sensitive animals. Contrasting with the progressive multiplication of mutant Schwann cells in BDIX trigeminal nerves, however, the numbers of mutant cells began to decrease during the intermediary phase of the carcinogenic process in BDIV animals, and premalignant neu-mutant cells were no longer detectable by the time BDIX rats developed full-blown trigeminal schwannomas. The resistance of BDIV rats thus involves the elimination of initiated neu-mutant Schwann cells during the postinitiation period of EtNU-induced schwannomagenesis via mechanisms that remain to be clarified.

Role of DNA repair in carcinogen-induced ras mutation.

In this contribution we discuss the gene- and cell type-specific repair of miscoding DNA alkylation products as a risk parameter in both mutation induction and malignant transformation by N-nitroso carcinogens. Upon exposure to N-nitroso compounds such as N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), about a dozen different alkylation products are formed in cellular DNA. Among these are O(6)-methylguanine (O(6)-MeGua) and O(6)-ethylguanine (O(6)-EtGua), respectively, which differ only by one CH(2) group in their alkyl residue and, when unrepaired, cause G:C-->A:T transition mutations by anomalous base pairing during DNA replication. We have analyzed the global and gene-specific repair of O(6)-MeGua and O(6)-EtGua in target cell DNA, ras gene mutation frequencies, and tumor incidence, in the model of mammary carcinogenesis induced in 50-day-old female Sprague-Dawley rats by a single application of MeNU or EtNU. Both carcinogens induce histologically indistinguishable mammary adenocarcinomas at high yield. In the target mammary epithelia, O(6)-MeGua is repaired at similar slow rates in both transcriptionally active genes (Ha-ras, beta-actin), silent genes (lgE heavy chain), and in bulk DNA, by the one-step repair protein O(6)-alkylguanine-DNA alkyltransferase (MGMT; low level of expression in the target cells). The slow repair of O(6)-MeGua translates into a high frequency of mutations at the central position of Ha-ras codon 12 (GGA) in MeNU-induced tumors. O(6)-EtGua, however, is removed approximately 20 times faster than O(6)-MeGua selectively from transcribed genes via an MGMT independent, as yet uncharacterized excision mechanism. Accordingly, no Ha-ras codon 12 mutations are found in the EtNU-induced mammary tumors. Neither MeNU- nor EtNU-induced tumors exhibit mutations at codons 13 and 61 of Ha-ras or at codons 12, 13 and 61 of Ki-ras. While a moderate surplus MGMT activity of the target cells - contributed by a bacterial MGMT transgene (ada) - significantly counteracts mammary tumorigenesis in MeNU-exposed rats, this is not the case in the EtNU-treated animals. Differential repair of structurally distinct DNA lesions in transcribed or (temporarily) silent genes thus determines the probability of mutation and, together with cell type-specific and interindividual differences in DNA repair capacity, influences carcinogenic risk.

DNA repair: counteragent in mutagenesis and carcinogenesis-- accomplice in cancer therapy resistance.

DNA-reactive carcinogens and anticancer drugs induce many structurally distinct mutagenic and cytotoxic DNA lesions. The varying capability of normal and malignant cells to recognize and repair specific DNA lesions influences both cancer risk and the relative sensitivity or resistance of cancer cells towards cytotoxic agents. Using monoclonal antibody-based immunoanalytical assays, very low amounts of defined carcinogen-DNA adducts can be quantified in bulk genomic DNA, in individual genes, and in the nuclear DNA of single cells. DNA repair kinetics can, thus, be measured in a lesion-, gene-, and cell type-specific manner, and the DNA repair profiles of malignant cells can be monitored in individual patients. Even structurally very similar DNA lesions may be repaired with strongly differing efficiency. The miscoding DNA alkylation products O(6)-methylguanine and O(6)-ethylguanine, for example, differ only by one CH(2) group. These lesions are formed in DNA upon exposure to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea, both of which induce mammary adenocarcinomas in female rats at high yield. Unrepaired O(6)-alkylguanines in DNA cause G:C-->A:T transition mutations via mispairing during DNA replication. O(6)-methylguanines are repaired at a similar slow rate in both transcriptionally active (H-ras, beta-actin) and inactive genes (IgE heavy chain; bulk DNA) of the target mammary epithelia (which express the repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) at a very low level). In contrast, O(6)-ethylguanines are repaired approximately 20 times faster than O(6)-methylguanines in both DNA strands of the transcribed genes selectively via an AGT-independent, as yet unclarified excision mechanism. Accordingly, G:C-->A:T transitions resulting from the misreplication of an O(6)-methylated guanine at the second position of codon 12 (GGA) of H-ras represent a frequent "signature" mutation in rat mammary adenocarcinomas that develop after exposure to N-methyl-N-nitrosourea. However, this mutation is not observed when these tumors are induced by N-ethyl-N-nitrosourea, due to the fast repair of O(6)-ethylguanines in the H-ras gene. The key importance of "conventional" and "conditional" gene knockout technology for resolving the intricacies of the complex network of DNA repair pathways is briefly discussed.

Allele-specific losses of heterozygosity on chromosomes 1 and 17 revealed by whole genome scan of ethylnitrosourea-induced BDIX x BDIV hybrid rat gliomas.

The induction of neural tumors by N-ethyl-N-nitrosourea (EtNU) in inbred strains of rats has evolved as a valuable model system of developmental stage- and cell type-dependent oncogenesis. Tumor yield and latency times are strongly influenced by genetic background. Compared with BDIX rats, BDIV rats are relatively resistant to the induction of brain tumors by EtNU, with a lower tumor incidence and latency periods prolonged by a factor of 3. To characterize genetic abnormalities associated with impaired tumor suppressor gene function in neuro-oncogenesis, losses of heterozygosity (LOHs) and microsatellite instability (MI) were investigated in brain tumors induced by EtNU in (BDIV x BDIX) F(1) and F(2) rats. The polymerase chain reaction was used to amplify 55 polymorphic microsatellite markers spanning the entire rat genome. The tumors displayed different histologies and grades of malignancy, corresponding to part of the spectrum of human gliomas. MI was not observed in any of the tumors. LOH of rat chromosome 1q was predominantly detected in oligodendrogliomas and mixed gliomas, with a 30% incidence in informative cases. 11p15.5, the human genome region syntenic to the consensus region of LOHs observed on rat chromosome 1, has been shown to be involved in the formation of gliomas in humans. Furthermore, rat brain tumors of different histologies often showed allelic imbalances on chromosome 17p. In both cases of LOH, there was a clear bias in favor of the parental BDIV allele, suggesting the involvement of tumor suppressor genes functionally polymorphic between the two rat strains.

Neural cell surface differentiation antigen gp130(RB13-6) induces fibroblasts and glioma cells to express astroglial proteins and invasive properties.

Transient expression of the differentiation and tumor cell surface antigen gp130(RB13-6) characterizes a subset of rat glial progenitor cells susceptible to ethylnitrosourea-induced neurooncogenesis. gp130(RB13-6) is as a member of an emerging protein family of ecto-phosphodiesterases/nucleotide pyrophosphatases that includes PC-1 and the tumor cell motility factor autotaxin. We have investigated the potential role of gp130(RB13-6) in glial differentiation by transfection of three cell lines of different origin that do not express endogenous gp130(RB13-6) (NIH-3T3 mouse fibroblasts; C6 and BT7Ca rat glioma cells) with the cDNA encoding gp130(RB13-6). The effect of gp130(RB13-6) expression was analyzed in terms of overall cell morphology, the expression of glial cell-specific marker proteins, and invasiveness. Transfectant sublines, consisting of 100% gp130(RB13-6)-positive cells, exhibited an altered, bipolar morphology. Fascicular aggregates of fibroblastoid cells subsequently developed into mesh-like patterns. Contrary to the parental NIH-3T3 and BT7Ca cells, the transfectant cells invaded into collagen type I. As shown by immunofluorescence staining of the transfectant sublines as well as of primary cultures composed of gp130(RB13-6)-positive and -negative cells, expression of gp130(RB13-6) induced coexpression of proteins typical for glial cells and their precursors, i.e., glial fibrillary acidic protein, the low affinity nerve growth factor receptor, and the neural proteins Thy-1, Ran-2, and S-100. In accordance with its expression in the immature rat nervous system, gp130(RB13-6) may thus have a significant role in the glial differentiation program and its subversion in neurooncogenesis.

Mutagenicities of N-nitrosodimethylamine and N-nitrosodiethylamine in Drosophila and their relationship to the levels of O-alkyl adducts in DNA.

N-Nitrosodialkylamines are potent carcinogens in experimental animals. Previously, we reported that the mutagenicity of N-nitrosodimethylamine (NDMA) was 10 times higher than that of N-nitrosodiethylamine (NDEA) in the Drosophila wing spot test. To find out how to explain this difference, we have measured the levels of O-alkylated bases in the DNA of exposed Drosophila larvae. Third instar larvae were fed for 3 or 6 h with NDMA or NDEA. Part of the treated larvae were grown to adult flies to score their wings for the presence of mutant spots. From the remaining larvae, DNA was isolated and digested to deoxyribonucleosides, and the digest fractionated by high-performance liquid chromatography (HPLC). The amounts of specific alkyldeoxyribonucleosides present in the fractions were quantified by a radioimmunoassay (RIA) using monoclonal antibodies. Dose-dependent O6-methylguanine, O6-ethylguanine and O4-ethylthymine formations were found to be correlated with the induction frequencies of mutant wing spots. At the same exposure dose, the values of O6-alkylde- oxyguanosine/106 deoxyguanosine were similar for NDMA and NDEA: on feeding 20 micromol/1.5 ml feeding solution, the values for NDMA were 4.0 with 3 h and 18.5 with 6 h of exposure; with 20 micromol NDEA, the corresponding values were 5.4 with 3 h and 14.6 with 6 h of exposure. The wing spot frequencies were very different; however, with NDMA, the total numbers of spots/wing were 3.5 (3 h) and 15 (6 h), and with NDEA 0.8 (3 h) and 0.9 (6 h). Similar discrepancies exist as well between the mutagenicities and the alkylation rates observed for O4-alkylthymidines. These results suggest that the difference between the mutagenic potencies of NDMA and NDEA cannot be explained by the amounts of O-alkyl adducts formed. Different mechanisms are considered by which NDMA and NDEA may produce the genetic effects observed.

Ethylnitrosourea-induced development of malignant schwannomas in the rat: two distinct loci on chromosome of 10 involved in tumor susceptibility and oncogenesis.

Inbred rodent strains with differing sensitivity to experimental tumor induction provide model systems for the detection of genes that either are responsible for cancer predisposition or modify the process of carcinogenesis. Rats of the inbred BD strains differ in their susceptibility to the induction of neural tumors by N-ethyl-N-nitrosourea (EtNU). Newborn BDIX rats that are exposed to EtNU (80 microg/g body weight; injected s.c.) develop malignant schwannomas predominantly of the trigeminal nerves with an incidence >85%, whereas BDIV rats are entirely resistant. A T:A-->A:T transversion mutation at nucleotide 2012 of the neu (erbB-2) gene on chromosome 10, presumably the initial event in EtNU-induced schwannoma development, is later followed by loss of the wild-type neu allele. Genetic crosses between BDIX and BDIV rats served: (a) to investigate the inheritance of susceptibility; (b) to obtain animals informative for the mapping of losses of heterozygosity (LOH) in tumors with polymorphic simple sequence length polymorphisms (SSLPs); and (c) to localize genes associated with schwannoma susceptibility by linkage analysis with SSLPs. Schwannoma development was strongly suppressed in F1 animals (20% incidence). All of the F1 schwannomas displayed LOH on chromosome 10, with a consensus region on the telomeric tip encompassing D10Rat3, D10Mgh16 and D10Rat2 but excluding neu. A strong bias toward losing the BDIV alleles suggests the involvement of a BDIV-specific tumor suppressor gene(s). Targeted linkage analysis with chromosome 10 SSLPs in F2 intercross and backcross animals localized schwannoma susceptibility to a region around D10Wox23, 30 cM centromeric to the tip. Ninety-four % of F1 tumors exhibited additional LOH at this region. Two distinct loci on chromosome 10 may thus be connected with susceptibility to the induction and development of schwannomas in rats exposed to EtNU.

Three-dimensional imaging of nerve tissue by x-ray phase-contrast microtomography.

We show that promising information about the three-dimensional (3D) structure of a peripheral nerve can be obtained by x-ray phase-contrast microtomography (p-microCT; Beckmann, F., U. Bonse, F. Busch, and O. Günnewig, 1997. J. Comp. Assist. Tomogr. 21:539-553). P-microCT measures electronic charge density, which for most substances is proportional to mass density in fairly good approximation. The true point-by-point variation of density is thus determined in 3D at presently 1 mg/cm3 standard error (SE). The intracranial part of the rat trigeminal nerve analyzed for the presence of early schwannoma "microtumors" displayed a detailed density structure on p-microCT density maps. The average density of brain and nerve tissue was measured to range from 0.990 to 0.994 g/cm3 and from 1.020 to 1.035 g/cm3, respectively. The brain-nerve interface was well delineated. Within the nerve tissue, a pattern of nerve fibers could be seen that followed the nerve axis and contrasted against the bulk by 7 to 10 mg/cm3 density modulation. Based on the fact that regions of tumor growth have an increased number density of cell nuclei, and hence of the higher z element phosphorus, it may become possible to detect very early neural "microtumors" through increases of average density on the order of 10 to 15 mg/cm3 by using this method.

Determinants of carcinogen-induced malignant conversion in rat brain: proliferative properties of neural precursor cell subpopulations analyzed by multi-parameter flow cytometry.

The induction of neuroectodermal tumors in BDIX rats by N-ethyl-N-nitrosourea (EtNU) is a model system for the analysis of transformation risk as a function of target cell properties. The yield of neural tumors induced by EtNU varies with the developmental window chosen for the carcinogen pulse; i.e. with the relative proportions of different neural precursor cells exposed to EtNU at distinct developmental stages. Different subsets of fetal brain cells have been characterized previously with respect to their relative risk of malignant transformation using monoclonal antibodies. As DNA replication of target cells is considered to be a prerequisite for malignant conversion, we analyzed the cell cycle distributions, using flow-cytometry, of 4 subsets of neural precursor cells considered to be at high or low risk, respectively, of malignant conversion by EtNU in vivo. Cell populations associated with an elevated risk of transformation exhibited higher proportions of cells in S-phase. One of the 2 putative low-risk populations exhibited a significantly lower fraction of S-phase cells, while the value of the second one exceeded those obtained for the 2 high-risk subpopulations. Therefore, a higher than average fraction of cells in S-phase appears to be positively correlated with the cellular risk of malignant transformation by EtNU, but does not represent a dominant risk determinant per se.

New modular delivery system for diagnostic and therapeutic pre-targeting using tautomer-specific monoclonal antibody EM-6-47 and 3-substituted adenines.

We have developed a new modular affinity system for the 2-step delivery of functional molecules to target cells. The system is based on the tautomer-specific monoclonal antibody (MAb) EM-6-47, which binds to 3- and 3,8-substituted adenines with high affinity (Ka > 10(9) l/mol) without cross-reacting with naturally occurring purine derivatives. This MAb serves as the hapten-specific fusion partner to produce bispecific MAbs (bs-MAbs) recognizing a target cell antigen and a low-m.w. hapten as carrier molecule for, e.g., radionuclides. Either the C-8 or the N-3 position of adenines can be used for conjugation with effector molecules; the remaining position may be substituted with different moieties to modulate the pharmacokinetics of the haptens. Different 3- and 3,8-substituted adenines conjugated to the chelates DOTA and DTPA or to the drug daunomycin were synthesized. Adenine-chelate derivatives were efficiently labeled with (111)In and 90Y, while high-affinity binding of 3-substituted adenines to MAb EM-6-47 remained almost unaffected by the conjugation to radiochelates. To confirm the validity of the delivery system, a prototype bs-MAb, EM-168-47, was generated by somatic cell fusion of MAb EM-6-47 and MAb EM-168-2, the latter recognizing a surface antigen on canine hematopoietic cells. Two-step targeting assays in vitro verified the bs-MAb-mediated, dose-dependent delivery of (111)In-labeled adenine-chelate derivatives to myeloid cells. This system represents a powerful tool for new pre-targeting approaches relying on bs-MAbs and low-m.w. haptens. Suitable cellular antigens can be targeted by fusing the appropriate MAbs with hapten-specific MAb EM-6-47, and tailor-made 3-substituted adenines may be labeled with diagnostic or therapeutic radionuclides, cytotoxic drugs or other functional molecules.

gp130RB13-6-positive neural progenitor cells are susceptible to the oncogenic effect of ethylnitrosourea in pre-natal rat brain.

Proliferation-competent rat brain precursor cells of glial lineages are thought to preferentially undergo malignant transformation after transplacental exposure to ethylnitrosourea (EtNU). We recently have reported that monoclonal antibody (mAb) RB13-6 recognizes a developmentally regulated 130 kDa cell surface glycoprotein (gp130RB13-6) transiently expressed by a small subpopulation of glial progenitor cells in pre-natal rat brain. The expression of gp130RB13-6 has now been analysed immunocytochemically in malignant gliomas induced on day 15, 18 or 21 of gestation and in long-term cultures of fetal brain cells (FBC) isolated after in vivo-exposure to EtNU on day 18 of gestation. Malignant gliomas induced at different gestational stages contained varying proportions of gp130RB13-6-positive cells, whereas a subpopulation of proliferative neural progenitor cells exhibiting sustained gp130RB13-6 expression persisted in long-term FBC cultures after 3 months. This subpopulation, which was not selected for in control cultures of FBC derived from buffer-treated rats, gave rise to malignant cell lines after a period of time similar to the latency period required for glioma development in vivo. These data suggest that gp130RB13-6-positive cells of the immature rat nervous system may represent a subset of neural progenitor cells particularly susceptible to the oncogenic effect of EtNU.

Fast repair of O6-ethylguanine, but not O6-methylguanine, in transcribed genes prevents mutation of H-ras in rat mammary tumorigenesis induced by ethylnitrosourea in place of methylnitrosourea.

Differential repair of structurally distinct mutagenic lesions in critical genes may influence the cellular risk of malignant conversion. We have investigated rat mammary tumorigenesis induced by N-ethyl-N-nitrosourea (EtNU) versus N-methyl-N-nitrosourea (MeNU) with respect to tumor incidence, ras gene mutation, and gene-specific repair. Both carcinogens induced mammary adenocarcinomas at high yield. In mammary epithelia (very low expression of O6-alkylguanine-DNA alkyltransferase, MGMT), O6-methylguanine (O6-MeGua) was eliminated from transcribed (H-ras and beta-actin) and inactive genes (IgE heavy chain) at the same slow rate as determined for bulk genomic DNA. The persistence of O6-MeGua in DNA correlated with a high frequency of G:C --> A:T transition mutations at codon 12 of the H-ras gene in MeNU-induced tumors. Repair of O6-ethylguanine (O6-EtGua), too, was slow in the IgE heavy chain gene as in bulk DNA. Contrasting with O6-MeGua, however, O6-EtGua was removed approximately 20 times faster from the active H-ras and beta-actin genes via MGMT-independent mechanism(s). Accordingly, no H-ras codon 12 mutations were found in EtNU-induced tumors, and 5- to 8-fold surplus alkyltransferase activity of the mammary epithelia-via a bacterial ada transgene-did not significantly counteract tumorigenesis in EtNU-exposed contrary to MeNU-treated animals. Neither MeNU- nor EtNU-induced tumors exhibited mutations at codons 13 and 61 of H-ras or codons 12, 13, and 61 of K-ras. Fast repair of O6-EtGua, but not O6-MeGua, in transcribed genes thus prevents mutational activation of H-ras when rat mammary carcinogenesis is initiated by EtNU in place of MeNU.

Rapid hydrolysis of amides under physiological conditions: influence of the microenvironment on the stability of the amide bond.

A new class of bicyclic carboxyamides 1a-9a differing with respect to substitution patterns and exo-endo geometry has been synthesized. These amides are characterized by a structure-dependent unusual rapid hydrolysis rate at physiological conditions. The corresponding bicyclic anhydrides might be used as tools for masking and modifying therapeutic agents containing amine functionalities.

Relevance of DNA repair to carcinogenesis and cancer therapy.

DNA-reactive carcinogens and anticancer drugs induce many structurally distinct cytotoxic and potentially mutagenic DNA lesions. The capability of normal and malignant cells to recognize and repair different DNA lesions is an important variable influencing the risk of mutation and cancer as well as therapy resistance. Using monoclonal antibody-based immunoanalytical assays, very low amounts of defined carcinogen-DNA adducts can be quantified in bulk genomic DNA, individual genes, and in the nuclear DNA of single cells. The kinetics of DNA repair can thus be measured in a lesion-, gene-, and cell type-specific manner, and the DNA repair profiles of malignant cells can be monitored in individual patients. Even structurally very similar DNa lesions may be repaired with extremely different efficiency. The miscoding DNA alkylation products O6-methylguanine (O6-MeGua) and O6-ethylguanine (O6-EtGua), for example, differ only by one CH2 group. These lesions are formed in DNA upon exposure to N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), both of which induce mammary adenocarcinomas in female rats at high yield. Unrepaired O6-alkylguanines cause transition mutations via mispairing during DNA replication. O6-MeGua is repaired at a similar slow rate in transcribed (H-ras, beta-actin) and inactive genes (IgE heavy chain; bulk DNA) of the target mammary epithelia (which express the repair protein O6-alkylguanine-DNA alkyltransferase at a very low level). O6-EtGua, however, via an alkyltransferase-independent mechanism, is excised approximately 20 times faster than O6-MeGua from the transcribed genes selectively. Correspondingly, G:C-->A:T transitions arising from unrepaired O6-MeGua at the second nucleotide of codon 12 (GGA) of the H-ras gene are frequently found in MeNU-induced mammary tumors, but are absent in their EtNU-induced counterparts.

Drug resistance and DNA repair in leukaemia.

Most cytotoxic agents exert their action via damage of DNA. Therefore, the repair of such lesions is of major importance for the sensitivity of malignant cells to chemotherapeutic agents. The underlying mechanisms of various DNA repair pathways have extensively been studied in yeast, bacteria and mammalian cells. Sensitive and drug resistant cancer cell lines have provided models for analysis of the contribution of DNA repair to chemosensitivity. However, the validity of results obtained by laboratory experiments with regard to the clinical situation is limited. In both acute and chronic leukaemias, the emergence of drug resistant cells is a major cause for treatment failure. Recently, assays have become available to measure cellular DNA repair capacity in clinical specimens at the single-cell level. Application of these assays to isolated lymphocytes from patients with chronic lymphatic leukaemia (CLL) revealed large interindividual differences in DNA repair rates. Accelerated O(6)-ethylguanine elimination from DNA and faster processing of repair-induced single-strand breaks were found in CLL lymphocytes from patients nonresponsive to chemotherapy with alkylating agents compared to untreated or treated sensitive patients. Moreover, modulators of DNA repair with different target mechanisms were identified which also influence the sensitivity of cancer cells to alkylating agents. In this article, we review the current knowledge about the contribution of DNA repair to drug resistance in human leukaemia.

Recombinant human O6-alkylguanine-DNA alkyltransferase (AGT), Cys145-alkylated AGT and Cys145 --> Met145 mutant AGT: comparison by isoelectric focusing, CD and time-resolved fluorescence spectroscopy.

Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 degrees C but remained monomeric at 4 degrees C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8. 4 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The alpha-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-benzylguanine did not alter the secondary structure of AGT, its alpha-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for polyubiquitination. Therefore the signal for AGT degradation remains to be discovered.