An XPG DNA repair defect causing mutagen hypersensitivity in mouse leukemia L1210 cells.

One of the most widely used antitumor drugs is cis-diamminedichloroplatinum(II) (cisplatin), and mechanisms of cisplatin resistance have been investigated in numerous model systems. Many studies have used mouse leukemia L1210/0 as a reference wild-type cell line, and cisplatin-resistant subclones have been derived from it. Increased DNA excision repair capacity is thought to play a key role in the acquired cisplatin resistance, and this has influenced development of drugs for clinical trials. We report here that the L1210/0 line is in fact severely deficient in nucleotide excision repair of damaged DNA in vivo and in vitro. L1210/0 cell extracts could be complemented by extracts from repair-defective human xeroderma pigmentosum (XP) or rodent excision repair cross-complementing (ERCC) mutant cells, except for XPG/ERCC5 mutants. Purified XPG protein could restore repair proficiency to L1210/0 extracts. Expression of mouse XPG mRNA was similar in all L1210 lines studied, suggesting a point mutation or small alteration of XPG in L1210/0 cells. The DNA repair capacity of a cisplatin-resistant subline, L1210/DDP10, is similar to that of type culture collection L1210 cells and to those of other normal mammalian cell lines. Nucleotide excision repair of DNA is thus clearly important in the intrinsic cellular defense against cisplatin. However, in contrast to what is generally believed, enhancement of DNA repair above the normal level in these rodent cells does not appear to be a mechanism of acquired resistance to the drug.

Reversal of deamination-related cytotoxicity of 5-methyl-2'-deoxycytidine by tetrahydrouridine in human leukemia cells.

The present experiments were conducted to test the effects of the potent cytidine deaminase inhibitor tetrahydrouridine (THU) on the metabolism and cytotoxicity of 5-methyl-2'-deoxycytidine (5-Med-Cyd) in several human leukemia cell lines in vitro. It was observed that 5-Med-Cyd exerts its effects via deamination to thymidine, which is particularly toxic to human promyelocytic (HL-60) and T-cell (JM) leukemia cell lines in vitro. The deamination and the cytotoxicity of 5-Med-Cyd were effectively hindered by 10(-3) M THU in 3-day cultures of HL-60 cells. Although the catabolism of [14C]5-Med-Cyd in the HL-60 cell cultures was blocked by THU, no radioactive 5-Med-Cyd was incorporated into DNA. The cytotoxicity and DNA incorporation of fluorodeoxycytidine are enhanced by THU. Unlike that compound 5-Med-Cyd resembled more bromodeoxycytidine and iododeoxycytidine; THU decreases the toxicity of both of these deoxycytidine analogues.

Metabolism, incorporation into DNA, and interactions with 1-beta-D-arabinofuranosylcytosine of 5-hydroxymethyl-2'-deoxyuridine in human promyelocytic leukemia cells (HL-60).

5-Hydroxymethyl-2'-deoxyuridine (5HmdUrd) and 1-beta-D-arabinofuranosylcytosine (Ara-C) had a dose-dependent synergistic or antagonistic action on growth of human promyelocytic leukemic (HL-60) cells in suspension culture. For instance, in 3-day cultures, the cell number was reduced from 100% (with either 100 nM Ara-C or 10 microM 5HmdUrd alone) to 65% (with 100 nM Ara-C plus 10 microM 5HmdUrd), or from 35% (with 1.0 microM Ara-C alone) to 10% (with 1.0 microM Ara-C plus 10 microM 5HmdUrd), compared to the control cultures without drugs. 1.0 and 10 microM 5HmdUrd potentiated the incorporation of radioactive Ara-C (1.0 microM) into HL-60 cell nucleic acids in 2-day cultures by 56 and 64%, respectively. 5HmdUrd-induced enhancement of Ara-C incorporation is one explanation for the synergism of these two drugs. On the other hand, 10 nM Ara-C partially inhibited the toxicity of 100 microM 5HmdUrd. Radioactive 5HmdUrd was incorporated into DNA, but not RNA, the rate being 5% of that observed with thymidine. [3H]5HmdUrd-derived radioactivity remained stable in DNA for at least 24 h, indicating that the compound was not excised to a significant extent from DNA in these conditions. The incorporation of Ara-C and 5HmdUrd into DNA appeared to take place via different pathways, which is a second explanation for their synergism. Ara-C is the most important drug in the clinical chemotherapy of acute nonlymphoblastic leukemia. Experience with 5HmdUrd in experimental antileukemia chemotherapy has been promising. This novel combination of antileukemic agents merits further evaluation.

Radioimmunoassay of 5-methyl-2'-deoxycytidine. A method for the quantitation of DNA methylation.

5-Methyl-2'-deoxycytidine (5MedCyd) is a minor constituent of mammalian cell DNA. We report the production and characterization of highly specific rabbit anti-5MedCyd antiserum. The antiserum was suitable for the radioimmunological measurement of 5MedCyd. This simple radioimmunoassay was capable of quantitating calf thymus DNA methylation at nanomolar levels of total DNA.

Time-resolved fluoroimmunoassay of 5-methyl-2'-deoxycytidine employing europium-labeled antigen as tracer.

We describe a solid-phase fluoroimmunoassay, based on competition between europium-labeled 5 MeCyd (5-methylcytidine) and sample 5MedCyd (5-methyl-2'-deoxycytidine) for polyclonal anti-5MedCyd antibodies (rabbit). Europium labeling of antigen was performed using a novel polylysine-5MeCyd conjugate. Standard and sample preparations containing 5MedCyd inhibited the binding of the europium-labeled 5MeCyd to the antibody molecules. A second antibody, directed against rabbit IgG, was coated on the solid phase, and bound the IgG-5MeCyd-polylysine-europium complex, giving rapid and complete separation of antibody-bound and free antigen. The measuring range was from 3.7 to 2500 pmol of 5MedCyd per assay. A good correlation between the results obtained with TR-FIA and HPLC was demonstrated when the methods were applied to the measurement of methylation in various DNA samples, enzymatically hydrolyzed to their constituent deoxyribonucleosides. This new TR-FIA possesses the same advantages (high sensitivity, wide assay range, rapidity, simplicity, and low cost) as the previous assay developed in our laboratories. The superiority of the new system is based on (i) its low inter- and intra-assay variation, (ii) low antiserum consumption, and (iii) a protocol, which permits the use of second-antibody-coated microtitration strips common to other assays.

Radioimmunoassay of 5-hydroxymethyl-2'-deoxyuridine.

5-Hydroxymethyl-2'-deoxyuridine is an antileukemic thymidine analogue. It is also a well known thymidine-derivative in DNA exposed to ionizing irradiation. We report the production and characterization of specific rabbit anti-5HmdUrd antisera. The antisera were used for the radioimmunological measurement of 5HmdUrd. The radioimmunoassay was capable of quantitating 2 pmol of 5 HmdUrd per tube corresponding to 0.2 mumol/l in a 10 microliter plasma sample. A good correlation between the results obtained with the radioimmunoassay and HPLC was demonstrated when the methods were applied to the measurement of plasma levels of 5HmdUrd in mice receiving experimental chemotherapy.

An improved radioimmunoassay for the quantitation of DNA methylation.

A novel radioimmunoassay of 5MedCyd is described. The assay, employing a highly specific antiserum raised in rabbits against BSA-conjugated 5MeCyd, used 5-125iodo-2'-deoxycytidine as the tracer. The measuring range for the assay was found to be 1-1000 pmol per assay of 5MedCyd. When the methods were applied to the measurement of methylation in DNA samples a good correlation between the results obtained with the radioimmunoassay and HPLC was demonstrated. The method has several advantages over the more laborious and sophisticated techniques previously available: high sensitivity, large assay range, rapidity and potential for large number of simultaneous assays, simplicity, and low cost provided that the laboratory has equipment for gamma counting.

6-Substituted and 5,6-disubstituted derivatives of uridine: stereoselective synthesis, interaction with uridine phosphorylase, and in vitro antitumor activity.

Stereoselective procedures are described for the synthesis of 6-alkyluridines by Lewis acid-catalyzed condensation of (a) trimethylsilylated 6-alkyl-4-alkylthiouracils with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (ABR) and (b) trimethylsilylated 6-alkyl-3-benzyluracils with ABR. The 4-methylthio group was subsequently removed with the use of 1 N trifluoroacetic acid and the 3-benzyl group by a new modified procedure with the use of the complex BBr3-THF. Furthermore, 6-(hydroxymethyl)uridine (39) and 5-fluoro-6-(hydroxymethyl)uridine (40) were obtained by sequential oxidation with SeO2 and reduction with tetrabutylammonium borohydride of the 6-methyl group of 6-methyluridine (5) and 5-fluoro-6-methyluridine (35), and their corresponding 6-fluoromethyl congeners 41 and 42 were obtained by DAST treatment of 39 and 40, respectively. For all the foregoing nucleosides in the fixed syn conformation about the glycosyl bond, 1H NMR spectroscopy further demonstrated that the pentose rings exist predominantly in the conformation N (3'-endo). Most of the nucleosides were weak substrates of Escherichia coli pyrimidine nucleoside phosphorylase. Enhanced susceptibility to phosphorolysis was exhibited by two of them, 39 and 41, with 6-CH2OH and 6-CH2F substituents capable of formation of an additional hydrogen bond with the enzyme. The 5-fluoro-6-substituted uridines were the poorest substrates. Cytotoxicities of the nucleosides were examined vs the human tumor cell lines MOLT-3, U-937, K-562, and IM-9, as well as PHA-stimulated human lymphocytes. Two of the analogues, 5-fluoro-6-(fluoromethyl)uridine (42) and 5-fluoro-6-(hydroxymethyl)uridine (40), exhibited cytotoxicities comparable to that of 5-fluorouracil.

In vitro and in vivo studies of a promising antileukemic thymidine analogue, 5-hydroxymethyl-2' deoxyuridine.

The toxicity and metabolism of a thymidine analogue, 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) were studied with human leukemia cells (HL-60) and with human platelets. 3 X 10(-5) M 5HmdUrd caused a 50% inhibition in the proliferation of HL-60 cells. The compound was hydrolyzed to 5-hydroxymethyluracil (5HmUra) by the enzyme thymidine phosphorylase (EC 2.4.2.4) present in leukemia cells; this catabolic product was non-toxic. The catabolism of 5HmdUrd by human platelet thymidine phosphorylase could be inhibited by 6-aminothymine. The toxicity of 5HmdUrd was effectively reversed by deoxycytidine and 5HmdUrd increased the incorporation of deoxycytidine into dCTP and DNA several fold. The two latter phenomena are explicable in terms of a feedback action to ribonucleotide reductase, resulting in deoxycytidylate starvation, which is a known effect of excess thymidine. We report here also our preliminary observations that 5HmdUrd is active against mouse leukemia in vivo.

Antileukemic activity against L1210 leukemia, pharmacokinetics and hematological side effects of 5-hydroxymethyl-2'-deoxyuridine.

The chemotherapeutic potential of 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) was examined in vitro and in vivo. The compound was toxic in 2-day cultures; 7, 66 and 88% inhibition in the growth of L1210 cells was achieved with 1, 10 and 100 microM 5HmdUrd, respectively. The maximal plasma concentration of 5HmdUrd at 15 min after a single i.p. injection (100 mg/kg) in DBA/2 mice was 193-244 mumol./l and the compound had a logarithmic disappearance curve with a half-life of 20 min. Chemotherapy given as two daily i.p. injections of 5HmdUrd (100 mg/kg) for five successive days resulted in a 239% increase in median lifespan and 2/6 long-term survivals among DBA/2 mice bearing leukemia L1210. This treatment resulted in temporary neutropenia and thrombocytopenia, which were followed by rebound thrombocytosis and neutrophilia of short duration. Our data indicate that 5HmdUrd can successfully be used in experimental cancer chemotherapy in vivo.

Uracil-DNA glycosylase activity in human acute leukemia.

The expression of the DNA excision repair enzyme uracil-DNA glycosylase was investigated in bone marrow and peripheral samples from seven patients with acute lymphoblastic leukemia (ALL), from 17 patients with acute non-lymphocytic leukemia (ANLL), and from one patient with chronic granulocytic leukemia (CGL) in blast crisis. In addition, uracil-DNA glycosylase activities were determined in nine human leukemia/lymphoma cell lines. There was a clear correlation between the percentage of blast cells and the enzyme activity when mononuclear cell fractions from patient samples were analysed. The following uracil-DNA glycosylase activities were recorded (mean +/- S.D., number of samples): ALL = 45.6 +/- 14.8 U/mg of protein, N = 10; ANLL = 41.1 +/- 13.8 U/mg of protein, N = 22; CGL (blast crisis) = 44.7 U/mg of protein. The uracil-DNA glycosylase activity in nine human leukemia/lymphoma cell lines ranged from 35.2 to 66.0 U/mg of protein, and no striking differences were observed between the T-ALL, B-ALL, null cell ALL or myeloid lines. Similarly, the various biological features, such as the common ALL surface antigen, the terminal deoxynucleotidyl transferase enzyme, the sub-type of leukemia, chromosomal aberrations, or previous chemotherapy, did not apparently affect the expression of uracil-DNA glycosylase. We propose that the integrity of the genetic information is well protected by uracil-DNA glycosylase in different forms of leukemia, including cases with a low proportion of S-phase blasts, as assessed by flow cytometry in the present work. When compared to the activities in benign hematopoietic progenitor cells, studied previously in this laboratory, no big differences between the benign and malignant hematopoiesis were demonstrated. Hence, it is unlikely that selectivity of chemotherapy towards malignant vs benign hematopoietic growth could be based on the enzyme uracil-DNA glycosylase.

Uracil-DNA glycosylase activity in chronic lymphoproliferative disorders.

The activity of uracil-DNA glycosylase, a repair enzyme for the excision of uracil from DNA, was studied in patients with chronic lymphoproliferative disorders and with malignant plasma cell dyscrasias. The biochemical assay was performed on mononuclear cells, isolated by density gradient centrifugation from peripheral blood, from bone marrow or from both. The activity of the uracil-DNA glycosylase of peripheral blood cells in 8/8 cases of myeloma and in 3/3 cases of Waldenström's macroglobulinemia was in the same range as in 22 non-hematological control patients, i.e. 2.4-25.1 U/mg of protein. Higher activities were found in 9/12 cases of chronic lymphocytic leukemia (CLL), in 2/4 cases of hairy cell leukemia (HCL), in 2/2 cases of chronic T-cell lymphocytosis and in the only case of small cell lymphocytic lymphoma. Follow-up of some CLL and HCL patients revealed that uracil-DNA glycosylase activity was fairly stable during the course of the disease. We conclude that malignant cells in chronic lymphoproliferative disorders are characterized by a normal or even increased capability to repair DNA, as exemplified by uracil-DNA glycosylase in this study.

Genetic aberrations in pediatric acute lymphoblastic leukemia by comparative genomic hybridization.

Classical cytogenetic analysis plays an important role in the diagnosis and classification of childhood acute lymphoblastic leukemia (ALL). However, poor in vitro growth of the malignant cells and suboptimal quality of metaphase spreads may sometimes cause false-negative findings (normal karyotype). We used comparative genomic hybridization (CGH) to study whether this new method is able to detect and characterize genetic aberrations not detected by karyotyping. CGH showed clonal genetic aberrations in 8 of 13 cases, most of which showed gains of several chromosomes, indicating hyperdiploidy. The sensitivity of CGH was sufficient to detect a small interstitial deletion of 6q. One karyotypically complex case was resolved by CGH showing a high-level amplification of DNA sequences originating from the 12p12-13. Interphase fluorescence in situ hybridization (FISH) analyses confirmed the CGH findings in 2 cases, validating the accuracy of CGH. In conclusion, CGH experiments established the known fact that hyperdiploidy is the most common finding in pediatric ALLs and that CGH may detect aberrations that are not seen in the G-banded karyotype. CGH was also able to further characterize genetic aberrations such as gene amplification, which is occasionally involved in pediatric ALL as well as in other leukemias.

Kinetics of chlorambucil in vitro: effects of fluid matrix, human gastric juice, plasma proteins and red cells.

The mechanisms involved in the bioavailability of chlorambucil or 4-[p-(bis[2-hydroxyethyl]amino)phenyl]-butyric acid are poorly understood. The effects of different matrices on the disintegration of chlorambucil were investigated by HPLC, 1H NMR, 31P NMR, and mass spectrometry. Cellular incorporation and protein binding of the drug in vitro was assessed with [3H]-chlorambucil. Decomposition of chlorambucil and its major metabolite, phenylacetic acid mustard, to mono- and dihydroxy derivatives, was significantly faster in water than in PBS, (phosphate-buffered saline, pH 7.4). The hydrolysis of chlorambucil was as fast in plasma ultrafiltrate as in PBS; plasma proteins, preferentially albumin, prevented this disintegration. In phosphate-buffered media, two additional stabile hydrolysis products were found which were characterised as the mono- and bis-phosphates of 4-[p-(bis[2-hydroxyethyl]amino)phenyl]butyric acid, results of the reaction of nucleophilic buffer species with the aziridinium ion intermediates. Chlorambucil bound covalently to plasma proteins and was incorporated into red cells. These interactions are likely to have a significant role in vivo, reducing the bioavailability of the drug. High H+ concentration associated with high chloride concentration in human gastric juice had a stabilizing effect on chlorambucil. Incorporation of [3H]-chlorambucil into red cells was inhibited in a concentration-dependent fashion by whole human plasma as well as by albumin. We conclude that the chemico-biological interactions demonstrated in the present investigation provide explanations for the remarkable pharmacokinetic differences observed intra- and inter-individually in the clinical use of chlorambucil. The present information is important, when clinical or in vitro evaluation of efficacy and bioavailability of chlorambucil is considered.

The DNA-repair enzyme uracil-DNA glycosylase in the human hematopoietic system.

The expression of the DNA base-excision-repair enzyme uracil-DNA glycosylase in the human hematopoietic system followed a tightly regulated pattern: high enzyme activities were recorded in proliferating bone marrow progenitor cells and in peripheral blood T- and B-cells, both groups of cells requiring the integrity of their genetic information for their proper function. The blood quiescent immunocompetent cells retained their DNA-uracil exclusion capacity, even in the oldest age groups. Peripheral blood mature end cells, granulocytes, platelets and red cells had little activity, consistent with the fact that these cells are anuclear or short-lived, so that no template-primer functions of their DNA are required. Uracil-DNA glycosylase expression is high in all types of human leukemia, providing a selective advantage for survival of leukemic cells. Overall results show that a deficiency of this DNA base-excision-repair pathway is not likely to be an etiopathogenetic factor in the formation of non-random or other chromosomal abnormalities or in the leukemogenesis itself.

Uracil-DNA glycosylase activity in human blood cells.

We studied uracil-DNA glycosylase activities systematically in all types of human peripheral blood cells. The highest amounts of uracil-DNA glycosylase activity were found in cells capable of using their genetic information either in DNA replicative or repair synthesis or in DNA transcription. These cells included cytotoxic/suppressor and inducer/helper T lymphocytes, B lymphocytes and monocytes. On the other hand, the peripheral blood mature end cells, erythrocytes, platelets and granulocytes, contained very little if any uracil-DNA glycosylase activity. In addition to this biological capacity, we show that the housekeeping excision repair capacity of uracil-DNA glycosylase is well maintained in human peripheral blood mononuclear cells throughout life from the neonatal period to old age.

Normal uracil-DNA glycosylase activity in Bloom's syndrome cells.

Cells from patients with Bloom's syndrome, a rare human disease with autosomal recessive mode of inheritance, exhibit cytological abnormalities involving DNA metabolism. Bloom's syndrome is characterized by a greatly increased cancer frequency which may reflect a specific defect in DNA repair and replication. Evidence has recently been presented of the existence in Bloom's syndrome of an abnormality of the DNA ligase involved in semiconservative DNA replication. Another abnormality, in the excision-repair pathway of Bloom's syndrome cells, is reportedly due to an aberrant immunological reactivity of the DNA-repair enzyme uracil-DNA glycosylase. In this investigation we show, however, that the catalytic activity of uracil-DNA glycosylase appears to be normal in Bloom's syndrome lymphoblastoid cells.

Uracil-DNA glycosylase in benign and malignant maturing human hematopoietic cells.

The expression of uracil-DNA glycosylase was studied in human normal hematopoietic bone marrow cells and in malignant counterparts obtained from patients with chronic granulocytic leukemia. We observed that the expression of the enzyme was highest in the proliferating granulocytic compartment (myeloblasts through myelocytes) and that it was diminished in more mature cells. Furthermore, we demonstrated that uracil-DNA glycosylase activity was higher in immature red blood cells or reticulocytes than in more mature red cells. The same tendency was also demonstrated in human malignant monoblasts, which were induced to terminal maturation by phorbol ester. It can be concluded from these results that uracil-DNA glycosylase expression is equal in benign and malignant hematopoietic progenitor cells; no selectivity towards malignant vs. benign progenitors can be expected in possible chemotherapeutic approaches relying on uracil-DNA glycosylase.

Biochemical mechanisms by which reutilization of DNA 5-methylcytosine is prevented in human cells.

The salvage metabolism of 5-methyldeoxycytidine 5'-monophosphate (5MedCMP) was studied in human promyelocytic leukemia (HL-60) cells and in PHA-stimulated human lymphocytes. To this end [5'-32P]5MedCMP was synthesized by a novel postlabeling procedure. At low substrate concentrations (less than 100 microM), the enzyme(s) present in crude HL-60 whole-cell extract deaminated 5MedCMP faster than they did dCMP. Although the phosphorylation of dCMP to dCDP was easily demonstrable with both kinds of cell extracts, no phosphorylation of 5MedCMP to 5MedCDP (5-methyldeoxycytidine 5'-diphosphate) was observed. This phenomenon was confirmed using HL-60 cells made permeable to nucleotides with Tween 80. In view of the substantial 5MeCyt (5-methylcytosine) content of DNA and the degradation of DNA that occurs in cells, it is conceivable that 5MedCyd (5-methyl-2'-deoxycytidine) and 5MedCMP are available for reutilization in DNA synthesis. This would have devastating effects on cellular control and gene expression. The results of the present investigation indicate that rapid deamination at the monophosphate level and, in particular, stringent discrimination of 5MedCMP by cellular monophosphokinase(s) are the key mechanisms by which reutilization of DNA 5MeCyt is prevented in human hematopoietic cells.

Repair of gamma-irradiation-induced DNA single-strand breaks in human bone marrow cells: effects of a second irradiation.

Human bone marrow mononuclear cells were isolated by density gradient centrifugation and irradiated with a 137Cs source. The extent of irradiation-induced single-strand breaks (SSBs) and alkali labile sites as well as their repair was investigated by using the alkaline single-cell gel electrophoresis (SCGE) technique, or comet assay. A dose-dependent increase in the length of DNA migration was seen when cells were exposed to 0, 2.43 and 5.43 Gy of gamma-irradiation. Complete repair of DNA SSBs was observed over 24 h after a dose of 2.43 Gy. Second challenges of 0, 2.43 and 5.43 Gy resulted in similar SSBs as with the first irradiation. Furthermore, the DNA repair kinetics of two cell populations, one previously unirradiated and the other having received 2.43 Gy 24 h earlier, was indistinguishable. This means that most human bone marrow cells retain their genetic stability after a dose of 2.43 Gy if SSBs are used as an endpoint.