Selective toxicity of vincristine against chronic lymphocytic leukemia cells in vitro.

The cytotoxicity of vincristine in vitro was investigated in B chronic lymphocytic leukemia (CLL) cells and in normal peripheral blood mononuclear cells. An approximately 25-fold selectivity towards leukemic vs. normal lymphocytes was demonstrated. Cells from patients having a mature subtype (CLL or CLL/mix) or a slowly progressing form of CLL were significantly more sensitive to vincristine in vitro than the cases with a CLL/PL phenotype or faster-progressing disease. Depending on the vincristine dose, the number of dead CLL cells accumulated slowly during the 4-d observation period. Our data indicate a marked individual variation in vincristine susceptibility among individual CLL cells. Vincristine induced annexin positivity, nuclear blebbing and DNA fragmentation in CLL cells. These indicate an "apoptosis-like" cell death. Since CLL cells are in the G0/G1 phase of the cell cycle, the only known mode of anticancer action of vinca alkaloids, i.e. anti-mitotic action, cannot explain the death of CLL cells. Furthermore, similar cellular uptake and efflux of vincristine by normal and CLL cells excluded pharmacokinetic differences as a cause of selectivity of vincristine towards leukemic lymphocytes. Immunostaining of filamentous structures of CLL cells revealed that vincristine brings about selective changes in alpha-tubulin but not in beta-actin or vimentin. Although the antitubulin action of vinca alkaloids in the biochemical sense is well demonstrated, this kind of anticancer effect has not previously been shown. Vincristine is used in several regimens for CLL, but its efficacy in CLL has never been demonstrated in a clinical context and its value in routine CLL chemotherapy has been questioned. The present data strongly support the need for further evaluation of the role and mode of action of vincristine in chemotherapy of CLL and other cancers as well.

Kinetics of excision repair of UV-induced DNA damage, measured using the comet assay.

The kinetics of UV- (254 nm) irradiation-induced DNA single-strand breaks (SSBs), generated during the excision repair of UV-induced DNA damage, in leukemic lymphocytes and in normal blood mononuclear cells (MNCs) were studied using the alkaline comet assay. The cells were isolated by density gradient centrifugation from peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy study subjects. The cytotoxicity of UV irradiation was determined in vitro in peripheral blood mononuclear lymphocytes from 36 CLL patients and from eight healthy donors using the incorporation of radioactive leucine in 4-day cultures. A remarkable difference in excision repair capability was observed between normal and leukemic lymphocytes. In contrast to normal lymphocytes, there was always a subpopulation of CLL cells that did not complete the repair of UV-induced DNA damage during the 24-h repair period. Furthermore, differences were also recorded between UV-sensitive and UV-resistant CLL cases. The differences in DNA migration between the maximum increase (59-77 microm) and that at 24 h after irradiation (21-66 microm) was statistically significant in two of three patients exhibiting UV-resistance. Correspondingly, only in one of three patients exhibiting UV-sensitivity was the difference in DNA migration statistically significant (maximum increase: 44-107 microm, vs. 24 h after: 42-100 microm). Our results confirm an abnormal pattern of the CLL cell response to UV irradiation. Furthermore, we identified defective processing of UV-induced DNA damage in CLL versus normal lymphocytes, particularly in UV-sensitive cases.

Gamma-irradiation-induced DNA single- and double-strand breaks and their repair in chronic lymphocytic leukemia cells of variable radiosensitivity.

Gamma-irradiation-induced DNA single- and double-strand break (SSB and DSB) formation and their repair kinetics in normal hematopoietic cells and in leukemic lymphocytes was investigated using alkaline and neutral comet assays. The cells were isolated by density gradient centrifugation from peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy study subjects. Furthermore, CD34+ progenitor cells isolated with immunomagnetic beads from bone marrow of non-leukemic persons were investigated. The cytotoxicity of 137Cs irradiation was determined in vitro in peripheral blood mononuclear lymphocytes from 36 CLL patients and from 8 healthy donors using radioactive leucine incorporation assay in 4-day culture. A dose-dependent increase in DNA migration was observed in alkaline (SSBs) and neutral (DSBs) gel electrophoresis when the cells were exposed to gamma-irradiation doses up to 10.4 Gy. After irradiation with doses of 2.4 and 5.4 Gy, the cells repaired their single- and double-strand breaks almost completely. The formation and repair of DNA strand breaks were essentially similar in all normal cell populations investigated and in CLL cells. The gamma-irradiation-induced cytotoxicity did not correlate with DNA strand break formation and repair capacity. According to these results, the differences of gamma-irradiation tolerance among individual CLL cases and among healthy persons are explicable in terms other than DNA strand break formation or repair.

Mode of cytostatic action of mesoionic oxatriazole nitric oxide donors in proliferating human hematopoietic cells.

The cytopathological effects of the novel nitric oxide (NO)-releasing, mesoionic 3-aryl-substituted oxatriazole-5-imine derivatives GEA 3162 and GEA 3175, and a reference NO donor SIN-1 were investigated in proliferating human hematopoietic cells. The GEA compounds (10-50 microM) induced rapid surface changes, which progressed as peculiar deep indentations and strictures in human leukemic T cells (MOLT-3) in 30 min. An excess of red cells partially prevented these surface changes. GEA 3162-treated MOLT-3 cells became permeable to ethidium bromide and lost their ability to be stained by acridine orange after 5 h of exposure. GEA 3162 and GEA 3175 suppressed thymidine and uridine incorporation in a dose-dependent manner, reflecting the inhibition of DNA and RNA synthesis respectively. In addition, the GEA compounds inhibited the growth of human bone marrow stem cells, CFU-GM colonies being more susceptible to the cytostatic action than BFU-E. The reference compound SIN-1 had comparative cytostatic effects at ten times greater concentrations (500 microM). We conclude that NO-releasing mesoionic oxatriazole derivatives have cytostatic action against human malignant and non-malignant hematopoietic cells, supporting the value of NO-releasing and NO-inducing compounds as anti-cancer agents.

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.

UV- and gamma-irradiation-induced DNA single-strand breaks and their repair in human blood granulocytes and lymphocytes.

Ionizing irradiation and UV-irradiation cause DNA damage. Ionizing irradiation induces single-strand breaks, much less abundantly double-strand breaks, alkali-labile sites, and various oxidized purines and pyrimidines. UV-irradiation, on the other hand, causes cyclobutane pyrimidine dimers, (6-4) photoproducts, and various monomeric base damages. The deposition of energy in DNA may result directly in single-strand breaks (predominant form after ionizing radiation), or the strand breaks may be generated during the repair process (predominant form after UV-irradiation). We investigated the formation and repair of DNA single-strand breaks in human blood granulocytes and lymphocytes by the single-cell gel electrophoresis or comet assay. The induction and repair of DNA lesions by gamma-irradiation was comparable in human blood granulocytes and lymphocytes. The finding is consistent with the expression of the pertinent base excision repair proteins in these cells. In contrast to gamma-irradiation, fewer single-strand breaks were observed immediately after UV-irradiation; the maximum number of breaks were seen when the cells were incubated for 30-60 min. After an incubation period of 150 min, a significant reduction of single-strand breaks was noted. It is conceivable that the first 30-60 min represented a period during which the incision-excision phase of nucleotide excision repair (NER) predominated. After that, strand joining was dominant, evidently representing the synthesis and ligation phase of NER. These results indicate that the approx. 30 different polypeptides required for complete NER are functional in these mature blood cells. This is the first demonstration of the expression of global NER in human granulocytes.

Prevention of DNA 5-methylcytosine reutilization in human cells.

DNA methylation is an important process contributing to transcriptional regulation in animal and plant cells. The well known reutilization of DNA nucleotide bases indicated that DNA degradation occurs in many cells and tissues. On the other hand, the reutilization of 5-methyl-2'-deoxycytidine monophosphate in the DNA synthesis would have deleterious effects on gene regulation. Recent molecular insights into the exclusion of exogenous 5-methylcytosine from DNA are the subject of this review.

Restriction, methylation and ligation of 5-hydroxymethyluracil-containing DNA.

Oxidation of DNA and its components can cause genetic mutations and chromosomal instability. These changes have generally been implicated in aging. Oxidation of the methyl group of thymidine residues in DNA is known to result in the formation 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd). We have utilized Bacillus subtilis phage SPO1 DNA as a model of oxidatively damaged DNA. In this phage, all thymine (Thy) residues are replaced by 5-hydroxymethyluracil (5HmUra), but the species is naturally devoid of other oxidatively-induced DNA lesions. Particular attention was paid to the behavior of 5HmUra-containing DNA as a target for several enzymes employing DNA as substrate; restriction endonucleases, dam DNA methylase and T4 DNA ligase. We noticed that susceptibility of SPO1 DNA varied when different restriction endonucleases having 5HmUra in the restriction sites were tested. Endonucleolytic cleavage brought about Sau3A proceeded as effectively with SPO1 DNA as with conventional DNA (lambda phage). The same was true when the ligation of Sau3A sites was performed with T4 DNA ligase. In contrast, both endonucleolytic cleavage and ligation were slower in SPO1 DNA, compared with lambda phage, when Taq I and T4 DNA ligase were used for restriction and ligation, respectively. We also noticed that SPO1 phage does not naturally contain N6-methyladenine (N6MeAde) opposite 5HmUra, i.e., no hydrolysis of SPO1 DNA was observed when assessed with methylation-dependent restriction endonuclease DpnI. Our results show that the presence of 5HmUra in the respective site of DNA does not, per se, prevent the activity of restriction endonucleases, ligases or DNA methylases. These data support the view that oxidation of Thy to 5HmUra in target DNA does not necessarily result in substantial deterioration in the functions of DNA processing enzymes.

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.

Nucleoside monophosphate kinase may be the key enzyme preventing salvage of DNA 5-methylcytosine.

Nucleoside monophosphate kinase (EC 2.7.4.4) catalyzes the phosphorylation of various nucleoside monophosphates to their corresponding diphosphates. We investigated whether 5-methyl-2'-deoxycytidine 5'-monophosphate (5MedCMP) could serve as a substrate for the enzyme isolated from bovine liver. Although the substrate activity of UMP, CMP and dCMP was readily demonstrable, no activity was recorded with 5MedCMP as the candidate substrate. Moreover, 5MedCMP did not affect the active site of the enzyme, since no inhibition in the phosphorylation of UMP was recorded in the presence of 5MedCMP. This metabolic step appears to be the key phase where the incorporation of exogenous 5-methylcytosine (5MeCyt) into DNA is prevented. Hence, very little or no salvage of DNA 5MeCyt can be expected.

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.

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.

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.

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-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.

Time-resolved fluoroimmunoassay of 5-methyl-2'-deoxycytidine.

We describe time-resolved fluoroimmunoassay of 5-methyl-2'-deoxycytidine (5MedCyd). The assay is based on the use of a highly specific antiserum raised in rabbits against BSA-conjugated 5-methylcytidine (5MeCyd). The tracer in the solid-phase time-resolved fluoroimmunoassay (TR-FIA) was antigen-selected anti-5MedCyd labeled with Europium. Thyroglobulin-linked 5MeCyd served as the solid-phase antigen. The measuring range for the fluoroimmunoassay was from less than 1 to 5000 pmol per assay of 5MedCyd. A good correlation between the results obtained with the TR-FIA and HPLC was demonstrated when the methods were applied to the measurement of methylation in human leukemic cells and other DNA samples. TR-FIA has several advantages over the more laborious techniques available so far: (i) high sensitivity, (ii) large assay ranges, (iii) rapidity and large number of simultaneous assays, (iv) simplicity, and (v) low cost provided that the laboratory has equipment for time-resolved fluorometry.

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.

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.