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.

Increased DNA single-strand break joining activity in UV-irradiated CD34+ versus CD34- bone marrow cells.

The kinetics of UV-irradiation-induced (254 nm) DNA single-strand breaks (SSBs) were studied in single human hematopoietic cells using alkaline comet assay. Three cell populations were investigated: (i) Bone marrow mononuclear cells (BMMNCs) isolated by density gradient centrifugation, (ii) CD34- cells, and (iii) CD34+ cells. The two latter populations were purified from BMMNCs by negative and positive selection, respectively, using anti-CD34 immunobeads. SSBs were induced faster by 10 and 50 J/m2 than by 2 J/m2 and those caused by 2 J/m2 were joined faster that those caused by 10 or 50 J/m2. During the first 1.5 h after irradiation with a dose of 10 J/m2, CD34+ cells joined SSBs faster than did BMMNCs. The superior joining capacity of CD34+ cells was further substantiated with a higher UV dose. The comet lengths, indicating the extent of DNA repair, among 8/8 study subjects were shorter in CD34+ than in CD34- cells when assessed 24 h after a dose of 50 J/m2. Overall, the comet lengths at 24 h after irradiation were: CD34+ cells; 39+/-12 *m, and CD34- cells; 65+/-18 *m (8 subjects, 50 cells measured from each donor, mean+/-S.D.; p=0.0087, Mann-Whitney U-test). These results strongly suggest that nucleotide excision repair, the major mechanism responsible for the repair of UV-irradiation-induced DNA lesions in mammalian cells, is increased in CD34+ cells compared with CD34- cells and with BMMNCs. These results may have implications in stem cell purging, clinical chemotherapy and carcinogenesis.

Optimized mitogen stimulation induces proliferation of neoplastic B cells in chronic lymphocytic leukemia: significance for cytogenetic analysis.The Tampere Chronic Lympocytic Leukemia group.

We tested the effects of interleukin-2 (IL-2), human recombinant tumor necrosis factor alpha (TNF-alpha), Staphylococcus aureus Cowan I (SAC), TPA, and their combinations, using a standard thymidine incorporation assay, in order to identify an optimal mitogen combination (OMC) for 24 consecutive patients with B-cell chronic lymphocytic leukemia (B-CLL). The combination that induced the highest thymidine incorporation was chosen as the OMC for each patient. Among 14 mitogen combinations tested, there were six different OMCs, of which the most frequent was TNF-alpha + IL-2. It was the OMC in 9 of 24 cases. The other OMCs were TNF-alpha + TPA1 (5/24), SAC + IL-2 (5/24), TPA1 + IL-2 (3/24), TPA10 + IL-2 (1/24), and TNF-alpha + TPA10 + IL-1 (1/24). The mitogenic power of the selected OMC in each case was then evaluated both by the combination of immunophenotyping and molecular cytogenetic techniques known as MAC (Morphology, Antibody, Chromosomes) and standard chromosome analysis. After OMC stimulation, the levels of DNA synthesis and B-cell proliferation (mitotic index) were, on average, 10-fold higher than those observed after standard TPA stimulation (P < 0. 0001). The proportion of mitotic B cells exceeded the proportion of mitotic T cells in 70.1% of the cases after OMC stimulation. After TPA stimulation, 7.7% +/- 2.5% of all mitoses were B-cell mitoses, whereas after OMC stimulation this proportion rose to 57.9% +/- 5.3%. The frequency of clonal chromosomal aberrations increased from 46% after TPA stimulation to 79% after OMC stimulation. The clonal aberrations del(6q), del(11q), and/or del(13q) were observed in 26%, 32%, and 42% of the patients with the respective clonal chromosomal aberrations, whereas the corresponding frequencies after TPA stimulation were only 4%, 21%, and 17%. When the lineage involvement of cells with clonal chromosomal aberrations from three patients was analyzed, the aberrations were found to be restricted to B cells only, and in one patient to a minor subset of B cells. The results demonstrate that an individually chosen OMC induces a high rate of proliferation in neoplastic B cells. We found deletions in 6q, 11q, and 13q at higher frequencies than reported previously, most probably as a result of an improved mitogenic response. The identification of an optimal mitogen stimulation for each patient, prior to chromosome analysis, can well be expected to reduce the rate of false-normal results in the future. This is essential for accurate evaluation of the prognostic significance of chromosomal aberrations in B-CLL.

Repair of gamma-irradiation-induced DNA single-strand breaks in human bone marrow cells: analysis of unfractionated and CD34+ cells using single-cell gel electrophoresis.

Human bone marrow mononuclear cells (BMMNCs) were separated by density gradient centrifugation, and a subpopulation of progenitor cells was further isolated using anti-CD34-coated magnetic beads. The cells were irradiated with gamma-rays (0.93-5.43 Gy) from a 137Cs source. The extent of DNA damage, i.e., single-strand breaks (SSBs) and alkali-labile lesions of individual cells, was investigated using the alkaline single-cell gel electrophoresis technique. The irradiation resulted in a dose-dependent increase in DNA migration, reflecting the number of detectable DNA lesions. An approximately similar extent of SSB formation was observed in BMMNCs and CD34 + cells. Damage was repaired when the cells were incubated at 37 degrees C: a fast initial repair phase was followed by a slower rejoining of SSBs in both BMMNC and CD34 + cell populations. A significantly longer time was required to repair the lesions caused by 5.43 Gy than those caused by 0.93 Gy. In the present work we report, for the first time, the induction and repair of DNA SSBs at the level of single human bone marrow cells when exposed to ionizing radiation at clinically relevant doses. These data, together with our previous results with human blood granulocytes and lymphocytes, indicate an approximately similar extent of formation and repair of gamma-irradiation-induced DNA SSBs in immature and mature human hematopoietic cells.

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.

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.

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.

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.

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.

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.

Mammalian DNA nucleotide excision repair reconstituted with purified protein components.

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

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.

Hemolysis. Which laboratory investigations and when?

The Scandinavian Society for Clinical Chemistry and NORDKEM have had a number of joint efforts to facilitate the optimal performance and use of clinical laboratory investigations. A new committee (DOOKK or Diagnosis and Organ Oriented Committee for Clinical Chemistry) was established (in 1984) in order to improve collaboration between clinicians and laboratory physicians. In particular, the approach has been to work out recommendations for different clinical working hypotheses concerning restricted diagnoses or organ-specific problems. The aim of our project ("Hemolysis") was to create a rational scheme for the use of clinical chemical and hematological laboratory investigations in suspected or known cases of hemolysis.

Template-primer activity of 5-(hydroxymethyl)uracil-containing DNA for prokaryotic and eukaryotic DNA and RNA polymerases.

We have utilized Bacillus subtilis phage SPO-1 DNA as a model of irradiated DNA. In this phage, all thymine (Thy) residues are replaced by 5-(hydroxymethyl)uracil (5HmUra), which is a known irradiation-induced derivative of DNA Thy. SPO-1 phage is naturally devoid of other such irradiation-induced DNA lesions. DNase I activated SPO-1 phage DNA served as well as, or even better than, the control DNAs (Bacillus subtilis DNA and calf thymus DNA) as a template-primer for Escherichia coli, Micrococcus luteus, and human HL-60 cell DNA polymerases. Furthermore, the template activity of SPO-1 phage DNA was also superior when transcription with E. coli RNA polymerase was investigated. The results reported here indicated that the replacement of Thy by 5HmUra is not deleterious to template and primer functions during DNA or RNA synthesis.