Entanglement-Guided Search for Parent Hamiltonians.

We introduce a method for the search of parent Hamiltonians of input wave functions based on the structure of their reduced density matrix. The two key elements of our recipe are an ansatz on the relation between the reduced density matrix and parent Hamiltonian that is exact at the field theory level, and a minimization procedure on the space of relative entropies, which is particularly convenient due to its convexity. As examples, we show how our method correctly reconstructs the parent Hamiltonian correspondent to several nontrivial ground state wave functions, including conformal and symmetry-protected-topological phases, and quantum critical points of two-dimensional antiferromagnets described by strongly coupled field theories. Our results show the entanglement structure of ground state wave functions considerably simplifies the search for parent Hamiltonians.

Metal Tolerance Capability of Helichrysum microphyllum Cambess. subsp. tyrrhenicum Bacch., Brullo & Giusso: A Candidate for Phytostabilization in Abandoned Mine Sites.

Sardinia was known as an important mine pole in Europe during his history. Still after decades from mine closure, 75.000.000 m3 of mine waste, rich in heavy metals, were left abandoned causing a huge environmental legacy on the mine district area. Consequently, cost effective remediation is required. In this frame, phytoremediation is considered a feasible candidate. This research was focused on Helichrysum microphyllum subsp. tyrrhenicum, which is pioneer in xeric soils with low-functions, like mine tailings. The aim of this study was to evaluate its ability to extract heavy metals from mine soils and accumulate them in plant tissues and its suitability for phytostabilization. Sundry samples of soil, roots and epigean organ were collected through field sampling and analysed in order to obtain metals concentration and mineralogical characteristics. Our results indicate that this species tolerates high concentration of zinc, lead and cadmium, behaving as a species suitable for phytostabilization.

Metals and metalloids in hair samples of children living near the abandoned mine sites of Sulcis-Inglesiente (Sardinia, Italy).

The Sulcis-Iglesiente district (SW Sardinia, Italy) is one of the oldest and most important polymetallic mining areas in Italy. Large outcrops of sulfide and oxide ores, as well as the products of the long-lasting mining activity, are present throughout the district releasing significant quantities of metals and metalloids into the surrounding environment. Here are reported concentrations of 21 elements determined in scalp hair samples from children (aged 11-13 years) living in different geochemical environments of southwestern Sardinia: Iglesias, hosting several abandoned mines, and the island of Sant׳Antioco, not affected by significant base metal mineralization events. Trace element determinations were performed by ICP-MS. Statistically significant differences (p<0.01) in elemental concentration levels between the two study sites were found. Hair of children from Iglesias exhibited higher concentration values for Ag, Ba, Cd, Cr, Ni, Pb, Rb, Sb, U, V, and Zn. Rubidium, V and U resulted more abundant at Sant׳Antioco. Hair samples from Iglesias showed gender-related differences for a larger number of elements (Ag, Ba, Cd, Co, Cu, Ni, Sr, U and Zn) than at Sant׳Antioco, where only U was significantly different. The above elemental concentrations in females were always higher than in male donors. Robust Principal Component Analysis operated on log-transformed elemental concentrations showed components indicative of a) sulfides ore minerals (PC1) reflecting the influence of the diffuse mineralization covering the entire study area, b) the presence of some bioavailable As sources (PC2) as As-rich pyrite and Fe-containing sphalerite and c) other sources of metals overlapping the diffuse mineralizations, as carbonate rocks and coal deposits (PC3). The results provided evidence of a potential risk of adverse effects on the health of the exposed population, with children living at Iglesias being greatly exposed to several metals and metalloids originated in mining tailings, enriched soils, waters and food.

Lead isotopic fingerprint in human scalp hair: The case study of Iglesias mining district (Sardinia, Italy).

The Sulcis-Iglesiente district (SW Sardinia, Italy) has been, until recently, one of the most important Italian polymetallic mining areas for the extraction of lead. Epidemiological studies conducted over several decades have indicated this site at high risk of environmental crisis with possible adverse effects on the public health. In the present paper we discuss Pb isotope signatures in human scalp hair and road dust collected from the Sulcis-Iglesiente area in order to trace the exposure of populations to potential Pb sources. A total of 23 determinations (20 on hair samples and 3 on road dust samples) of lead isotope ratios (206Pb/207Pb and 208Pb/206Pb) were carried out. The obtained results were integrate with literature data regarding the total content of Pb in hair samples from the same study area. Hair from children living in Sant'Antioco exhibited lead isotope ratios in the ranges 1.152-1.165 for 206Pb/207Pb and 2.101-2.108 for 208Pb/206Pb, while hair samples from Iglesias resulted less radiogenic: 206Pb/207Pb~1.147-1.154 and 208Pb/206Pb~2.106-2.118. These values pointed to a multi-source mixing between the less radiogenic sources, corresponding to the Pb ore deposits, and the more radiogenic sources identified in local background.

Comparison of histone variant synthesis in human lymphocytic leukemia cells and in normal lymphocytes.

The synthesis of core histone variants and of histone H1 variants was determined in fresh leukemic cells of eight patients with leukemia [seven acute lymphoblastic (ALL) and one chronic lymphocytic (CLL)], in normal lymphocytes from healthy donors or from ALL patients in complete remission. Histone variant synthesis was evaluated by incubating cells with [14C]Lys and [3H]Arg in medium without Lys and Arg and then by two-dimensional polyacrylamide gel electrophoretic separations (acetic acid-urea-Triton x-100 acetic acid-urea-hexadecyltrimethylammonium bromide for core histone variants; sodium dodecyl sulfate/acetic acid-urea-hexadecyltrimethyl ammonium bromide for H1 variants). As previously reported, quiescent lymphocytes and lymphocytes stimulated with phytohaemagglutinin (PHA) showed clearcut changes in the proportions of synthesis of core histone variants and H1 variants. Leukemic lymphocytes freshly obtained from blood showed a pattern of core histone synthesis and H1 synthesis intermediate between that of quiescent and PHA-stimulated lymphocytes; this is probably due to the presence of a mixture of resting and growing cells. When leukemic cells were stimulated to grow by mitogens, the pattern of core histone and H1 variant synthesis was similar to that in mitogen-stimulated normal lymphocytes. Histone variants whose synthesis is associated with the S-phase were not synthesized in leukemic cells treated with the DNA synthesis inhibitors hydroxyurea and 1-beta-D-arabinofuranosylcytosine (Ara-C). The pattern of acetylation of histone H4 was also apparently similar in leukemic cells and normal lymphocytes. The radioactivity associated with the ubiquitinated forms of H2A increased in nongrowing lymphocytes and in leukemic cells treated with DNA synthesis inhibitors whereas they decreased after mitogenic stimulation. Variability was wide in the synthesis of ubiquitinated H2A in different cases of leukemia. The only clear-cut difference between leukemic cells and normal lymphocytes was that leukemic cells from ALL patients, but not lymphocytes from normal donors or from ALL patients in complete remission, synthesized appreciable amounts of H1 degrees, increasing after hydroxyurea/Ara-C treatment and decreasing after PHA-stimulation. In leukemic cells from a CLL patient H1 degrees synthesis was undetectable.

The paired box domain gene PAX5 is fused to ETV6/TEL in an acute lymphoblastic leukemia case.

The PAX5 gene, encoding the B-cell-specific activator protein, is a critical determinant of commitment to the B-lymphocyte pathway. This gene, mapped at 9p13, is juxtaposed to the immunoglobulin heavy chain (IgH) gene as a result of the t(9;14)(p13;q32), a rare but recurring translocation found in a subset of B-cell non-Hodgkin's lymphoma cases. In all of these, this translocation results in deregulated expression of the gene product because of the proximity of IgH. We present here the molecular characterization of a previously reported acute lymphoblastic leukemia case carrying a t(9;12)(q11;p13) translocation. Using 5' rapid amplification of cDNA ends PCR, a novel chimeric transcript was identified that contained the NH(2)-terminal region of PAX5 and most of the ETV6/TEL gene on 12p13. According to the fusion transcript, the resulting chimeric protein would retain the PAX5 paired-box domain and both the helix-loop-helix and DNA binding domains of TEL. Thus, it is reasonable to hypothesize that this protein could act as an aberrant transcription factor. This is the first report of PAX5 rearrangement in a human malignancy resulting in a chimeric transcript.

Malignant behavior and resistance to cisplatin of human ovarian carcinoma xenografts established from the same patient at different stages of the disease.

Three human ovarian carcinoma lines (HOC8) derived from the same patient before (P-HOC8) and after (R-HOC8 and Y-HOC8) cycles of chemotherapy were established i.p. in nude mice. The biological characterization showed that these tumor lines had various features in common. Cytological and histopathological characteristics and the expression of tumor-associated antigens OC125 and MOV18 were maintained in the three variants and were comparable to the patient's primary tumor. The HOC8 variants were aneuploid with a chromosome mode number of 80-81. All three tumor lines grew better i.p. than s.c. in nude mice. After i.p. injection the HOC8 lines produced ascites in all the mice, infiltration of pancreas, liver, diaphragm, and lung metastases. The sensitivity to cisplatin was evaluated for HOC8 lines growing in nude mice and mirrored the clinical development of resistance. Treatment with cisplatin of mice transplanted i.p. with P-HOC8 (obtained before the patient received chemotherapy) resulted in a significant increase in survival time; the R-HOC8 and Y-HOC8 lines (obtained after chemotherapy) were less sensitive. HOC8 xenografts, which represent the course of a single patient's disease, are a useful model for investigating the development of drug resistance in ovarian carcinoma.

Microgranular variant of acute promyelocytic leukemia in children.

PURPOSE: The microgranular variant (M3v) of acute promyelocytic leukemia (APL) rarely has been reported in a pediatric series of acute nonlymphoblastic leukemia (AnLL). We reviewed the clinical and biologic features of childhood M3v cases in our AnLL series. PATIENTS AND METHODS: From January 1970 to January 1991, 11 children with M3v were admitted and treated at our center. A diagnosis was made according to French-American-British (FAB) criteria. Morphologic examination, cytochemical analysis, and immunophenotyping were performed by a single pathologist. From January 1984, the diagnosis was confirmed by cytogenetic and, subsequently, by molecular analysis on frozen material. RESULTS: In our series, the overall incidence of children with APL was unusually high, 31.2% of the AnLL and M3v constituted one case in every four cases of APL. Even restriction of the analysis to the time when either cytogenetic and DNA studies confirmed the diagnosis, the incidence did not change. The immunophenotype of M3v cases was identical to that described for the hypergranular type, but an unexpected association of CD2 with M3v was shown. The onset was characterized by marked hyperleukocytosis (median WBC count, 87 x 10(9)/L) unlike classic APL. Disseminated intravascular coagulation (DIC) was always present and severe. Hyperleukocytosis and DIC were responsible for the high incidence of deaths for hemorrhagic events in the first days after onset (eight of 11 patients). CONCLUSIONS: In our experience, for unknown reasons, M3v may occur in childhood more than generally was considered. The clinical course and prognosis seem worse in M3v than in typical APL cases.

The first ambulatory screening on thromboembolism: a multicentre, cross-sectional, observational study on risk factors for venous thromboembolism.

OBJECTIVES: To assess the prevalence of risk factors for venous thromboembolism (VTE) and the prevalence of recent (<1 year) VTE [including superficial vein thrombosis (SVT), deep vein thrombosis (DVT) and pulmonary embolism (PE)] amongst patients attending general practitioner (GP) surgeries. DESIGN: Multicentre, cross-sectional, observational study. SETTING: A total of 1536 GP surgeries. PARTICIPANTS: A total of 15 180 adult, co-operative subjects, who had consulted their GP for a health disorder and signed the informed consent form. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Prevalence of known VTE risk factors graded according to importance and prevalence of recent (<1 year) VTE events (including SVT), based on interviews. RESULTS: About 1:5 patients had at least one strong risk factor and about 1:20 had at least two risk factors, with no difference between sexes. The prevalence of strong risk factors increased with age. Most were related to medical conditions: history of SVT and/or DVT/PE, heart failure and malignancy. About 3:4 women and 2:3 men had at least one moderate to weak risk factor; nearly 1:2 women and 1:3 men had at least two moderate to weak risk factors. The most common were: history of VTE, smoking, history of miscarriage, estrogen therapy, obesity, and varicose veins. Overall, 80% women and 67% men had at least one risk factor, and 50% women and 35% men had at least two risk factors. The prevalence of recent (<1 year) VTE was 3.4% in women and 2.4% in men, and increased with age. The majority of cases were SVT in both sexes (2.5% in women and 1.5% in men). CONCLUSIONS: The prevalence of risk factors for VTE amongst patients attending GP surgeries is high. GPs should bear this in mind during their daily practice.

Unbalanced t(3;12) in a case of juvenile myelomonocytic leukemia (JMML) results in partial trisomy of 3q as defined by FISH.

Juvenile myelomonocytic leukemia (JMML) is a rare disorder of early childhood, to which no recurrent chromosome rearrangement has been yet associated. We report a case where leukemic cells harbored a 46,XX,der(12)t(3;12) (q21 approximately 22;p13.33) karyotype, resulting in partial trisomy of 3q. The origin of chromosome material translocated to chromosome 12 was assessed by chromosome painting using a whole chromosome 3-specific probe. The breakpoint regions were defined by FISH using YAC probes from 3q and 12p chromosomal regions. Interestingly, partial trisomy of 3q has been detected in a previously reported JMML case, consequent to the presence of a der(15)t(3;15)(q13.1;q26). The involvement of a similar chromosome 3 rearrangement in these two JMML cases suggests the hypothesis that either the resulting duplication of some gene/s on 3q or the loss of heterozygosity (LOH) of some gene/s on 3p may be involved in one of the steps leading to JMML. On the other hand, it cannot be ruled out that the relevant mutation in our case might be consequent to the particular breakpoints at bands 3q21 approximately 22 and 12p13.3, that may alter the structure and/or expression of the involved gene/s.

Human T-cell lymphoblastic lymphoma expressing the T-cell receptor gamma/delta established in immune-deficient (bg/nu/xid) mice.

A small subgroup of human CD3-positive T-cell lymphoblastic lymphoma (T-LL) has been recently identified to express the T-cell receptor (TCR) gamma/delta heterodimer. Moreover peculiar clinical and histologic patterns of spleen and liver involvement have been associated with the TCR gamma/delta phenotype of tumor cells. In this paper we describe a human T-LL cell line (LL-DP) established in beige-nude-xid (BNX) mice, that by immunophenotype, molecular, and karyotype analyses, maintained most of the features of the patient. After serial transplants in BNX mice, LL-DP acquired quite a stable phenotype, producing a visible tumor in about 5 weeks in all the intravenously injected animals. The minimum number of transplanted cells that produce a tumor in all mice is 1 x 10(6). BNX mice bearing LL-DP lymphoma cells presented marked abdominal distension and splenomegaly. Diffuse lymphadenopathy with large tumor deposits in various lymph nodes that produce architectural effacement with a diffuse growth pattern was documented. The bone marrow was completely replaced, and spleen, liver, and kidneys were involved. Invasion of the central nervous system was leptomeningeal and perivascular. Overall this model might be useful for understanding mechanisms supporting lymphoma growth and progression as well as for testing new therapeutic strategies.

Minimal residual disease in childhood acute lymphoblastic leukemia: analysis of patients in continuous complete remission or with consecutive relapse.

Differences in tumor cell burden among acute lymphoblastic leukemia (ALL) patients are largely unexplored, because methods of detecting residual malignant cells have not been sufficiently sensitive. Using the polymerase chain reaction (PCR) amplification of rearranged T-cell receptor delta(TCR delta)-chain junctional sequences for the preparation of clonospecific probes, we performed a retrospective PCR study of remission bone marrow (BM) samples in seven pediatric patients with ALL who subsequently relapsed (the largest series studied so far) and in 10 patients who were in longterm (greater than 39 to greater than 72 months) remission. Following two rounds of PCR primed by nested amplimers, 1 x 10(-4) to 1 x 10(-6) cells could be identified in 16 out of 17 cases. PCR analysis of 39 BM and peripheral blood samples obtained from ALL patients considered to be in complete remission according to morphological criteria revealed the following results. In BM remission specimens of all 10 patients in continuous complete remission for a long time (median 55 months), no residual leukemic cells could be identified in the latest remission sample available for PCR analysis. In three patients the persistence of residual leukemic cells, or the continuous increase of residual blasts to the point of clinical manifestation, were indicative of impending relapse. In three patients PCR analysis failed to identify residual leukemic cells in BM samples obtained 2, 6 and 16 months respectively before clinical relapse. Differences in the duration of minimal residual disease were not associated with distinct clinical-hematological features. In one patient a different pattern of V delta 2 recombination occurred in leukemic cells from diagnosis to relapse, thus preventing the further monitoring of the patient by the initial clonospecific probe.

Identification of a novel molecular partner of the E2A gene in childhood leukemia.

The 'promiscuous' E2A gene, at 19p13.3, is fused with two different molecular partners, PBX1 and HLF, following two chromosome translocations recurrent in childhood pre-B ALL. We have identified a novel gene, FB1, by virtue of its fusion with E2A and by a combination of molecular techniques. FB1 was localized on 19q13.4, suggesting that the novel chimera originated by a cryptic rearrangement of chromosome 19. Two FB1 transcripts, of 1.2 kb and 1.1 kb, are differentially expressed at low level in a variety of human tissues, including hemopoietic cell lines from different lineages. Accordingly, FB1 cDNA displays high homology with a number of cDNA clones from different human tissues. High homology was found also with cDNA clones from mouse and rat, suggesting that the sequence might be conserved at least among mammals. The function of the putative FB1 protein, however, is currently unknown as database sequence comparisons have failed to reveal strong homology with known proteins. The E2A/FB1 fusion appears to be a recurrent feature of pre-B ALLs, suggesting that it might have a role in the development and/or progression of leukemogenesis.

Treatment Efficacy and Resistance Mechanisms Using the Second-Generation ALK Inhibitor AP26113 in Human NPM-ALK-Positive Anaplastic Large Cell Lymphoma.

UNLABELLED: ALK is a tyrosine kinase receptor involved in a broad range of solid and hematologic tumors. Among 70% to 80% of ALK(+) anaplastic large cell lymphomas (ALCL) are caused by the aberrant oncogenic fusion protein NPM-ALK. Crizotinib was the first clinically relevant ALK inhibitor, now approved for the treatment of late-stage and metastatic cases of lung cancer. However, patients frequently develop drug resistance to Crizotinib, mainly due to the appearance of point mutations located in the ALK kinase domain. Fortunately, other inhibitors are available and in clinical trial, suggesting the potential for second-line therapies to overcome Crizotinib resistance. This study focuses on the ongoing phase I/II trial small-molecule tyrosine kinase inhibitor (TKI) AP26113, by Ariad Pharmaceuticals, which targets both ALK and EGFR. Two NPM-ALK(+) human cell lines, KARPAS-299 and SUP-M2, were grown in the presence of increasing concentrations of AP26113, and eight lines were selected that demonstrated resistance. All lines show IC50 values higher (130 to 1,000-fold) than the parental line. Mechanistically, KARPAS-299 populations resistant to AP26113 show NPM-ALK overexpression, whereas SUP-M2-resistant cells harbor several point mutations spanning the entire ALK kinase domain. In particular, amino acid substitutions: L1196M, S1206C, the double F1174V+L1198F and L1122V+L1196M mutations were identified. The knowledge of the possible appearance of new clinically relevant mechanisms of drug resistance is a useful tool for the management of new TKI-resistant cases. IMPLICATIONS: This work defines reliable ALCL model systems of AP26113 resistance and provides a valuable tool in the management of all cases of relapse upon NPM-ALK-targeted therapy.

Microscopic biomineralization processes and Zn bioavailability: a synchrotron-based investigation of Pistacia lentiscus L. roots.

Plants growing on polluted soils need to control the bioavailability of pollutants to reduce their toxicity. This study aims to reveal processes occurring at the soil-root interface of Pistacia lentiscus L. growing on the highly Zn-contaminated tailings of Campo Pisano mine (SW Sardinia, Italy), in order to shed light on possible mechanisms allowing for plant adaptation. The study combines conventional X-ray diffraction (XRD) and scanning electron microscopy (SEM) with advanced synchrotron-based techniques, micro-X-ray fluorescence mapping (µ-XRF) and X-ray absorption spectroscopy (XAS). Data analysis elucidates a mechanism used by P. lentiscus L. as response to high Zn concentration in soil. In particular, P. lentiscus roots take up Al, Si and Zn from the rhizosphere minerals in order to build biomineralizations that are part of survival strategy of the species, leading to formation of a Si-Al biomineralization coating the root epidermis. XAS analysis rules out Zn binding to organic molecules and indicates that Zn coordinates Si atoms stored in root epidermis leading to the precipitation of an amorphous Zn-silicate. These findings represent a step forward in understanding biological mechanisms and the resulting behaviour of minor and trace elements during plant-soil interaction and will have significant implications for development of phytoremediation techniques.

Free radicals promote modifications in plasma high-density lipoprotein: nuclear magnetic resonance analysis.

The behavior of high-density lipoprotein (HDL) after free-radical-mediated oxidation was studied by incubating plasma HDL with chemical oxidizing systems (Cu++) in conditions similar to those used for low-density lipoprotein (LDL) chemical oxidation. Nuclear magnetic resonance (NMR) spectroscopy (1H and 31P) was used to evaluate the degree of oxidation and to characterize the oxidized products. The almost complete loss of polyunsaturated systems together with an appreciable decrease in choline peak demonstrates large-scale HDL-lipid degradation. The appearance of epoxide systems on fatty chains and the identification of oxidized cholesterol derivatives as cholesterol 5 alpha,6 alpha-epoxide, 5 beta,6 beta-epoxide, 7-keto, and 25-hydroxy confirm this picture. Phospholipid analysis indicates an alteration of the phospholipid profile in lyso-phosphatidylcholine (Lyso-PC) production and the disappearance of phosphatidylethanolamine (PE). This study shows that HDL is extensively degraded although there are no large variations in the classical oxidative monitors, lipid hydroperoxide (LPO) and thiobarbituric acid reactive substance (TBARS). Our results suggest that HDL is significantly modified when submitted to an oxidative process.

Organ-specific growth of a murine lymphoma of spontaneous origin in nude mice.

The MOC-25 tumour arose spontaneously in a female nude mouse and was established as a continuous line intraperitoneally in nude mice, where it reproduces the topological features of its origin, growing preferentially in the uterus, ovaries and liver. Karyotype analysis showed that MOC-25 cells are hyperdiploid. Tumorigenicity and malignant behaviour were studied by transplanting tumour cells into different sites in nude mice. The comparison of tumour take after i.p. and s.c. injections of scaled concentrations of MOC-25 cell suspension showed preferential growth in the peritoneum. Regardless of the route of implantation (s.c., i.v., i.p.), this tumour rapidly and preferentially disseminated to the liver, uterus, ovaries, spleen and bone marrow. No significant differences in tumour growth and metastatic behaviour were observed when MOC-25 was injected in ovariectomized nude mice or in male nude mice. Morphology studies using light and electron microscopy, immunophenotyping and molecular analysis indicated a B-lymphoid origin of the MOC-25 tumour.

Restriction-fragment-length polymorphisms in the A-I-C-III gene complex occurring in a family with hypoalphalipoproteinemia.

Two restriction-fragment-length polymorphisms in the apolipoprotein A-I-C-III gene complex were defined by digestion with PstI and SacI in a family with hypoalphalipoproteinemia. These polymorphisms established a PstI + /SacI - haplotype which may constitute a linkage marker for this condition within the family.

Characterization of genetic markers in the 3' end of the apo B gene and their use in family and population studies.

The 3' end of the apo B gene is highly polymorphic. Two point mutations in the coding sequence of the gene create EcoRI (E+, E-) and XbaI (X+, X-) RFLPs. The two loci are in random association and the frequency of the four haplotypes, E+X+, E+X-, E-X+ and E-X- in the normolipidaemic population are 0.68, 0.30, 0.02 and 0.00, respectively. Although the polymorphic nucleotide underlying the EcoRI RFLP creates an amino acid substitution in the apo B protein (Glu----Lys) in a region close to a putative LDL-receptor recognition site(s), we find no statistically significant difference in the frequency of the apo BGlu and apo BLys alleles in hyperlipidaemic patients (familial hypercholesterolaemia, type IIA with no tendon xanthomas, IIB and probably IV) and the normolipidaemic population. In contrast, we confirm previous findings, that the X+ allele is in linkage disequilibrium with a genetic locus that predisposes to the development of higher fasting plasma triglyceride levels than the X- allele. We have characterized a highly polymorphic region immediately 3' to the apo B gene. At least 5 alleles of this locus exist in the population and our family studies show it should be an extremely informative locus to use in studies where polymorphic or mutant apo B alleles are suspected to underly certain forms of familial hyperlipidaemia. DNA sequence analysis of this highly polymorphic locus shows that the variation is entirely attributable to the number of times the simple repeating sequence 5'-TTTATAATTAAAATATTTATAATTAAATAT-3' is present.

Cytotoxic activity and mechanism of action of 5-Aza-2'-deoxycytidine in human CML cells.

We investigated the cytotoxic activity and some aspects of the mode of action of 5-aza-2'-deoxycytidine (Aza-dC) in 21 primary cultures of leukemic cells freshly obtained from patients with chronic myeloid leukemia (CML) in blast crisis. The cytotoxic potency of Aza-dC was comparable or even greater than that of 1-beta-D-arabinofuranosylcytosine (Ara-C) in most cases, suggesting that this drug has potential in the therapy of blast crisis of CML. Drug incorporation into DNA was evaluated by exposing leukemic cells simultaneously to 3H-Aza-dC at the concentration of 0.1 micrograms/ml and 14C-thymidine (TdR) used as internal standard. Incorporation of Aza-dC into DNA was detectable in all cases. In 17 samples we evaluated the DNA integrity of leukemic cells exposed to Aza-dC using alkaline elution techniques. The drug caused a detectable amount of DNA alkali labile sites (ALS). DNA-ALS increased in cells exposed to Aza-dC concentrations from 0.1 to 1 microgram/ml but did not further increase at 10 micrograms/ml. A plateau in the levels of DNA-ALS was also seen in human K562 cells exposed to increasing concentrations of Aza-dC from 5 to 10 micrograms/ml, whereas in these cells Aza-dC incorporation into DNA increased with increasing Aza-dC concentrations. Therefore, DNA-ALS caused by Aza-dC are not simply the result of the chemical decomposition of azacytosine molecules incorporated into DNA, but are presumably the result of a saturable DNA repair mechanism (e.g., glycosylases) leading to formation of the apyrimidinic sites.