Cytogenetic heterogeneity in blast crisis (BC) of chronic myelogenous leukemia (CML)

New cytogenetic findings are reported in a patient who entered into an accelerated blastic phase of chronic myelogenous leukemia (CML). The cytogenetic findings of this case can be described as 46, xy, t (5;7) (q 31;q 11), t (9;22) (q 34;q 11), Ph'. The prognostic implications in such patients with rare and unusual cytogenetic findings are discussed.     

VP-16-induced nucleotide pool changes and poly(ADP-ribose) synthesis: the role of VP-16 in interphase death

Exposure of human promyelocytic leukemia cell line (HL-60) to VP-16 resulted in accumulation of DNA strand breaks. Concomitantly, intracellular NAD levels fell at 1 h, followed by declines in ATP at 2 h and in GTP, CTP, and UTP at 3 h. Furthermore, marked morphological changes, such as loss of microvilli or bleb formation, appeared at 4 h and cell death by 8-10 h. The addition of an inhibitor of poly(ADP-ribose) polymerase, 3-aminobenzamide (5 mM), theophylline (2 mM), or thymidine (1 mM), prevented these sequential reductions of nucleotide pools and cell death. In fact, the activation of poly(ADP-ribose) synthesis was detectable within a few hours after treatment with VP-16, although it was smaller than that induced by N-methyl-N'-nitro-N-nitrosoguanidine. These results may suggest the possible role of activation of poly(ADP-ribosyl)ation in VP-16-induced nucleotide pool changes and subsequent interphase death.     

Peroxidative free radical formation and O-demethylation of etoposide(VP-16) and teniposide(VM-26)

The peroxidative activation of the antitumor drugs, etoposide (VP-16) and teniposide (VM-26), has been studied in vitro. Both of these drugs, in the presence of horseradish peroxidase or prostaglandin synthetase, formed phenoxy radical intermediates. Furthermore, this activation also resulted in the formation of two metabolites from each of the drugs. Using HPLC and mass spectrometry, one of the metabolites was shown to be the reactive o-quinone derivative of the parent drug which resulted from the peroxidative O-demethylation. It appears that O-demethylation catalyzed by peroxidases may be an important mechanism for the formation of reactive intermediates and may play a role in the mechanism of action of VP-16 and VM-26.     

Dysregulated calcium homeostasis and oxidative stress in chronic myeloid leukemia (CML) cells

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by the oncogenic activity of the Bcr‐Abl protein, a deregulated tyrosine kinase. Calcium may act directly on cellular enzymes and in conjunction with other cellular metabolites, such as cyclic nucleotides, to regulate cell functions. Alteration in the ionized calcium concentration in the cytosol has been implicated in the initiation of secretion, contraction, and cell proliferation as well as the production of reactive oxygen species (ROS) has been correlates with normal cell proliferation through activation of growth‐related signaling pathways. In this study we evaluated in peripheral blood leukocytes from CML patients the role of the balance between intracellular calcium and oxidative stress in CML disease in order to identify possible therapeutic targets in patients affected by this pathology. Our results demonstrated that peripheral blood mononuclear cells derived from CML patients displayed decreased intracellular calcium [Ca²⁺]_i fluxes both after InsP₃ as well as ATP and ionomycin (IONO) administration. CML cells showed lower levels of superoxide dismutase (SOD) activity and significantly higher malondialdehyde levels (MDA) than peripheral blood mononuclear cells derived from control patients. Finally we showed that resveratrol is able to down‐regulate InsP3 and ATP effects on intracellular calcium [Ca²⁺]_i fluxes as well as the effects of ATP and IONO on oxidative stress in CML cells. J. Cell. Physiol. 224: 443–453, 2010. © 2010 Wiley‐Liss, Inc.     

Investigations on karyotype evolution in patients with chronic myeloid leukemia (CML)

In an attempt to relate karyotype evolution to clinical and hematological data serial chromosomal analyses were performed in 31 patients with chronic myeloid leukemia (CML), both in chronic and acute phases. Our results in Philadelphia chromosome (Ph1)-positive CML are in line with karyotype profiles described in the literature. In addition, we report on chromosomal findings in 4 cases of Ph1-negative disease, one presenting with an iso17q chromosome in the positive CML. The same chromosomal abnormality was observed in a small population of Ph1-negative cells present in one of two patients with mixed Ph1-positive/Ph1-negative CML. The first case of a female patient with the loss of a sex chromosome in Ph1-positive cells is reported. Two patients with unusually long and mild chronic phases despite the presence of trisomy 8 in their karyotypes are described. Our findings suggest that the order of appearance of additional chromosomal changes of CML is of prognostic significance for the progression and the clinical picture of the disease.     

Phenotypic and gene expression diversity of malignant cells in human blast crisis chronic myeloid leukemia

Chronic myeloid leukemia (CML) is considered as a paradigm of neoplasias developing through multistep track. It is believed that in the blast crisis (BC) terminal phase of the disease, blood-circulating blasts represent an expansion of a single CML clone. However, although these blasts grow mostly in suspension under standard culture conditions, a relatively small cell-fraction adheres to the plastic dish. Yet, it is unknown whether these two cell-fractions are distinct sub-populations that originated from a common CML clone and whether they have different biological and malignant properties. To address these questions, we have characterized the plastic-adherent and non-adherent sub-populations of various cell lines and primary cells derived from patients with CML in BC. This study indicated that the adherent-subsets retain repopulating ability with indications of increased malignant properties as greater anchorage-independent clonogenicity, impairment of cell-cell contact inhibition, loss of serum-dependent attenuation of plastic-adhesion, and a significant up-regulation of the oncogenes BCR-ABL, c-JUN, and c-FOS along with the adhesion-related genes KiSS-1, THBS3, and ITGB5. The adherent blasts stably retain their unique properties even after elimination of the adherence selection pressure. Sub-cloning analyses indicated that the adherent cells could be continuously evolved from any parental non-adherent clone in a unidirectional manner. This study provides new insights into the biology and the malignant evolution of CML, indicating that at the BC phase, circulating blasts are heterogeneous and consisting of at least two distinct populations of a common clonal origin. The existence of a minor "pool" of blasts of greater clonogenic capacity along with significantly higher expression level of BCR-ABL, individually or in conjunction with other cancer and adhesion-related genes, might also signify clonal evolution toward subsequent increased malignancy and lower therapeutic sensitivity.     

Higher-order interactions of Bcr-Abl can broaden chronic myeloid leukemia (CML) drug repertoire

Bcr-Abl, a nonreceptor tyrosine kinase, is associated with leukemias, especially chronic myeloid leukemia (CML). Deletion of Abl's N-terminal region, to which myristoyl is linked, renders the Bcr-Abl fusion oncoprotein constitutively active. The substitution of Abl's N-terminal region by Bcr enables Bcr-Abl oligomerization. Oligomerization is critical: it promotes clustering on the membrane, which is essential for potent MAPK signaling and cell proliferation. Here we decipher the Bcr-Abl specific, step-by-step oligomerization process, identify a specific packing surface, determine exactly how the process is structured and identify its key elements. Bcr's coiled coil (CC) domain at the N-terminal controls Bcr-Abl oligomerization. Crystallography validated oligomerization via Bcr-Abl dimerization between two Bcr CC domains, with tetramerization via tight packing between two binary assemblies. However, the structural principles guiding Bcr CC domain oligomerization are unknown, hindering mechanistic understanding and drugs exploiting it. Using molecular dynamics (MD) simulations, we determine that the binary complex of the Bcr CC domain serves as a basic unit in the quaternary complex providing a specific surface for dimer–dimer packing and higher-order oligomerization. We discover that the small α1-helix is the key. In the binary assembly, the helix forms interchain aromatic dimeric packing, and in the quaternary assembly, it contributes to the specific dimer–dimer packing. Our mechanism is supported by the experimental literature. It offers the key elements controlling this process which can expand the drug discovery strategy, including by Bcr CC-derived peptides, and candidate residues for small covalent drugs, toward quenching oligomerization, supplementing competitive and allosteric tyrosine kinase inhibitors.     

Experience with using aclarubicin in the treatment of acute leukemia and blast crisis of chronic myeloid leukemia]

The clinical efficacy of aclarubicin, an anthracycline antibiotic, was studied in 48 patients with leukemia. The antibiotic was used in the following combinations with cytarabine: "7 + 7", "5 + 5" and "7 & 3". A complete remission was stated in 14 (42.4 per cent) out of 33 patients with acute nonlymphoid leukemia, 6 (43 per cent) out of the 14 patients having relapses. The combined therapy was effective in 4 out of 5 pre-resistant patients. The "7 + 3" scheme was the most beneficial. The most common adverse reactions were nausea and vomiting.     

Inhibition of small GTPase RalA regulates growth and arsenic-induced apoptosis in chronic myeloid leukemia (CML) cells

Chronic myelogenous leukemia (CML) results from the transformation of a primitive hematopoietic cell by the bcr-abl gene. RalA, one of the Ras superfamily of small GTPases, is a downstream molecule of bcr-abl fusion protein in ras signaling pathway, but its role in CML is poorly understood. Here, we first detected RalA level in CML cells, which is highly expressed and distributed mainly in the cytoplasm and/or partially in endomembrane. Next, siRNA was used to deplete RalA expression for elucidating its function. The results showed that siRNA RalA effectively inhibited cell viability, induced apoptosis and enhanced sensitivity of arsenic trioxide (ATO), and there are some synergistic effects of anti-CML between RalA siRNA and ATO. Finally, we found that ATO also could downregulate protein level of bcr-abl in K562 and KCL-22. Our research provides evidence that RalA might also serve as linchpin modulators in leukemia, and combinatorial therapies of dual inhibition of bcr-abl and ras signaling pathways have a great potential in treatment of CML.     

Ultrastructure of neutrophilic granulopoiesis in the bone marrow of patients with chronic myeloid leukemia (CML)

In seven patients with chronic myeloid leukemia (CML) an ultrastructural and morphometric study was performed on neutrophilic granulopoiesis in bone marrow trephine biopsies. Bone marrow specimens from five patients without hematological abnormalities served as controls. In stable phases of CML, abnormalities of the maturing granulocytic lineage were most conspicuously expressed by an infrequently occurring nuclear disfiguration (blebs and disturbed bridging of segments). Morphometric evaluation included the numbers of azurphil (primary) and specific (secondary) granules, the cisternal length of the endoplasmic reticulum and the area of the mitochondrial profiles. These variables could be determined in early and late myeloblasts, promyelocytes, metamyelocytes, band cells and mature polymorphonuclear granulocytes. Statistical analysis with regard to control specimens demonstrated no significant differences in the total amount of neutrophil granules or of the other cell organelles. Partly supported by a grant from the Maria-Pesch Foundation, Cologne, Federal Republic of Germany     

Apoptosis of unstimulated human lymphocytes and DNA strand breaks induced by the topoisomerase II inhibitor etoposide (VP-16)

Etoposide (VP-16)-induced DNA strand breaks and repair and apoptosis of unstimulated human lymphocytes have been studied using DNA comet assay, electrophoresis of low-molecular-weight DNA extracts, and fluorescence microscopy. Incubation of unstimulated human lymphocytes with VP-16 (50-200 microg/ml) for 3 or 24 h induced apoptosis. This conclusion is supported by results of morphological studies, evaluation of the proportion of hypodiploidy and internucleosomal degradation of DNA in lymphocytes. Etoposide-induced formation of DNA strand breaks preceded the appearance of these conventional apoptotic manifestations. The number of single-strand breaks depended on VP-16 concentration, and 2-3 h after its removal from the incubation medium they were repaired. The hydroxyl group at the C-4; position of the etoposide dimethoxyphenol ring may be responsible for the formation of single-strand breaks. Double-strand breaks were unrepaired 20 h after the change of the incubation medium. The number of double-strand breaks and a proportion of apoptotic cells did not exhibit any dependence on VP-16 concentration and/or duration of cell exposure to this agent. We suggest that the cytotoxic effect of VP-16 on unstimulated lymphocytes is mediated by a topoisomerase II isoform, topoisomerase II-beta, which is localized in the nucleolus and is not related to the cell cycle.     

Higher‐order interactions of Bcr‐Abl can broaden chronic myeloid leukemia (CML) drug repertoire

Bcr‐Abl, a nonreceptor tyrosine kinase, is associated with leukemias, especially chronic myeloid leukemia (CML). Deletion of Abl''s N‐terminal region, to which myristoyl is linked, renders the Bcr‐Abl fusion oncoprotein constitutively active. The substitution of Abl''s N‐terminal region by Bcr enables Bcr‐Abl oligomerization. Oligomerization is critical: it promotes clustering on the membrane, which is essential for potent MAPK signaling and cell proliferation. Here we decipher the Bcr‐Abl specific, step‐by‐step oligomerization process, identify a specific packing surface, determine exactly how the process is structured and identify its key elements. Bcr''s coiled coil (CC) domain at the N‐terminal controls Bcr‐Abl oligomerization. Crystallography validated oligomerization via Bcr‐Abl dimerization between two Bcr CC domains, with tetramerization via tight packing between two binary assemblies. However, the structural principles guiding Bcr CC domain oligomerization are unknown, hindering mechanistic understanding and drugs exploiting it. Using molecular dynamics (MD) simulations, we determine that the binary complex of the Bcr CC domain serves as a basic unit in the quaternary complex providing a specific surface for dimer–dimer packing and higher‐order oligomerization. We discover that the small α1‐helix is the key. In the binary assembly, the helix forms interchain aromatic dimeric packing, and in the quaternary assembly, it contributes to the specific dimer–dimer packing. Our mechanism is supported by the experimental literature. It offers the key elements controlling this process which can expand the drug discovery strategy, including by Bcr CC‐derived peptides, and candidate residues for small covalent drugs, toward quenching oligomerization, supplementing competitive and allosteric tyrosine kinase inhibitors.     

Cytofluorometric studies on the action of podophyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts

Flow microfluorometric analysis of human lymphoid cells exposed in vitro to cytostatic concentrations of podophyllotoxin (0.01-5 mug/ml for 24 h) shows that a major part of this population (40-60%) has the DNA content of cells in the G2-M part of the cell cycle, and that approximately 60% of these cells are arrested in mitosis. Although a similar pattern of DNA distribution is seen in cultures exposed to cytostatic concentrations of VM-26(0.01 mug/ml) and VP--16-213(0.1 mug/ml), no mitotic cells are seen in these cultures. Exposure to higher concentrations: of VM-26 (0.1 mug/ml) and VP-16-213 (1.0 mug/ml) inhibits cell cycle traverse, and after 24 hr of exposure a major part of the population is arrested with the DNA content of cell in the S part of the cell cycle. Exposure to higher drug concentrations leads to a reduction in the number of cells with the late S-G2DNA content. Whereas the cell cycle block induced by cytostatic concentrations of podophyllotoxin (0.01 mug/ml) is readily reversible by reincubation of cells in drug-free medium, cells blocked by VM-26 and VP-16-213 are unable to resume cell-cycle traverse under similar conditions.     

Blast crisis of Philadelphia chromosome-positive chronic myelocytic leukemia (CML). Treatment results of 69 patients

We have studied the clinical courses of 69 patients with blastic crises of Philadelphia chromosome positive CML to identify parameters that were associated with an increased response rate or survival. Cytogenetic analysis at the time of blastic transformation revealed additional chromosome changes in 70% of the patients tested. Bone marrow fibrosis was detected in 58% of evaluable patients. Lymphoblastic transformation was seen in 28% of the patients tested with cell surface marker analysis. The value of 5'-nucleotidase as a marker for distinguishing lymphoid from non-lymphoid blast crisis was confirmed. Of 57 evaluable patients, 23 (40%) responded to therapy (CR/PR longer than 14 days). Median survival was 75 days. Longer survival was related to the following factors: Ph1-chromosome as the only detectable cytogenetic abnormality; lymphoblastic transformation; no bone marrow fibrosis; high percentage of blasts and promyelocytes in the bone marrow, and response to therapy. No prognostic significance was associated with age, sex, Tdt, LDH, spleen size, duration of the chronic phase of the disease, white blood cell count, Hb, platelet count and percentages of basophils, eosinophils, erythroblasts and blasts and promyelocytes in the peripheral blood. These data confirm the poor prognosis of patients with blastic crisis of CML treated by conventional chemotherapy.     

Combination vincristine and VP-16-213: evaluation of drug sequence

The combination of vincristine and VP-16-213 has been found to have synergistic antitumor activity in a murine system in vivo when the sequence of drug administration was vincristine followed by VP-16-213. To investigate the potential influence of drug scheduling on this synergistic combination, the reverse sequence of drug administration was evaluated. DBA/2 mice were inoculated with 10(6) P-388 murine leukemia cells, after which saline only, VP-16-213 only, vincristine only, or VP-16-213 followed at various time intervals by vincristine, were administered. Probable cure (survival greater than 60 days) was observed in 0/20, 0/20, 0/120, and 46/115 (40%), respectively (p less than 0.001). The proportion of animals attaining probable cure was greatest in the group receiving vincristine 4-72 hours after VP-16-213 (40-50%). Similar results had been obtained previously with the reverse drug sequence. In this animal model, the synergistic antitumor activity of vincristine and VP-16-213 does not appear to be schedule-dependent with respect to the sequence of drug administration.