demecolcine and Leukemia

Using Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) as a model, our aim has been to develop a molecular cytogenetic method of high resolution analysis for monitoring the frequency of cells with nonrandom chromosome rearrangements in the bone marrow of patients receiving treatment for hematologic malignancies. Long-term exposure (24 hours) of bone marrow cultures to colcemid (0.1 microgram/mL) maximized a high frequency of metaphase collection. Such preparations were subjected to fluorescence in situ hybridization (FISH) using a 5 Mb probe that overlapped the region of the translocation at chromosome 9q34. This detected the Ph translocation in the resultant large number of overly contracted chromosome spreads. The procedure was validated and verified by studying 70 double-blind marrow samples from patients in different stages of Ph+ CML and from patients with Ph- hematologic malignancies (controls). This hypermetaphase FISH (HMF) method clearly identified Ph+ metaphases and allowed the analysis of 500 hypermetaphases per sample in less than 1 hour after FISH. HMF (1) identified statistically significant differences between the frequencies of Ph+ cells in samples that differed by less than 4%; (2) resolved such differences among patient samples that were all judged 100% Ph+ by standard G-band cytogenetics (CG); (3) resulted in the reclassification of response status in 23% of the patients initially classified by CG; (4) recognized Ph+ cells in 16% of patients characterized as having a complete cytogenetic response and in one patient with an original diagnosis of Ph- CML; and in one patient with an original diagnosis of Ph- CML; and (5) was informative where insufficient metaphases were obtainable for analysis by CG. HMF appears to be uniquely suitable for monitoring the status of patients with CML receiving treatment. It should also be applicable for patients with any hematologic diseases where chromosomal alterations are known and appropriate FISH probes are available.

Topics: Azure Stains; Bone Marrow; Bone Marrow Examination; Chromosome Banding; Demecolcine; Humans; Immunologic Factors; In Situ Hybridization, Fluorescence; Interferon-alpha; Karyotyping; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Metaphase; Neoplasm, Residual; Philadelphia Chromosome; Single-Blind Method; Treatment Outcome; Tumor Cells, Cultured

The proportions of aneuploid/polyploid versus euploid cells formed after treatment with spindle poisons like nocodazole are of course dependent on the relative survival of cells with numerical chromosome aberrations. This work aimed at studying the survival of polyploid cells formed after treatment with a nocodazole concentration sufficient to significantly decrease tubulin polymerization (0.1 microg/ml). First, normal primary lymphocytes were analysed and the following complementary chromosomal parameters were quantified: mitotic index, frequency of abnormal mitoses, polyploid metaphases and apoptotic cells. The results clearly indicate a positive correlation between abnormal mitotic figures, apoptosis and the induction of polyploidy. They therefore led to a single cell approach in which both apoptosis and polyploidy induction could be scored in the same cell. For this purpose, actively proliferating cells are required and two human leukaemic cell lines were used, KS (p53-positive) and K562 (p53-negative), which have a near-triploid karyotype. Cells were separated into an apoptotic and a viable fraction by means of annexin-V staining and flow cytometry. In KS, treatment with nocodazole induced a similar fraction of hexaploid cells in both the viable and apoptotic fraction, but no dodecaploid cells were ever observed. In contrast, a population of dodecaploid cells (essentially viable) was clearly observed in the K562 cell line. The results in KS, as compared with K562, confirm that wild-type p53 can prevent further cycling of polyploid cells by blocking rereplication. The most probable explanation for these data is that not only the mitotic spindle but also interphase microtubules are sensitive to nocodazole treatment. Our data thus strongly suggest that besides the G(1)/S checkpoint under the control of p53, the G(2)/M transition may be sensitive to depolymerization of microtubules, possibly under the control of Cdc2, Bcl-2, Raf-1 and/or Rho.

Topics: Adult; Antineoplastic Agents; Apoptosis; Cell Survival; Cells, Cultured; Demecolcine; Dose-Response Relationship, Drug; Gene Expression; Humans; In Situ Nick-End Labeling; K562 Cells; Leukemia; Lymphocytes; Nocodazole; Phytohemagglutinins; Polyploidy; Spindle Apparatus; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Cells undergoing apoptosis (programmed cell death) display profound morphologic and biochemical changes in the nucleus, cytoplasm, and plasma membrane. We have shown a direct temporal relationship between the onset of apoptosis in Jurkat T-cell lymphoblast cultures and a greater than two-fold increase in the signal intensity of the methylene resonance (at 1.3 ppm) as observed by proton nuclear magnetic resonance spectroscopy (1H NMR). The increase in the methylene resonance intensity was seen when apoptosis was induced by serum deprivation, glucocorticoid, and doxorubicin treatment but not in necrotic (nonapoptotic) cell death. We have found similar changes in a variety of other cell lines undergoing apoptosis including the Hut 78 T-cell leukemia, JY natural killer T-cell leukemia, Daudi B-cell lymphoma, HeLa, and 3T3 fibroblast cell lines. Furthermore, this spectral change was diminished in Bcl-2 overexpressing HL-60 cell cultures treated with doxorubicin, which were relatively resistant to apoptosis, as compared to apoptotic HL-60 cultures. 1H NMR spectroscopy therefore may be useful in detecting apoptotic cell death in vivo.

Topics: 3T3 Cells; Animals; Apoptosis; Burkitt Lymphoma; Cell Line; Culture Media, Serum-Free; Demecolcine; Dexamethasone; DNA Damage; Doxorubicin; HL-60 Cells; Humans; Leukemia; Lymphocyte Subsets; Magnetic Resonance Spectroscopy; Mice; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Protons; Tumor Cells, Cultured

The development of drug resistance in cancer cells is a significant clinical problem for the successful cancer chemotherapy. Since the cytoskeleton, including microtubules, may be involved in modulating cellular signal transduction, morphological and structural changes, the microtubules assembly of multidrug resistant cells was examined using Confocal Laser Microscope MRC500 system (Bio Rad). In this study, multidrug resistant cells were established by the continuous exposure to ADR(adriamycin) starting with 20 nM up to 1 microM. The expression of MDR-1 (multidrug resistance) gene was detected in K562 leukemia cells and to more extent in the multidrug resistant K562/ADR cells, but not in HL-60 leukemia cells and multidrug resistant HL-60/ADR cells by RT-PCR method. The chronological features of microtubules assembly in the parent cell lines were lost on day 3, after incubation with 20nM of ADR. In accordance with development of drug resistance, the microtubules assembly appeared to be more dense and stronger than that of parent cells. During the development of drug resistant cells, the ADR-accumulation in the nucleus was decreased according to the increase of microtubules assembly. In the case of incubation with 0.5 microM colcemid, an inhibitor of microtubules polymerization, for 3 hours, the stainings of microtubules were lost their fine network and appeared to be diffuse and dot-like pattern. At the same time, both untreated HL-60/ADR and K562/ADR showed the decrease of ADR-accumulation, but the accumulations both colcemid treated resistant cells were increased the same level of their parent cells at the point of 120 min. These results suggested that the resistance to ADR in human leukemia cells correlated with microtubules assembly, and the microtubules assembly played an important role of drug resistance with or without MDR-1 gene overexpression.

Topics: Demecolcine; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Gene Expression; Humans; Leukemia; Microscopy, Confocal; Microtubules; Polymerase Chain Reaction; Tumor Cells, Cultured

In most eukaryotic cells the regular alternation of chromosome reduplication and cell division is controlled by interdependent relationships which prevent progression to the next cell-cycle phase unless the preceding phase has been completed. Megakaryocytes become polyploid by allowing many rounds of DNA replication without completion of intervening mitoses. To assess the role of cell-cycle dependencies in megakaryocytopoiesis we examined human cell lines which express megakaryocytic features for their ability to continue DNA synthesis and undergo polyploidization in the presence of mitotic poisons. Treatment of HEL cells with colcemid blocked cell division but not cellular DNA synthesis. DNA content distributions of cells treated with colcemid for 48 h showed a marked increase in the proportion of polyploid cells (57.6% +/- 9.9%, n = 16), an increase in cellular size and nuclear lobation. Identical effects were observed in HEL cells treated with colchicine, nocodazole or taxol but not with the inactive compound lumicolchicine. Induction of polyploidization by antimicrotubule agents was also observed in the megakaryoblastic cell lines MEG-01, DAMI and UT-7 but not in the T-cell line MOLT-4 or the promyelocytic cell line HL-60. These results suggest that dependency of DNA replication on completion of the previous mitosis is suppressed in the megakaryocytic lineage.

Topics: Cell Division; Colchicine; Demecolcine; DNA; Flow Cytometry; Humans; Leukemia; Lumicolchicines; Megakaryocytes; Microtubules; Nocodazole; Paclitaxel; Polyploidy; Tumor Cells, Cultured

Studies were undertaken to determine whether EDTA was a satisfactory anticoagulant for tissue specimens for cytogenetic analysis and to investigate a modification of a currently used culture technique for obtaining metaphases. The latter involved to prolonged exposure to very low-dose colcemid and was successful in qualitative or quantitative enhancement, or both, of the temperature yield over that obtained from direct harvest in 53% of the patients studied. EDTA is a suitable anticoagulant for cytogenetic studies of specimens from either direct harvest or short-term culture if the specimen is either processed within 24 hr after collection or diluted 1:1 with Eagles minimal essential media, supplemented with fetal bovine serum and refrigerated until processed. Success has been obtained with specimens stored up to 144 hr.

Topics: Bone Marrow; Culture Media; Culture Techniques; Demecolcine; Drug Evaluation, Preclinical; Edetic Acid; Humans; Karyotyping; Leukemia; Metaphase; Mitotic Index; Time Factors

Short incubation of heparinized human leukemic bone-marrow cells in phosphate buffered saline containing colcemid and overnight chilling of fixed cells yields metaphases with elongated and well-spread chromosomes. This technique enables us to do trypsin-Giemsa banding of chromosomes obtained from leukemic marrow cells otherwise difficult to band.

Topics: Azure Stains; Bone Marrow; Chromosome Banding; Cytological Techniques; Demecolcine; Humans; Karyotyping; Leukemia; Metaphase; Mitotic Index

Spontaneous mitotic cells of the mouse leukemic cell line GF7 are preferentially included in cell fusion products after treatment with polyethylene glycol (PEG). This implies a unique configuration of the natural mitotic membrane which is particularly vulnerable to induction of fusion by PEG. Colcemid-arrested GF7 mitotic cells, however, are excluded from PEG-induced cell fusion products, suggesting that colcemid reverses the membrane configuration which is susceptible to the action of PEG. When Sendai virus is used as the fusogenic agent, both colcemid-arrested and spontaneous mitotic cells are selectively fused. There must, therefore, be an essential membrane-fusogen reaction which is characteristically different for each of these agents.

Topics: Cell Fusion; Cell Line; Cell Membrane; Demecolcine; Leukemia; Lymphocyte Activation; Mitosis; Parainfluenza Virus 1, Human; Polyethylene Glycols; Thymidine

Topics: Blood Cells; Cells, Cultured; Demecolcine; Humans; In Vitro Techniques; Lectins; Leukemia; Lymphocyte Activation; Lymphocytes; Mitosis; Platinum

Topics: Alkaloids; Animals; Antifungal Agents; Antineoplastic Agents; Ascites; Colchicine; Demecolcine; Hydrazines; Indicators and Reagents; Iron; Leukemia; Mice; Radiation-Sensitizing Agents; Riboflavin; Triacetin; Triglycerides; Urethane; Vitamin K

Topics: Colchicine; Demecolcine; Drug Therapy; Leukemia; Neoplasms

Topics: Chronic Disease; Demecolcine; Humans; Leukemia; Leukemia, Myeloid; Polycythemia

Topics: Colchicine; Demecolcine; Disease; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Medical Records; Pregnancy; Ribs

Topics: Colchicine; Demecolcine; Leukemia; Leukemia, Myeloid

Topics: Busulfan; Colchicine; Demecolcine; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid

Topics: Colchicine; Demecolcine; Leukemia

Topics: Colchicine; Demecolcine; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid

Topics: Colchicine; Demecolcine; Leukemia; Leukemia, Myeloid; Pregnancy

Topics: Colchicine; Demecolcine; Hodgkin Disease; Humans; Leukemia

Topics: Colchicine; Demecolcine; Hematologic Diseases; Leukemia; Neoplasms

Topics: Colchicine; Demecolcine; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid

Topics: Antineoplastic Agents; Busulfan; Colchicine; Demecolcine; Glycols; Leukemia; Leukemia, Myeloid

Topics: Colchicine; Demecolcine; Leukemia; Leukemia, Myeloid; Neoplasms

Topics: Antineoplastic Agents; Colchicine; Demecolcine; Leukemia; Leukemia, Myeloid