aphidicolin and Leukemia--Myeloid

The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway. Importantly, we provide evidence that these recurrent deletions originate in pre-leukemic stem cells. While DNA polymerase theta (POLQ) is considered a key component in MMEJ repair, we provide evidence that pre-leukemic MMEJ (preL-MMEJ) deletions can be generated in POLQ knockout cells. In contrast, aphidicolin (an inhibitor of replicative polymerases and replication) treatment resulted in a significant reduction in preL-MMEJ. Altogether, our data indicate an association between POLQ independent MMEJ and clonal hematopoiesis and elucidate mutational mechanisms involved in the very first steps of leukemia evolution.

Topics: Aphidicolin; Calreticulin; Clonal Hematopoiesis; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Polymerase theta; DNA-Directed DNA Polymerase; Enzyme Inhibitors; Humans; Leukemia, Myeloid; Myeloid Progenitor Cells; Poly (ADP-Ribose) Polymerase-1; Repressor Proteins; Sequence Deletion; Serine-Arginine Splicing Factors

Double strand breaks (DSBs) are considered the most lethal form of DNA damage for eukaryotic cells, and misrepair of DSB can cause cell death, chromosome instability, and cancer. Nonhomologous end-joining (NHEJ) is a major mechanism for the repair of DSBs. We previously reported that the cancer predisposition Bloom's syndrome and myeloid leukemias demonstrate increased NHEJ activity and consequent misrepair. In this study, we link this increased NHEJ activity and infidelity to ongoing or induced DNA damage at sites that recruit key NHEJ proteins. We show here that in myeloid leukemia cells and normal hemopoietic cells, agents that induce DSBs produce an up to 2-fold increase in this DSB misrepair activity, whereas an alkylating agent produces little or no increases. Furthermore, NHEJ overactivity after induction of DSBs is dependent on the presence of Ku70/Ku86. We also present data to explain the constitutively activated NHEJ in myeloid leukemias. Using an immunofluorescence-based assay for DNA damage, myeloid leukemias demonstrate constitutive DNA damage in the absence of treatment with DSB-inducing agents compared with normal hemopoietic cells. Importantly, damaged foci from myeloid leukemia and normal cells colocalize with NHEJ proteins Ku70 and Ku86. These data suggest that the generation of increased constitutive DNA damage may be a common pathway for the creation of NHEJ-dependent genomic instability.

Topics: Antibodies; Antigens, Nuclear; Aphidicolin; DNA Damage; DNA Helicases; DNA Repair; DNA-Binding Proteins; DNA, Neoplasm; HL-60 Cells; Humans; K562 Cells; Ku Autoantigen; Leukemia, Myeloid

The efficiency of retinoic acid (RA)-induced differentiation was dependent on the position of HL-60 cells in the cell cycle. Our results demonstrated that cells at the G1/S border were more efficiently induced to differentiate by short exposure to RA than cells at other phases of the cell cycle. Synchronization of cells in G1/S phase by aphidicolin (APH) or mimosine (MIMO) increased the sensitivity of cells to RA short exposure treatment. Pretreatment with sphinganine (SP), a protein kinase C (PKC) inhibitor, potentiated RA-induced cell differentiation. By cell cycle analysis, SP was found to block the cell progression through the G1/S phase. Consequently, cells accumulated in the G1/S phase of the cell cycle. The present data therefore suggest a possible mechanism of action of SP to enhance RA-induced differentiation.

Topics: Aphidicolin; Cell Cycle; Cell Differentiation; DNA; Granulocytes; Humans; In Vitro Techniques; Leukemia, Myeloid; Mimosine; RNA; Sphingosine; Tretinoin; Tumor Cells, Cultured

We investigated the effect of aphidicolin, an inhibitor of DNA polymerase alpha and delta, on the induction of apoptosis by arabinosyl nucleosides in a human promyelocytic leukemia cell line, HL-60. Pretreatment of HL-60 cells with aphidicolin (2 microM) significantly increased the number of morphologically apoptotic cells induced by 1-beta-D arabinofuranosylcytosine (ara-C) during 4 hr of incubation. This is consistent with the appearance of DNA fragmentation as determined quantitatively by diphenylamine or by agarose gel electrophoresis. The inhibition of cell growth on day 3 after drug exposure was correlated with the degree of apoptosis: Such synergistic interaction between aphidicolin and ara-C has also been observed in other human myeloid leukemia cell lines, U937 and KG-1. In addition, the induction of apoptosis by 9-beta-D arabinofuranosyladenine or 9-beta-D arabinofuranosylguanine is augmented by aphidicolin.

Topics: Aphidicolin; Apoptosis; Arabinonucleosides; DNA; Drug Synergism; Electrophoresis, Agar Gel; Humans; Leukemia, Myeloid; Tumor Cells, Cultured; Vidarabine

Proliferation of the human monocytic leukemia cell line JOSK-I is inhibited by transforming growth factor-beta (TGF-beta). Growth inhibition by TGF-beta was not due to either a toxic effect or to induction of differentiation. TGF-beta induced a cell cycle arrest at late G1 phase and was not found to be inhibitory to JOSK-I cells in S phase or G2/M. This G1 cell cycle arrest was associated with an accumulation of the unphosphorylated form of the retinoblastoma susceptibility gene product (Rb) in good correlation with inhibition of DNA synthesis. In contrast to the effects of TGF-beta, two other agents which induced a G1 arrest of JOSK-I cells had a different effect on Rb. Aphidicolin blocked cells at G1/S but could not reduce Rb phosphorylation as great as that seen with TGF-beta. 12-O-Tetradecanoylphorbol-13-acetate, an inducer of differentiation, did reduce Rb phosphorylation, but not until 72 h, when differentiation had already occurred. The identities of the Rb kinases are unknown, but recent evidence suggests that the cdc2 gene product could participate in Rb phosphorylation. Although cdc2 mRNA and total protein levels were not affected, TGF-beta inhibited the rate of translation and kinase activity of cdc2 in JOSK-I cells. These results suggest that growth inhibition of hematopoietic cells by TGF-beta is linked to suppression of Rb phosphorylation to retain Rb in an unphosphorylated, growth-inhibitory state. The suppression of Rb phosphorylation is suggested to be mediated through inhibition of cdc2 kinase activity by TGF-beta.

Topics: Aphidicolin; Blotting, Northern; Cell Cycle; Cell Division; Cell Line; DNA Replication; Genes, Retinoblastoma; HLA-DR Antigens; Humans; Kinetics; Leukemia, Myeloid; Phosphorylation; Receptors, Transferrin; Retinoblastoma Protein; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Thymidine Kinase; Transforming Growth Factor beta

Of 175 CML patients studied, 14 variants were found, seven of which are presently described. The breakpoints involved in the translocation, other than 9q34 and 22q11, are 3p21, 5q13, 6p21, 7q22, 10q22, and 11p13. Fragile sites were investigated in some of these patients. In two cases a coincidence between fragile site location and breakpoint of the third chromosome involved in Philadelphia formation was found. This observation suggests that the fragile sites can lead to Ph variants in patients developing CML.

Topics: Adult; Aged; Aphidicolin; Chromosome Banding; Chromosome Fragile Sites; Chromosome Fragility; Diterpenes; Genetic Markers; Humans; Karyotyping; Leukemia, Myeloid; Middle Aged; Philadelphia Chromosome; Translocation, Genetic

Enhanced expression of the proto-oncogene c-fos is reported to be involved in the differentiation of monomyelocytic cells into mature macrophages. Such differentiated cells usually accumulate in the G1 phase of the cell cycle. M1 murine myeloid leukemia cells, which can be synchronized in early and late G1 and can also differentiate into mature macrophages when stimulated with granulocyte colony-stimulating factor (G-CSF), were used to investigate whether the enhanced expression of c-fos in differentiated monomyelocytic cells is associated with the position of the cells in the cell cycle or with their state of differentiation. Our data show that the enhanced expression of the c-fos gene occurs only in fully differentiated cells and not in proliferating or G1-arrested cells. These data imply that the enhanced expression of c-fos in M1 cells is restricted to functionally differentiated cells and not related to the position of the differentiated cells in the cell cycle.

Topics: Animals; Aphidicolin; Cell Cycle; Cell Differentiation; Cell Line; Cell Separation; Colony-Stimulating Factors; Diterpenes; Granulocyte Colony-Stimulating Factor; Growth; Leukemia, Myeloid; Macrophages; Mice; Proto-Oncogenes; Receptors, Fc

A statistical analysis of the 327 cases of variant Philadelphia chromosome so far reported in chronic myeloid leukemia was performed. The results showed 28 bands to be significantly rearranged; 11 bands are known to contain a fragile site; 12 are included in the smallest region of overlap of 14 proto-oncogenes. Also, 23 bands are known to be significantly involved in structural rearrangements in other malignancies.

Topics: Aphidicolin; Caffeine; Chromosome Banding; Chromosome Fragile Sites; Chromosome Fragility; Diterpenes; Folic Acid; Humans; Leukemia, Myeloid; Oncogenes; Philadelphia Chromosome

Topics: Antineoplastic Agents; Aphidicolin; Bone Marrow; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Cytarabine; Diterpenes; DNA Replication; Formamides; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Mercaptopurine; Myeloproliferative Disorders; Reference Values; Tretinoin