GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.
The E2A-HLF (for hepatic leukaemia factor) fusion gene, formed by action of the t(17;19) (q22;p13) chromosomal translocation, drives the leukaemic transformation of early B-cell precursors, but the mechanism of this activity remains unknown. Here we report that human leukaemia cells carrying the translocation t(17;19) rapidly died by apoptosis when programmed to express a dominant-negative suppressor of the fusion protein E2A-HLF, indicating that the chimaeric oncoprotein probably affects cell survival rather than cell growth. Moreover, when introduced into murine pro-B lymphocytes, the oncogenic E2A-HLF fusion protein reversed both interleukin-3-dependent and p53-mediated apoptosis. The close homology of the basic region/leucine zipper (bZIP) DNA-binding and dimerization domain of HLF to that of the CES-2 cell-death specification protein of Caenorhabditis elegans suggests a model of leukaemogenesis in which E2A-HLF blocks an early step within an evolutionarily conserved cell-death pathway.
Relative levels of cytoplasmic aldehyde dehydrogenase (ALDH) were determined in selected subpopulations of normal human bone marrow cells using a flow cytometric assay that simultaneously detects a cell surface antigen (as a marker of cell lineage and developmental stage) and the level of ALDH. The intracellular level of this enzyme has been shown to be directly related to cellular resistance to activated cyclophosphamide and is believed to be important in the survival of cells capable of repopulating marrow in autologous bone marrow transplant procedures. Western blot analysis and flow cytometric analysis of four murine cell lines with known ALDH levels were used to establish the relation between ALDH content and fluorescence with an affinity-purified anti-mouse ALDH antibody. An affinity purified anti- human ALDH antibody, characterized by immunoblotting of cytosolic extracts of cell lines with known ALDH content, was used to determine relative ALDH levels in the marrow subpopulations. We found that hematopoietic progenitor cells express the highest level of ALDH, while lymphocytes express the lowest level. Immature erythroid cells express ALDH at a level intermediate between progenitor cells and lymphocytes.
The aromatic fatty acid phenylbutyrate (PB) induces cytostasis, differentiation, and apoptosis in primary myeloid leukemic cells at clinically achievable concentrations. In the present study, we have investigated the structural and cellular basis for PBinduced cytostasis, using the ML-1 human myeloid leukemia cell line as a model system. PB induced a dose-dependent increase in cells in G1 with a corresponding decrease in cells in S-phase of the cell cycle. At comparable doses, PB induced expression of CD11b, indicating myeloid differentiation. At higher doses, the drug induced apoptosis. The antitumor activity was independent of the aromatic ring, as butyric acid (BA) was of equal or greater potency at producing these biological changes. In contrast, shortening of the fatty acid carbon chain length, as demonstrated with phenylacetate (PA), significantly diminished drug potency. Consistent with their effects on cell cycle, PB and BA, but not PA, induced the cyclindependent kinase inhibitor, p21 WAF1/CIP1 , and led to the appearance of hypophosphorylated Rb, suggesting a role for p21 WAF1/CIP1 in PB-induced cytostasis. Therefore, it appears that the fatty acid moiety of PB, rather than its aromatic ring, is critical for its activity in myeloid leukemic cells. These data provide a potential mechanistic basis for the increased potency of PB over PA previously demonstrated in primary leukemic samples, and support the further clinical development of PB in the treatment of hematologic malignancies.
Relative levels of cytoplasmic aldehyde dehydrogenase (ALDH) were determined in selected subpopulations of normal human bone marrow cells using a flow cytometric assay that simultaneously detects a cell surface antigen (as a marker of cell lineage and developmental stage) and the level of ALDH. The intracellular level of this enzyme has been shown to be directly related to cellular resistance to activated cyclophosphamide and is believed to be important in the survival of cells capable of repopulating marrow in autologous bone marrow transplant procedures. Western blot analysis and flow cytometric analysis of four murine cell lines with known ALDH levels were used to establish the relation between ALDH content and fluorescence with an affinity-purified anti-mouse ALDH antibody. An affinity purified anti- human ALDH antibody, characterized by immunoblotting of cytosolic extracts of cell lines with known ALDH content, was used to determine relative ALDH levels in the marrow subpopulations. We found that hematopoietic progenitor cells express the highest level of ALDH, while lymphocytes express the lowest level. Immature erythroid cells express ALDH at a level intermediate between progenitor cells and lymphocytes.
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