ABSTRACT
Contact with bone marrow stromal cells maintains normal and leukemic hematopoietic progenitors in an undifferentiated state. Recently, stromal contact has been shown to diminish the yield of megakaryocytes in cultures of primary human hematopoietic stem cells. This inhibition may explain the poor megakaryocytic engraftment frequently observed after bone marrow transplantation. In the current study, stromal co-culture is shown to render K562 cells refractory to megakaryocytic induction. This stromal inhibition correlated with the selective down-regulation in K562 cells of protein kinase C-epsilon (PKC-epsilon), which has recently been implicated in regulation of megakaryocytic lineage commitment. In addition, the stromal inhibition correlated with inactivation of the ERK/MAPK pathway, which has also been implicated in promoting megakaryocytic development. Forced expression of PKC-epsilon by retroviral transduction was insufficient to reverse the stromal blockade of ERK/MAPK signaling or of megakaryocytic induction. Thus stromal interruption of ERK/MAPK signaling occurred independently of PKC-epsilon levels and correlated more closely with megakaryocytic blockade. These findings provide potential mechanisms for stromal inhibition of hematopoietic differentiation and possibly for the poor megakaryocytic engraftment seen after bone marrow transplantation.
Subject(s)
Hematopoietic Stem Cells/physiology , Isoenzymes/metabolism , MAP Kinase Signaling System/physiology , Megakaryocytes/cytology , Mitogen-Activated Protein Kinases/metabolism , Protein Kinase C/metabolism , Signal Transduction/physiology , Stromal Cells/physiology , Bone Marrow Cells/cytology , Cell Line , Coculture Techniques , Flow Cytometry , Genes, Reporter , Green Fluorescent Proteins , Hematopoietic Stem Cells/cytology , Humans , Immunophenotyping , Isoenzymes/genetics , K562 Cells , Luminescent Proteins/genetics , Phosphorylation , Protein Kinase C/genetics , Protein Kinase C-epsilon , Recombinant Proteins/metabolism , Stromal Cells/cytology , TransfectionABSTRACT
The Fas (CD95) antigen plays a key role in regulating T-cell activation and survival. We have generated a Fas-resistant subclone of the human T-cell leukaemia line, H9, which is still able to undergo apoptosis in response to T-cell receptor ligation. Molecular analyses revealed that resistance to Fas-mediated apoptosis was due to a heterozygous mutation in the death domain of the Fas gene which generates a stop codon, and thus encodes a truncated Fas molecule. Fas ligation was able to induce apoptosis in the presence of cycloheximide, indicating that the mutant Fas molecule retained some signalling capability, which is death-domain independent. These cells will provide a useful tool for dissecting the complexities of Fas signalling pathways.
Subject(s)
Apoptosis/immunology , Leukemia, T-Cell/immunology , Receptors, Antigen, T-Cell/immunology , fas Receptor/immunology , Amino Acid Sequence , Apoptosis/drug effects , Apoptosis/genetics , Cycloheximide/pharmacology , Humans , Molecular Sequence Data , Mutation , Neoplastic Stem Cells/immunology , Tacrolimus/pharmacology , Tumor Cells, Cultured , fas Receptor/geneticsABSTRACT
Benzoin (ZOIN) and caprolactam (CAP) were administered by gavage to Fischer 344 rats at a dose of 750 mg/kg and the hepatocytes isolated 12, 24 or 48 h after treatment. The isolated hepatocytes were subsequently examined for the induction of DNA-strand breaks (SB) and unscheduled DNA synthesis (UDS). Neither ZOIN nor CAP induced SB or UDS in hepatocytes, however ZOIN did induce an increase in the fraction of cells in S-phase 24 h after treatment. These results correlate well with the observed lack of carcinogenicity of these compounds.
