ABSTRACT
Induction of specific gene expression may provide an alternative or a support to conventional cytotoxic chemotherapy of cancer, as well as to therapy for sickle cell diseases. In this respect, pharmacological induction of expression of the endogenous gamma-globin gene is a realistic approach to therapy of beta-globin disorders. Erythroid differentiation and inhibition of proliferation of the human CML K562 cell line was induced by guanosine 5'-triphosphate (GTP). The hemoglobin production in cells was correlated to an increase in alpha- and gamma-globin mRNA expression. At the transcriptional level, we showed that both the expression of the major erythroid transcription factor GATA-1 (protein and mRNA) and its binding capacity to the gamma-globin gene promoter was transiently increased. Moreover, GTP moderately stimulated the gamma-globin gene promoter after 48 h of treatment. At the post-transcriptional level, GTP treatment led to a drastic increase of the gamma-globin mRNA half-life. This stabilizing effect of GTP was mediated via the 3'-untranslated region (3'-UTR) of the gamma-globin mRNA. In conclusion, mechanism of GTP-mediated differentiation of K562 cells is linked to an early activation of gamma-globin gene transcription followed by a stabilization of its mRNA.
Subject(s)
DNA-Binding Proteins/biosynthesis , Erythropoiesis/physiology , Globins/metabolism , Guanosine Triphosphate/physiology , Transcription Factors/biosynthesis , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , Erythropoiesis/genetics , GATA1 Transcription Factor , Gene Expression Regulation, Neoplastic , Globins/genetics , Humans , K562 Cells , Promoter Regions, Genetic/genetics , RNA Stability , RNA, Messenger/metabolism , Transcription Factors/geneticsABSTRACT
The activity of numerous antineoplasic drugs is correlated with their capacity to induce the apoptotic process. In this study, apoptosis induced by the topoisomerase I (Topo I) inhibitors camptothecin (CPT) and the CPT-11 active metabolite SN-38 was evaluated on HL-60 cells and their multidrug resistant variant HL-60-Vincristine cells. Both CPT and SN-38 induced high levels of apoptosis in sensitive cells but very low levels in MDR cells. The role of the different genes and proteins usually implicated in the drug resistance phenomenon was studied. The Pgp independence of the two drugs was suggested by the lack of modulation of anti-Topo I effects with verapamil. Moreover CPT and SN-38 induced a strong decrease of mdr1 mRNA in MDR treated cells. MRP mRNA expression was very low in drug sensitive and resistant cells and decreased during treatments in both cell lines. However, MRP protein was not detected in control and MDR cells suggesting that this pump was probably not implicated in this resistance phenomenon. Topo I and BCL-2 proteins displayed a higher expression in MDR cells but only Topo I proteins decreased during treatments in the two cell lines. These data suggest that in addition to the classical multidrug resistance phenotype, dysregulation of proteins associated with DNA replication and apoptotic process could contribute to acquired resistance to a large panel of drugs, including those which are not considered as substrates for Pgp.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antineoplastic Agents, Phytogenic/toxicity , Apoptosis/physiology , Camptothecin/analogs & derivatives , Camptothecin/toxicity , Drug Resistance, Multiple , Enzyme Inhibitors/toxicity , Topoisomerase I Inhibitors , Vincristine/toxicity , Apoptosis/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genes, MDR , Genes, bcl-2 , HL-60 Cells , Humans , Irinotecan , Proto-Oncogene Proteins c-bcl-2/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Apoptosis is a genetically regulated cell death process which results in a variety of morphological changes like chromatin condensation and DNA fragmentation. The decision between survival or death in response to an apoptotic stimulus is determined and regulated in part by oncoproteins which include proteins of the Bcl-2 family (bcl-2, bax, bcl-xL) and bcr-abl. We investigated the effect of these proteins on the induction of this phenomenon in human promyelocytic leukemic HL60 cells and two multidrug resistant homologues selected respectively with vincristine (HL60/VCR) and daunorubicin (HL60R/DNR). We show that sensitive cells at 1 micron and HL60/VCR cells at DNR IC50 were able to undergo apoptosis while HL60R/DNR did not even at much higher concentration of DNR. However, treatment with synthetic C2-ceramide did not sensitize HL60/DNR cells to apoptosis. Cell death through apoptosis or necrosis was accompanied by acidification of the cytosol without mitochondrial membrane depolarization. Western blotting analysis shows that bax is expressed at slightly elevated level in HL60S/VCR in comparison with the other cells lines. Bcl-2 is overexpressed in HL60/VCR but not in HL60R/DNR. However, this cell line displayed a higher expression of bcl-xL. Interestingly, bcr-abl, a dysregulated tyrosine kinase was detected only in HL60R/DNR cells. DNR at the IC50, has no effect on expression of the oncoproteins. These data suggest that in addition of the multidrug resistance phenotype, bcr-abl translocation and bcl-xL overexpression could also account for the development of resistance to cell death induced by anthracyclines in leukemic cells.
Subject(s)
Apoptosis/physiology , Daunorubicin/toxicity , Drug Resistance, Multiple , Gene Expression Regulation, Neoplastic/drug effects , Genes, bcl-2/drug effects , Vincristine/toxicity , Apoptosis/drug effects , Carbonyl Cyanide m-Chlorophenyl Hydrazone/toxicity , Fusion Proteins, bcr-abl/genetics , HL-60 Cells , Humans , Hydrogen-Ion Concentration , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Mitochondria/drug effects , Mitochondria/physiology , Proto-Oncogene Proteins c-bcl-2/genetics , bcl-X Protein , fas Receptor/geneticsABSTRACT
The induction of apoptosis by topoisomerase I inhibitors, camptothecin and SN38, was evaluated in drug-sensitive HL60 and multidrug-resistant (MDR) HL60-Vinc leukemic cells. MDR cells displayed a partial resistance to these apoptotic stimuli and this phenomenon was not modulated by verapamil. Basal free calcium concentrations were similar in both cell sublines and were not modified during treatment. Cytoplasmic pH was more acidic in sensitive cells than in MDR cells. Moreover, a significant acidification was obtained during the early stage of apoptosis in sensitive HL60 cells only. Basal Bcl-2 protein expression was found to be greater in MDR than in sensitive cells and was not modulated by apoptosis inducers. This increase of Bcl-2 in MDR cells could be due to the selection process as vincristine enhances Bcl-2 phosphorylation and expression in HL60 sensitive cells. MDR HL60-Vincristine cells therefore display a resistance to apoptosis induced by non-MDR drugs, possibly by Bcl-2 overexpression and inability of these drugs to mediate intracellular pH changes in these drug-resistant cells.
Subject(s)
Apoptosis , Drug Resistance, Multiple , Enzyme Inhibitors/pharmacology , Topoisomerase I Inhibitors , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , HL-60 Cells , Humans , Hydrogen-Ion Concentration , Irinotecan , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Vincristine/pharmacologyABSTRACT
The nuclear morphological alterations occurring during apoptosis induced in human HL-60 cells by camptothecin were analyzed quantitatively by image cytometry. Two separate phases were identified during the apoptotic process. The first phase, observed between 0-2 h of incubation, consisted in the appearance of cells with an apparently decondensed chromatin. This phenomenon was blocked by the inhibitors of DNA fragmentation, TLCK and H7. In contrast, staurosporine and cytochalasin B, which inhibit apoptosis without any effect on DNA fragmentation in this system, did not prevent this morphological change. The second phase, observed after 3 h of culture, corresponded to the appearance of cells with very homogeneous and condensed chromatin. This phenomenon correlated with the detection of typical apoptotic cells with fragmented nuclei and was inhibited by all drugs (TLCK, H7, staurosporine, and cytochalasin B). These observations suggest that image cytometry allows the detection of subvisual microscopic events within the first hour after the induction of an apoptotic process and that the dissection of this process into several different phases might be associated with DNA fragmentation.
Subject(s)
Apoptosis/physiology , Camptothecin/pharmacology , Image Cytometry/methods , Topoisomerase I Inhibitors , Cell Division , Cell Nucleus/physiology , DNA Fragmentation , HL-60 Cells , Humans , Multivariate AnalysisABSTRACT
Nuclear DNA content was assessed in multidrug-resistant (MDR) cells by image and flow cytometry. Two human MDR cell lines (K562-Dox and CEM-VLB) obtained by in vitro drug selection and overexpressing mdr1 gene were compared to their respective sensitive counterparts (K562 and CCRF-CEM) and to the MDR hamster LR73-R cell line obtained by transfection of mouse mdr1 cDNA. Both cell lines obtained by selection displayed a decreased DNA content, as measured by image cytometry after Feulgen staining, or by flow cytometry after staining with propidium iodide, ethidium bromide, or Hoechst 33342. This decrease was not accompanied by changes in cell cycle phase distribution of cells. Moreover, image cytometry of cells stained after various hydrolysis times in 5 M HCl indicated that MDR cells displayed the same hydrolysis kinetics and sensitivity as drug-sensitive cells with a well-preserved stoichiometry of the Feulgen reaction. LR73-R cells transfected with mdr1 cDNA exhibited only a very limited change in propidium iodide staining as compared with sensitive LR73 cells, suggesting that mdr1 gene overexpression alone could not account for the alterations in DNA content observed in the selected MDR cells.
