title: GM-CSF-DFF40: a novel humanized immunotoxin induces apoptosis in acute myeloid leukemia cells.
abstract: DNA fragmentation factor 40 (DFF40) is an endonuclease that acts downstream in the apoptotic cascade. The objective of this study was to generate a novel humanized chimeric protein with human DFF40 fused with GM-CSF for targeting acute myeloid leukemia (AML) cells. cDNA cloning of human DFF40 and GM-CSF was done and the chimeric gene GM-CSF-DFF40 was generated by overlap extension PCR. The fusion protein was expressed in E.coli, purified, refolded and characterized. In vitro cytotoxicity was evaluated on various AML cell lines. Treated cell lines were screened for various morphological and biochemical changes that are characteristic of apoptosis, by different assays like annexin V-FITC staining, TUNEL assay, JC-1 staining and immunocytochemistry of pro-apoptotic proteins and caspases. Cell cycle analysis of treated cells was done to quantify the percentage of apoptotic cells. The chimeric protein was found to be cytotoxic to AML cells in a dose and time dependent manner. Morphological changes such as formation of apoptotic bodies were revealed by microscopic examination of treated cells after staining. Immunocytochemical staining demonstrated biochemical changes such as changes in mitochondrial membrane potential, mitochondrial co-localization of Bax, cytochrome c release, presence of activated caspase-3 and DNA fragmentation. FACS analysis proved the presence of apoptotic cells following treatment. The chimeric protein GM-CSF-DFF40 was found to mediate targeted killing of AML cells by inducing apoptosis. Thus, this chimeric construct can act as a prospective candidate for targeted therapy of AML and other malignancies where GM-CSF receptor expression is upregulated.
authors.affiliation: Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, Tamil Nadu, India.
authors.forename: Mrudula
authors.initials: M
authors.lastname: Mathew
authors.forename: K C
authors.initials: KC
authors.lastname: Zaineb
authors.forename: Rama Shanker
authors.initials: RS
authors.lastname: Verma
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000494:pharmacology*; D007938:Leukemia / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D058849:Protein Refolding; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D015398:Signal Transduction
chemical_list: D000922:Immunotoxins; D009363:Neoplasm Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C113696:DFFB protein, human; D003851:Deoxyribonucleases