Diphtheria toxin fused to GM-CSF in combination with ARA-C exerts synergistic toxicity to human AML (HL60) cells

K. M. Bhalia, R. J. Kreitman, P. Hall, A. Frankel

Research output: Contribution to journalArticle

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Abstract

Human GM-CSF fused to a truncated diphtheria toxin (DT388-GM-CSF) showed a dramatic synergistic effect (combination index of 0.07) with Ara-C on HL60 human leukemia cells which was not seen with native GM-CSF. The enhanced cell kill occurred within 30 hr of exposure to both agents and did not involve detectable changes in Ara-C incorporation into cellular DNA. The synergistic toxic effect of Ara-C was not seen with 2 other protein synthesis inhibitors: ricin and cycloheximide. Enhanced cell kill with the DT388-GM-CSF/Ara-C combination was observed using both an 18 hr thymidine incorporation and a CFC assay to measure loss of proliferative potential and DAPI staining to detect nuclear fragmentation, but was not seen using the MTT assay to measure mitochondrial metabolism. Exposure to DT388-GM-CSF alone induced mitochondrial release and cytosolic accumulation of cytochrome c, and increased the PARP cleavage activity of caspase-3. Co-treatment with Ara-C enhanced both of these effects as well as the apoptosis induced by exposure to DT388-GM-CSF. However, the degree of enhancement of the effects on cytochrome c, caspase-3, and apoptosis of HL60 cells did not correlate with their reduced clonogenicity, Neither DT388-GM-CSF nor Ara-C treatment altered Bcl-2, Bcl-xl, Bax or FasR levels in HL60 cells, although DT388-GM-CSF increased Fas ligand expression by approximately 50%. Furthermore, enforced overexpression of Bcl-2 in the HL60 cells did not block the synergistic effect of DT388-GM-CSF and AraC. These findings suggest that ADP-ribosylation of elongation factor 2 by DT388-GM-CSF leads to a lowered apoptotic threshold to Ara-C and significantly enhances its activity against human AML cells. These observations also suggest clinical trials of DT388-GM-CSF in combination with Ara-C may be warranted in patients with AML.

Original languageEnglish (US)
Pages (from-to)696
Number of pages1
JournalExperimental Hematology
Volume26
Issue number8
StatePublished - 1998

Fingerprint

Diphtheria Toxin
HL-60 Cells
Granulocyte-Macrophage Colony-Stimulating Factor
Cytarabine
Caspase 3
ADP-Ribosylation Factors
Peptide Elongation Factor 2
Apoptosis
Ricin
Protein Synthesis Inhibitors
Fas Ligand Protein
Poisons
Cycloheximide
Cytochromes c
Human Activities
Thymidine
Leukemia
Clinical Trials

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

Diphtheria toxin fused to GM-CSF in combination with ARA-C exerts synergistic toxicity to human AML (HL60) cells. / Bhalia, K. M.; Kreitman, R. J.; Hall, P.; Frankel, A.

In: Experimental Hematology, Vol. 26, No. 8, 1998, p. 696.

Research output: Contribution to journalArticle

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abstract = "Human GM-CSF fused to a truncated diphtheria toxin (DT388-GM-CSF) showed a dramatic synergistic effect (combination index of 0.07) with Ara-C on HL60 human leukemia cells which was not seen with native GM-CSF. The enhanced cell kill occurred within 30 hr of exposure to both agents and did not involve detectable changes in Ara-C incorporation into cellular DNA. The synergistic toxic effect of Ara-C was not seen with 2 other protein synthesis inhibitors: ricin and cycloheximide. Enhanced cell kill with the DT388-GM-CSF/Ara-C combination was observed using both an 18 hr thymidine incorporation and a CFC assay to measure loss of proliferative potential and DAPI staining to detect nuclear fragmentation, but was not seen using the MTT assay to measure mitochondrial metabolism. Exposure to DT388-GM-CSF alone induced mitochondrial release and cytosolic accumulation of cytochrome c, and increased the PARP cleavage activity of caspase-3. Co-treatment with Ara-C enhanced both of these effects as well as the apoptosis induced by exposure to DT388-GM-CSF. However, the degree of enhancement of the effects on cytochrome c, caspase-3, and apoptosis of HL60 cells did not correlate with their reduced clonogenicity, Neither DT388-GM-CSF nor Ara-C treatment altered Bcl-2, Bcl-xl, Bax or FasR levels in HL60 cells, although DT388-GM-CSF increased Fas ligand expression by approximately 50{\%}. Furthermore, enforced overexpression of Bcl-2 in the HL60 cells did not block the synergistic effect of DT388-GM-CSF and AraC. These findings suggest that ADP-ribosylation of elongation factor 2 by DT388-GM-CSF leads to a lowered apoptotic threshold to Ara-C and significantly enhances its activity against human AML cells. These observations also suggest clinical trials of DT388-GM-CSF in combination with Ara-C may be warranted in patients with AML.",
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