TY - JOUR
T1 - 1-β-d-arabinofuranosylcytosine-, mitoxantrone-, and paclitaxel-induced apoptosis in HL-60 cells
T2 - improved method for detection of internucleosomal DNA fragmentation
AU - Ray, Swapan
AU - Ponnathpur, Vidya
AU - Huang, Yue
AU - Tang, Caroline
AU - Mahoney, Mary Ella
AU - Ibrado, Ana Maria
AU - Bullock, Gloria
AU - Bhalla, Kapil
PY - 1994/9/1
Y1 - 1994/9/1
N2 - We investigated the ability of different doses and durations of exposure to the chemotherapeutic drugs 1-β-d-arabinofuranosylcytosine (Ara-C), mitoxantrone (MTN), and paclitaxel (taxol, TXL) to induce internucleosomal DNA fragmentation and apoptosis in human acute myeloid leukemia (AML) HL-60 cells in suspension culture. At clinically achievable concentrations, all three drugs have been shown to induced apoptosis in HL-60 cells. An improved method was developed for the isolation of pure genomic DNA and the detection of drug-induced intergenomic DNA and the detection of drug-induced internucleosomal DNA fragmentation in <1.0 μg of DNA sample by agarose gel electrophoresis. Morphologic evidence for apoptosis was determined by light microscopy following Wright staining, and cell viability was assessed by trypan blue dye exclusion. Internucleosomal DNA fragmentation was observed following exposure to 1.0 μM Ara-C for 4 h, which increased with 10 and 50 μM Ara-C. Incubation with 100 μM Ara-C produced internucleosomal DNA fragmentation starting at 3 h, which increased with longer periods of exposure to Ara-C. Utilizing a schedule of 1-h exposure followed by 3-h suspension in drug-free medium, 0.25 μM MTN was found to initiate DNA fragmentation, which increased with exposure to 1.0 and 5.0 μM MTN. However, identical treatment with higher concentrations of MTN resulted in random DNA degradation. Alternatively, continuous exposure to 1.0 μM MTN for 3 h was necessary to initiate internucleosomal DNA fragmentation. This increased with exposure intervals of up to 6 h. Exposure to TXL concentrations as low as 0.01 μM for 24 h caused internucleosomal DNA fragmentation, which increased with dose escalation (0.05, 0.1, 0.5, and 1.0 μM) of TXL. Although continuous exposure to 1.0 μM TXL for a period as short as 8 h produced internucleosomal DNA fragmentation, this increased significantly with longer exposure intervals. In general there appears to be a threshold concentration and duration of exposure below which non of these three drugs activates endonucleolytic internucleosomal DNA fragmentation and apoptosis. This threshold is lower for the DNA-interactive drugs MTN and Ara-C but higher for the non-DNA-interactive drug TXL. Higher doses or prolonged treatments with the drugs produce random DNA fragmentation associated with necrotic cell death. These in vitro results may further improve our understanding of the antileukemic cytotoxic effects of these drugs, which may enable a more rational design of drug regimens for optimal treatment of AML.
AB - We investigated the ability of different doses and durations of exposure to the chemotherapeutic drugs 1-β-d-arabinofuranosylcytosine (Ara-C), mitoxantrone (MTN), and paclitaxel (taxol, TXL) to induce internucleosomal DNA fragmentation and apoptosis in human acute myeloid leukemia (AML) HL-60 cells in suspension culture. At clinically achievable concentrations, all three drugs have been shown to induced apoptosis in HL-60 cells. An improved method was developed for the isolation of pure genomic DNA and the detection of drug-induced intergenomic DNA and the detection of drug-induced internucleosomal DNA fragmentation in <1.0 μg of DNA sample by agarose gel electrophoresis. Morphologic evidence for apoptosis was determined by light microscopy following Wright staining, and cell viability was assessed by trypan blue dye exclusion. Internucleosomal DNA fragmentation was observed following exposure to 1.0 μM Ara-C for 4 h, which increased with 10 and 50 μM Ara-C. Incubation with 100 μM Ara-C produced internucleosomal DNA fragmentation starting at 3 h, which increased with longer periods of exposure to Ara-C. Utilizing a schedule of 1-h exposure followed by 3-h suspension in drug-free medium, 0.25 μM MTN was found to initiate DNA fragmentation, which increased with exposure to 1.0 and 5.0 μM MTN. However, identical treatment with higher concentrations of MTN resulted in random DNA degradation. Alternatively, continuous exposure to 1.0 μM MTN for 3 h was necessary to initiate internucleosomal DNA fragmentation. This increased with exposure intervals of up to 6 h. Exposure to TXL concentrations as low as 0.01 μM for 24 h caused internucleosomal DNA fragmentation, which increased with dose escalation (0.05, 0.1, 0.5, and 1.0 μM) of TXL. Although continuous exposure to 1.0 μM TXL for a period as short as 8 h produced internucleosomal DNA fragmentation, this increased significantly with longer exposure intervals. In general there appears to be a threshold concentration and duration of exposure below which non of these three drugs activates endonucleolytic internucleosomal DNA fragmentation and apoptosis. This threshold is lower for the DNA-interactive drugs MTN and Ara-C but higher for the non-DNA-interactive drug TXL. Higher doses or prolonged treatments with the drugs produce random DNA fragmentation associated with necrotic cell death. These in vitro results may further improve our understanding of the antileukemic cytotoxic effects of these drugs, which may enable a more rational design of drug regimens for optimal treatment of AML.
KW - Apoptosis
KW - Ara-C
KW - DNA fragmentation
KW - HL-60 cells
KW - Mitoxantrone
KW - Taxol
UR - http://www.scopus.com/inward/record.url?scp=0028167771&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028167771&partnerID=8YFLogxK
U2 - 10.1007/BF00685559
DO - 10.1007/BF00685559
M3 - Article
C2 - 7915211
AN - SCOPUS:0028167771
VL - 34
SP - 365
EP - 371
JO - Cancer Chemotherapy and Pharmacology
JF - Cancer Chemotherapy and Pharmacology
SN - 0344-5704
IS - 5
ER -