Korean J Pediatr Hematol Oncol.  1999 Apr;6(1):57-67.

Apoptosis Induced by Chemotherapeutic Agents in Differentiated HL-60 Human Leukemic Cell Line

Affiliations
  • 1Department of Pediatrics, College of Medicine, Hanyang University, Seoul, Korea.
  • 2Department of Pediatrics, Gachon Medical School, Gil Medical Center, Incheon, Korea.
  • 3Department of Pharmacology, Catholic University Medical College, Seoul, Korea.

Abstract

PURPOSE: Chemotherapeutic agents are known to induce cell death in cancer cells by apoptotic mechanisms. This study was to investigate the influence of the differentiation on the apoptotic potential of chemotherapeutic agents.
METHODS
Etoposide and cytosine arabinoside (Ara-C) were chosen as chemotherapeutic agents, and human promyelocytic leukemia cell line, HL-60, was used as target cells.
RESULTS
Etoposide or Ara-C treated HL-60 cells showed cytoplasmic blebbing and nuclear condensation and fragmentation under fluorescence microscope when stained with acridine orange/ethidium bromide. In addition, the cellular DNA of HL-60 cells was found to cleave into internucleosomal fragments after treatment with chemotherapeutic agents. These findings were the characteristics of apoptosis and suggested the induction of apoptotic cell death of HL-60 cells by etoposide or Ara-C treatment. HL-60 cells are known to differentiate into myeloid or monocytic lineage by retinoic acid, phorbol 12-myristate acetate (PMA) and dimethyl sulfoxide (DMSO), and this differentiation itself can activate apoptosis program, so-called 'apoptosis by terminal differentiation'. The effect of terminal differentiation by PMA or DMSO on the apoptosis induced by etoposide or Ara-C was also investigated, utilizing qualitative and quantitative DNA fragmentation assay. HL-60 cells treated with PMA (100 nM) were adherent to culture dish and formed cellular processes. DMSO (1.25%) treated HL-60 cells instead recovered the ability to reduce nitroblue tetrazolium to blue-to-purple formazan, indicating its differentiation. After induction of differentiation by PMA or DMSO, differentiated HL-60 cells were treated with etoposide (10 muM) and Ara-C (50 muM) to compare its apoptotic potential with that of undifferentiated HL-60 cells. The ladder DNA induced by etoposide and Ara-C was decreased in differentiated HL-60 cells. On quantitative analysis of DNA fragmentation, PMA reduced DNA fragmentation induced by etoposide and Ara-C to 73% and 69%, respectively, and DMSO reduced it to 74% and 56%, respectively. In western blot analysis, the expression of Bcl-2, which is known to inhibit etoposide and Ara-C induced apoptosis, decreased significantly in HL-60 cells differentiated by PMA or DMSO.
CONCLUSION
These results suggest that the differentiation of HL-60 cells by PMA or DMSO prevents apoptosis by etoposide and Ara-C, but bcl-2 proto-oncogene may have only minor role in inhibiting apoptosis by chemotherapeutic agents in differentiated HL-60 cell.

Keyword

Apoptosis; Chemotherapeutic agents; Differentiation; HL-60; Leukemia

MeSH Terms

Apoptosis*
Blister
Blotting, Western
Cell Death
Cell Line*
Cytarabine
Cytoplasm
Dimethyl Sulfoxide
DNA
DNA Fragmentation
Etoposide
Fluorescence
HL-60 Cells
Humans*
Leukemia
Nitroblue Tetrazolium
Proto-Oncogenes
Tretinoin
Cytarabine
DNA
Dimethyl Sulfoxide
Etoposide
Nitroblue Tetrazolium
Tretinoin
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