Apoptosis Induced by Chemotherapeutic Agents in Differentiated HL-60 Human Leukemic Cell Line.
- Author:
Hee Jeong AHN
1
;
Ho Joon IM
;
Ki Wug SUNG
;
Sang Bok LEE
;
Hahng LEE
Author Information
1. Department of Pediatrics, College of Medicine, Hanyang University, Seoul, Korea.
- Publication Type:Original Article
- Keywords:
Apoptosis;
Chemotherapeutic agents;
Differentiation;
HL-60;
Leukemia
- MeSH:
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
- From:Korean Journal of Pediatric Hematology-Oncology
1999;6(1):57-67
- CountryRepublic of Korea
- Language:Korean
-
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.
