Time sequential expression of markers of apoptosis induced by 1-beta-D-arabinofuranosylcytosine in human T-lymphoblastic leukemia CCRF-CEM cells.
A wide variety of chemotherapeutic agents including 1-beta-D-arabinofuranosylcytosine can induce cell death by apoptosis. However, an appropriate method for early detection of apoptosis that can be clinically applicable to peripheral blood samples freshly obtained from leukemic patients under chemotherapy has not yet been established. We investigated the chronology of ara-C induced apoptosis in CCRF-CEM cells by monitoring the expression of a number of apoptotic markers in a time- and concentration-dependent manner with the aim of estimating the earliest reliable marker of apoptosis to apply to clinical drug sensitivity assays. We employed the following techniques: 1) Pulsed field gel electrophoresis to detect high molecular weight DNA fragmentation; 2) FACS analysis for evaluation of APO 2.7 expression and phosphatidylserine externalization; 3) May-Grunwald-Giemsa staining for studying gross morphology; and 4) TUNEL assay to detect multitudes of terminal 3'-OH groups of oligonucleosomal DNA fragments. At 10 microM ara-C, high molecular weight DNA fragmentation was observed after 4 hours incubation being the earliest marker to manifest. APO 2.7 expression and phosphatidylserine externalization were detected almost simultaneously at 6 hours, with marked similarity in their kinetics. Emergence of apoptotic bodies then followed after 12 hours incubation and, finally, oligonucleosomal DNA fragments were demonstrated by positive TUNEL assay at 48 hours. The results suggest the time-sequential expression of each individual marker and introduce high molecular weight DNA fragmentation assay as the most suitable candidate for early detection of sensitivity of malignant cells to apoptosis-inducing anticancer agents, including ara-C.
Badran
A
A
First Department of Internal Medicine, Fukui Medical University, Matsuoka, Japan.
Iwasaki
H
H
Wano
Y
Y
Kishi
S
S
Ueda
T
T
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D002469:Cell Separation; D003561:Cytarabine / Q000494:pharmacology*; D053938:DNA Fragmentation; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D007700:Kinetics; D007945:Leukemia, Lymphoid / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D003561:Cytarabine