Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis.
It has been suggested that the ratio of Bcl-2 family proapoptotic proteins to antiapoptotic proteins determines the sensitivity of leukemic cells to apoptosis. However, it is believed that Bcl-2 family proteins exert their function on apoptosis only when they target to the mitochondrial outer membrane. The vinblastine-resistant T-lymphoblastic leukemic cell line CEM/VLB100 has increased sensitivity to tumor necrosis factor-alpha (TNF-alpha)-induced cytochrome c release, mitochondrial respiratory inhibition, and consequently apoptosis, compared with parental CEM cells. However, there was no difference between the two cell lines in the expression of Bcl-2 family proteins Bcl-2, Bcl-XL, Bcl-XS, Bad, and Bax at the whole cell level, as analyzed by Western blotting. Bcl-2 mainly located to mitochondria and light membrane as a membrane-bound protein, whereas Bcl-XL was located in both mitochondria and cytosol. Similar levels of both Bcl-2 and Bcl-XL were present in the resting mitochondria of the two cell lines. Although the proapoptotic proteins Bcl-XS, Bad, and Bax were mainly located in the cytosol, CEM/VLB100 mitochondria expressed higher levels of these proapoptotic proteins. Subcellular redistribution of the Bcl-2 family proteins was detected in a cell-free system by both Western blotting and flow cytometry after exposure to TNF-alpha. The levels of Bcl-2 family proteins were not altered at the whole cell level by TNF-alpha. However, after exposure to TNF-alpha, Bax, Bad, and Bcl-XS translocated from the cytosol to the mitochondria of both cell lines. An increase in Bcl-2 levels was observed in CEM mitochondria, which showed resistance to TNF-alpha-induced cytochrome c release. By contrast, decreased mitochondrial Bcl-2 was observed in CEM/VLB100 cells, which released cytochrome c from the mitochondria and underwent apoptosis as detected by fluorescence microscopy. We conclude that mitochondrial levels of Bcl-2 family proteins may determine the sensitivity of leukemic cells to apoptosis and that, furthermore, these levels may change rapidly after exposure of cells to toxic stimuli.
Jia
L
L
Department of Haematology, St Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK.
Macey
M G
MG
Yin
Y
Y
Newland
A C
AC
Kelsey
S M
SM
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis; D001692:Biological Transport; D002352:Carrier Proteins / Q000032:analysis; D002462:Cell Membrane / Q000737:chemistry; D003574:Cytochrome c Group / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D019008:Drug Resistance, Neoplasm; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000378:metabolism / Q000473:pathology*; D008565:Membrane Proteins / Q000032:analysis; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D009363:Neoplasm Proteins / Q000032:analysis*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis*; D013347:Subcellular Fractions / Q000737:chemistry*; D013601:T-Lymphocytes / Q000737:chemistry* / Q000473:pathology / Q000648:ultrastructure; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
D000972:Antineoplastic Agents, Phytogenic; C494819:BAD protein, human; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D003574:Cytochrome c Group; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D014747:Vinblastine