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C‐Terminal 15kDa Fragment from β‐Actin Causes Apoptotic Cell Death
Author(s) -
Ito Nanako,
Tone Shigenobu,
Tanaka Masato
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb159
Subject(s) - transfection , hela , microbiology and biotechnology , jurkat cells , hek 293 cells , apoptosis , biology , actin , cell culture , complementary dna , t cell , gene , genetics , immune system
Truncated 15kDa‐β‐actin fragment was originally detected in apoptotic cancer cells treated with etoposide and its over‐expression of the fragment induced apoptotic morphological changes (Mashima, T. et al. Oncogene 18 , 2423–2430, 1999). Our final goal is to reveal how the 15kDa‐β‐actin fragment induces the apoptotic morphological changes. To distinguish the transfected cells from non‐transfected ones, we have developed a new expression vector in which two coding sequences are able to be simultaneously expressed in a mammalian cell. This vector enabled to express a fluorescent protein with the 15kDa‐β‐actin in an identical cell. Using these co‐expression plasmids, we have investigated the effects of the 15kDa‐β‐actin on the apoptotic processes of HEK293, COS‐7 HeLa S3, and Jurkat cells. The over‐expression of 15kDa‐β‐actin fragment was seemed to cause the loss of adhesive properties of COS‐7 cells onto culture dish, since the GFP‐labeled cells were observed to detach from dishes by losing the adhesion ability. The numbers of detached cells from HEK293, COS‐7 and HeLa S3 transfected with the 15kDa‐β‐actin cDNA were determined by counting chamber. The numbers of detached cells were significantly increased by transfection of 15kDa‐β‐actin cDNA, but not by any mock transfections. To examine the relationship between the detachment and cell death, the detached cells were stained with Hoechst33342 and NucView488 caspase‐3 vital staining. The almost detached cells showed caspase‐3 activation and nuclear condensation in HEK293, COS‐7 and HeLa S3 cells. The cell death is not seemed to result from anoikis, since the transfection of the 15kDa‐β‐actin cDNA gave the similar effects on Jurkat cells. We also confirmed that the DNA fragmentations in HEK293, COS‐7, HeLa S3 and Jurkat cells by TUNEL assay. Cytoskeletal β‐actin has three targets of caspase‐3 motif but β‐actin has been so far considered to be a poor target of caspase‐3. We showed that the 15kDa‐β‐actin C‐terminal fragment actually activated caspase‐3. These results suggest that the cleaved molecule of cytoskeletal β‐actin is not an innocent bystander but an apoptogeneic inducer. Furthermore, we found that an actin binding protein, profilin suppressed the cell death by either 15kDa‐β‐actin or etoposide, VP‐16. The cleavage of cytoskeletal β‐actin would play a key role in the process of apoptotic cell death.