The role of actin in the apoptotic cell death of P19 embryonal carcinoma cells.
The P19 mouse embryonal carcinoma cell line was used as a model for a study of apoptosis accompanying differentiation induced by all-trans retinoic acid (ATRA). Apoptosis was detected both on the basis of morphological features (nuclear fragmentation, blebbing of plasma membrane, and formation of apoptotic bodies), and by using DNA electrophoresis and flow-cytometric measurement of DNA content. Actin cytoskeleton was studied both on morphological and submicroscopic levels. ATRA-treated cells manifested apoptosis-specific changes in the distribution of actin foremost in association with their entry into executive phase of apoptosis, when F-actin cables participated in cell disintegration into apoptotic bodies. Using immunogold labeling, actin was also identified in centers of fragmenting apoptotic nuclei, in the disintegration of which it is likely involved as well. At the same time, a cleavage of actin by active caspase-3 was proved, resulting in the emergence of 32 kDa fragment, termed fractin. Measurement of F-actin and fractin content using flow cytometry showed an unequivocal decrease of F-actin and synchronous increase of fractin in the apoptotic population as compared to non-treated cells. Therefore, our results proved both actin proteolysis and active involvement of specific actin structures in the final cell disintegration during apoptosis in the P19 cells.
Neradil
Jakub
J
Cell Culture Laboratory, Department of Biology, Medical Faculty, Masaryk University in Brno, Brno, Czech Republic.
Veselská
Renata
R
Svoboda
Augustin
A
D000199:Actins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D018236:Carcinoma, Embryonal / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D004586:Electrophoresis; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D018922:HL-60 Cells; D006801:Humans; D007150:Immunohistochemistry; D008027:Light; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000737:chemistry / Q000502:physiology*; D012542:Scattering, Radiation; D014212:Tretinoin / Q000378:metabolism
D000199:Actins; D010446:Peptide Fragments; D014212:Tretinoin; D004247:DNA; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases