Herbimycin A, a tyrosine kinase inhibitor,
induces cellular differentiation and delayed apoptosis
in Colo-205 cells, a poorly differentiated human colon
carcinoma cell line. Cell cycle analysis in conjunction
with end labeling of DNA fragments revealed that G2
arrest preceded apoptotic cell death. Ultrastructural examination of herbimycin-treated cells demonstrated
morphologic features of epithelial differentiation, including formation of a microvillar apical membrane
and lateral desmosome adhesions. A marked accumulation of mitochondria was also observed. Fluorometric
analysis using the mitochondrial probes nonyl-acridine
orange and JC-1 confirmed a progressive increase in
mitochondrial mass. However these cells also demonstrated a progressive decline in unit mitochondrial
transmembrane potential (ΔΨm) as determined by the
ΔΨm-sensitive fluorescent probes rhodamine 123 and
JC-1 analyzed for red fluorescence. In concert with
these mitochondrial changes, Colo-205 cells treated
with herbimycin A produced increased levels of reactive oxygen species as evidenced by oxidation of both
dichlorodihydrofluorescein diacetate and dihydroethidium. Cell-free assays for apoptosis using rat-liver nuclei
and extracts of Colo-205 cells at 24 h showed that apoptotic activity of Colo-205 lysates requires the early action of mitochondria. Morphological and functional mitochondrial changes were observed at early time points,
preceding cleavage of poly (ADP-ribose) polymerase.These results suggest that apoptosis in differentiated
Colo-205 cells involves unrestrained mitochondrial proliferation and progressive membrane dysfunction, a
novel mechanism in apoptosis.
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