TY - JOUR
T1 - Vitamin K3 (menadione)-induced oncosis associated with keratin 8 phosphorylation and histone H3 arylation
AU - Scott, Gary K.
AU - Atsriku, Christian
AU - Kaminker, Patrick
AU - Held, Jason
AU - Gibson, Brad
AU - Baldwin, Michael A.
AU - Benz, Christopher C.
PY - 2005/9
Y1 - 2005/9
N2 - The vitamin K analog menadione (K3), capable of both redox cycling and arylating nucleophilic substrates by Michael addition, has been extensively studied as a model stress-inducing quinone in both cell culture and animal model systems. Exposure of keratin 8 (k-8) expressing human breast cancer cells (MCF7, T47D, SKBr3) to K3 (50-100 μM) induced rapid, sustained, and site-specific k-8 serine phosphorylation (pSer73) dependent on signaling by a single mitogen activated protein kinase (MAPK) pathway, MEK1/2. Normal nuclear morphology and k-8 immunofluorescence coupled with the lack of DNA laddering or other features of apoptosis indicated that K3-induced cytotoxicity, evident within 4 h of treatment and delayed but not prevented by MEK1/2 inhibition, was due to a form of stress-activated cell death known as oncosis. Independent of MAPK signaling was the progressive appearance of K3-induced cellular fluorescence, principally nuclear in origin and suggested by in vitro fluorimetry to have been caused by K3 thiol arylation. Imaging by UV transillumination of protein gels containing nuclear extracts from K3-treated cells revealed a prominent 17-kDa band shown to be histone H3 by immunoblotting and mass spectrometry (MS). K3 arylation of histones in vitro followed by electrospray ionization-tandem MS analyses identified the unique Cys110 residue within H3, exposed only in the open chromatin of transcriptionally active genes, as a K3 arylation target. These findings delineate new pathways associated with K3-induced stress and suggest a potentially novel role for H3 Cys110 as a nuclear stress sensor.
AB - The vitamin K analog menadione (K3), capable of both redox cycling and arylating nucleophilic substrates by Michael addition, has been extensively studied as a model stress-inducing quinone in both cell culture and animal model systems. Exposure of keratin 8 (k-8) expressing human breast cancer cells (MCF7, T47D, SKBr3) to K3 (50-100 μM) induced rapid, sustained, and site-specific k-8 serine phosphorylation (pSer73) dependent on signaling by a single mitogen activated protein kinase (MAPK) pathway, MEK1/2. Normal nuclear morphology and k-8 immunofluorescence coupled with the lack of DNA laddering or other features of apoptosis indicated that K3-induced cytotoxicity, evident within 4 h of treatment and delayed but not prevented by MEK1/2 inhibition, was due to a form of stress-activated cell death known as oncosis. Independent of MAPK signaling was the progressive appearance of K3-induced cellular fluorescence, principally nuclear in origin and suggested by in vitro fluorimetry to have been caused by K3 thiol arylation. Imaging by UV transillumination of protein gels containing nuclear extracts from K3-treated cells revealed a prominent 17-kDa band shown to be histone H3 by immunoblotting and mass spectrometry (MS). K3 arylation of histones in vitro followed by electrospray ionization-tandem MS analyses identified the unique Cys110 residue within H3, exposed only in the open chromatin of transcriptionally active genes, as a K3 arylation target. These findings delineate new pathways associated with K3-induced stress and suggest a potentially novel role for H3 Cys110 as a nuclear stress sensor.
UR - http://www.scopus.com/inward/record.url?scp=23944509337&partnerID=8YFLogxK
U2 - 10.1124/mol.105.013474
DO - 10.1124/mol.105.013474
M3 - Article
C2 - 15939799
AN - SCOPUS:23944509337
SN - 0026-895X
VL - 68
SP - 606
EP - 615
JO - Molecular pharmacology
JF - Molecular pharmacology
IS - 3
ER -