Previous studies have shown that human papillomavirus (HPV) 16 E6 inhibits apoptosis induced during terminal differentiation of primary human keratinocytes (PHKs) triggered by serum and calcium. E6 inhibition of apoptosis was accompanied with prolonged expression of Bcl-2 and reduced elevation of Bax levels. In the present study, the effect of E6 on Bax mRNA expression and protein stability was investigated. These studies indicate that stable E6 expression in differentiating keratinocytes reduced the steady-state levels of Bax mRNA and shortened the half-life of Bax protein. These results were confirmed in transiently transfected 293T cells where E6 degraded Bax in a dose-dependent manner. Bax degradation was also exhibited in Saos-2 cells that lack p53, indicating its p53 independence. E6 did not form complexes with Bax and did not induce Bax degradation in vitro under experimental conditions where p53 was degraded. Finally, E6 aa 120–132 were shown to be necessary for Bax destabilization and, more importantly, for abrogating the ability of Bax to induce cellular apoptosis, highlighting the functional consequences of the E6-induced alterations in Bax expression.
BargouR. C.,
DanielP. T.,
MaparaM. Y.,
BommertK.,
WagenerC.,
KallinichB.,
RoyerH. D.,
DorkenB.1995; Expression of the bcl-2 gene family in normal and malignant breast tissue: low bax-alpha expression in tumor cells correlates with resistance towards apoptosis. Int J Cancer 60:854–859[CrossRef]
BudtzP. E.1994; Epidermal homeostasis: a new model that includes apoptosis. In Apoptosis II: the Molecular Basis of Apoptosis in Disease pp 165–184 Edited by
TomeiL. D.,
CopeF. O.
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
ChangY.-C.,
LeeY.-S.,
TejimaT.,
TanakaK.,
OmuraS.,
HeintzN. H.,
MitsuiY.,
MagaeJ.1998; mdm2 and bax, downstream mediators of the p53 response, are degraded by the ubiquitin-proteasome pathway. Cell Growth Differ 9:79–84
ChenJ. J.,
ReidC. E.,
BandV.,
AndrophyE. J.1995; Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein. Science 269:529–531[CrossRef]
DeleheddeM.,
ChoS. H.,
SarkissM.,
BrisbayS.,
DaviesM.,
El-NaggarA. K.,
McDonnellT. J.1999; Altered expression of bcl-2 family member proteins in nonmelanoma skin cancer. Cancer 85:1514–1522[CrossRef]
GandarillasA.,
GoldsmithL. A.,
GschmeissnerS.,
LeighI. M.,
WattF. M.1999; Evidence that apoptosis and terminal differentiation of epidermal keratinocytes are distinct processes. Exp Dermatol 8:71–79[CrossRef]
GaoG.,
DouQ. P.2000; N-terminal cleavage of Bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes Bcl-2-independent cytochrome c release and apoptotic cell death. J Cell Biochem 80:53–72
GaoQ.,
SrinivasanS.,
BoyerS. N.,
WazerD. E.,
BandV.1999; The E6 oncoprotein of high-risk papillomaviruses bind to a novel putative GAP protein E6TP1, and target it for degradation. Mol Cell Biol 19:733–744
GewinL.,
GallowayD.2001; E-Box-dependent activation of telomerase by human papillomavirus, type 16 E6 does not require induction of c- myc
. J Virol 75:7198–7201[CrossRef]
GlaunsingerB. A.,
LeeS. S.,
ThomasM.,
BanksL.,
JavierR.2000; Interaction of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins. Oncogene 19:5270–5280[CrossRef]
Gross-MesilatyS.,
ReinsteinE.,
BercovichB.,
TobiasK. E.,
SchwartzA. L.,
KahanaC.,
CiechanoverA.1998; Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway. Proc Natl Acad Sci U S A 95:8058–8063[CrossRef]
HanJ.,
ModhaD.,
WhiteE.1998; Interaction of E1B 19K with Bax is required to block Bax-induced loss of mitochrondrial membrane potential and apoptosis. Oncogene 17:2993–3005[CrossRef]
HockenberyD. M.,
ZutterM.,
HickeyW.,
NahmM.,
KorsmeyerS. J.1991; BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci U S A 88:6961–6965[CrossRef]
HuibregtseJ. M.,
ScheffnerM.,
HowleyP. M.1991; A cellular protein mediates association of p53 with E6 oncoprotein of human papillomavirus tyes 16 or 18. EMBO J 10:4129–4135
HuibregtseJ. M.,
ScheffnerM.,
HowleyP. M.1993; Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papilllomavirus E6 oncoprotein with p53. Mol Cell Biol 13:775–784
JordanR. C. K.,
CatzavelosG. C.,
BarrettA. W.,
SpeightP. M.1996; Differential expression of bcl-2 and bax in squamous cell carcinomas of the oral cavity. Eur J Cancer B Oral Oncol 32B:394–400
KitanakaC.,
NamikiT.,
NoguchiK.7 other authors1997; Caspase-dependent apoptosis of COS-7 cells induced by Bax overexpression: differential effects of Bcl-2 and Bcl-xL on Bax-induced caspase activation and apoptosis. Oncogene 15:1763–1772[CrossRef]
KiyonoT.,
HiraiwaH.,
FujitaM.,
HayashiY.,
AkiyamaT.,
IshibashiM.1997; Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein. Proc Natl Acad Sci U S A 94:11612–11616[CrossRef]
KuhneC.,
BanksL.1998; E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G Box. J Biol Chem 273:34302–34309[CrossRef]
KukimotoI.,
AiharaS.,
YoshiikeK.,
KandaT.1998; Human papillomavirus oncoprotein E6 binds to the C-terminal region of human minichromosome maintenance 7 protein. Biochem Biophys Res Commun 249:258–262[CrossRef]
KumarA.,
ZhaoY.,
MengG.9 other authors2002; Human papillomavirus oncoprotein E6 inactivates the transcriptional coactivator human ADA3. Mol Cell Biol 22:5801–5812[CrossRef]
LeeS. S.,
WeissR. S.,
JavierR. T.1997; Binding of human virus oncoproteins to hDlg/SAP97, a mammalian homolog of the Drosophila discs large tumor suppressor protein. Proc Natl Acad Sci U S A 94:6670–6675[CrossRef]
LeeS. S.,
GlaunsingerB.,
MantovaniF.,
BanksL.,
JavierR. T.2000; Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins. J Virol 74:9680–9693[CrossRef]
LiB.,
DouQ. P.2000; Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc Natl Acad Sci U S A 97:3850–3855[CrossRef]
MarshallW. L.,
YimC.,
GustafsonE.,
GrafT.,
SageD. R.,
HanifyK.,
WilliamsL.,
FingerothJ.,
FinbergR. W.1999; Epstein-Barr virus encodes a novel homolog of the bcl-2 oncogene that inhibits apoptosis and associates with Bax and Bak. J Virol 73:5181–5185
MarshanskyV.,
WangX.,
BertrandR.,
LuoH.,
DuguidW.,
ChinnaduraiG.,
KanaanN.,
VuM. D.,
WuJ.2001; Proteasomes modulate balance among proapoptotic and antiapoptotic Bcl-2 family members and compromise functioning of the electron transport chain in leukemic cells. J Immunol 166:3130–3142[CrossRef]
MaruokaY.,
HaradaH.,
MitsuyasuT.,
SetaY.,
KurokawaH.,
KajiyamaM.,
ToyoshimaK.1997; Keratinocytes become terminally differentiated in a process involving programmed cell death. Biochem Biophys Res Commun 238:886–890[CrossRef]
MitchellK. O.,
RicciM. S.,
MiyashitaT.,
DickerD. T.,
JinZ.,
ReedJ. C.,
El-DeiryW. S.2000; Bax is a transcriptional target and mediator of c-Myc-induced apoptosis. Cancer Res 60:6318–6325
MitraR. S.,
Wrone-SmithT.,
SimonianP.,
ForemanK. E.,
NunezG.,
NickoloffB. J.1997; Apoptosis in keratinocytes is not dependent on induction of differentiation. Lab Invest 76:99–107
MiyashitaT.,
KrajewskiS.,
KrajewskaM.,
WangH. G.,
LinH. K.,
LeibermannD. A.,
HoffmanB.,
ReedJ. C.1994; Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo
. Oncogene 9:1799–1805
NakagawaS.,
HuibregtseJ. M.2000; Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase. Mol Cell Biol 20:8244–8253[CrossRef]
OhS. T.,
KyoS.,
LaiminsL. A.2001; Telomerase activation by human papillomavirus type 16 E6 protein: induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites. J Virol 75:5559–5566[CrossRef]
PatelD.,
HuangS. M.,
BagliaL. A.,
McCanceD. J.1999; The E6 protein of human papillomavirus type 16 binds to and inhibits co-activation by CBP and p300. EMBO J 18:5061–5072[CrossRef]
PerezD.,
WhiteE.2000; TNF- α signals apoptosis through a bid-dependent conformational change in bax that is inhibited by E1B 19K. Mol Cell 6:53–63[CrossRef]
PolakowskaR. R.,
PiacentiniM.,
BarlettR.,
GoldsmithL. A.,
HaakeA. R.1994; Apoptosis in human skin development: morphogenesis periderm and stem cells. Dev Dyn 199:176–188[CrossRef]
RoncoL. V.,
KarpovaA. Y.,
VidalM.,
HowleyP. M.1998; Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes Dev 12:2061–2072[CrossRef]
ScheffnerM.,
WernessB. A.,
HuibregtseJ. M.,
LevineA. J.,
HowleyP. M.1990; The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129–1136[CrossRef]
ScorranoL.,
KorsmeyerS. J.2003; Mechanisms of cytochrome c release by proapoptotic BCL-2 family members. Biochem Biophys Res Commun 304:437–444[CrossRef]
ShermanL.,
SchlegelR.1996; Serum- and calcium-induced differentiation of human keratinocytes is inhibited by the E6 oncoprotein of human papillomavirus type 16. J Virol 70:3269–3279
ShermanL.,
GazitA.,
YanivA.,
KawakamiT.,
DahlbergJ. E.,
TronickS. R.1988; Localization of sequences responsible for trans-activation of the equine infectious anemia virus long terminal repeat. J Virol 62:120–126
ShermanL.,
JackmanA.,
ItzhakiH.,
StopplerM. C.,
KovalD.,
SchlegelR.1997; Inhibition of serum- and calcium-induced differentiation of human keratinocytes by HPV 16 E6 oncoprotein: role of p53 inactivation. Virology 237:296–306[CrossRef]
ShermanL.,
ItzhakiH.,
JackmanA.,
ChenJ. J.,
KovalD.,
SchlegelR.2002; Inhibition of serum- and calcium-induced terminal differentiation of human keratinocytes by HPV 16 E6: study of the association with p53 degradation, inhibition of p53 transactivation and binding to E6BP. Virology 292:309–320[CrossRef]
SrivenugopalK. S.,
Ali-OsmanF.2002; The DNA repair protein, 06-methylguanine-DNA methyltransferase is a proteolytic target for the E6 human papillomavirus oncoprotein. Oncogene 21:5940–5945[CrossRef]
ThomasM.,
BanksL.1999; Human papillomavirus (HPV) E6 interactions with Bak are conserved amongst E6 proteins from high and low risk HPV types. J Gen Virol 80:1513–1517
TongX.,
HowleyP. M.1997; The bovine papillomavirus E6 oncoprotein interacts with paxillin and disrupts the actin cytoskeleton. Proc Natl Acad Sci U S A 94:4412–4417[CrossRef]
VeldmanT.,
HorikawaI.,
BarrettJ. C.,
SchlegelR.2001; Transcriptional activation of the telomerase hTERT gene by human papillomavirus type 16 E6 oncoprotein. J Virol 75:4467–4472[CrossRef]
VeldmanT.,
LiuX.,
YuanH.,
SchlegelR.2003; Human papillomavirus E6 and Myc proteins associate in vivo and bind to and cooperatively activate the telomerase reverse-transcriptase. Proc Natl Acad Sci U S A 100:8211–8216[CrossRef]
WoodD. E.,
ThomasA.,
DeviL. A.,
BermanY.,
BeavisR. C.,
ReedJ. C.,
NewcombE. W.1998; Bax cleavage is mediated by calpain during drug-induced apoptosis. Oncogene 17:1069–1078[CrossRef]
ZimmermannH.,
DegenkolbeR.,
BernardH. U.,
O'ConnorM. J.1999; The human papillomavirus type 16 E6 oncoprotein can down-regulate p53 activity by targeting the transcriptional coactivator CBP/p300. J Virol 73:6209–6219
ZwerschkeW.,
Jansen-DurrP.2000; Cell transformation by the E7 oncoprotein of human papillomavirus type 16: interactions with nuclear and cytoplasmic target proteins. Adv Cancer Res 78:1–29