Increased p50/p50 NF-kappaB activation in human papillomavirus type 6- or type 11-induced laryngeal papilloma tissue

Abstract

We have observed elevated NF-kappaB DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-kappaB activity could be correlated with a reduced level of cytoplasmic IkappaBbeta and could be associated with the overexpression of p21(CIP1/WAF1) in papilloma cells. Increased NF-kappaB activity and cytoplasmic accumulation of p21(CIP1/WAF1) might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues.

FIG. 1.

Increased p50 NF-κB DNA-binding activity is observed in papilloma nuclear extracts. NF-κB DNA-binding activity was assessed by EMSA. (A) Normal extract was derived from a pool of eight normal tissues. Five papilloma nuclear extracts were analyzed. All papillomas were typed by PCR and by Southern blot analysis. Papilloma 1 is HPV 6 positive. Papillomas 2 to 5 are HPV 11 positive. Papillomas 3 to 5 were analyzed on a different gel. (B) In competition or supershift experiments, papilloma nuclear extract was incubated with a 30-fold molar excess of wild-type (WT) or mutant (Mut) oligonucleotides or 1 μg of anti-p50 or anti-p65 antibodies (Ab) before adding ³²P-labeled NF-κB oligonucleotide. Papillomas 3 and 4 were analyzed on a different gel.

FIG. 2.

Elevated nuclear p50 in papilloma extracts is associated with reduced cytoplasmic IκBβ. Thirty micrograms of cytoplasmic or nuclear extracts prepared from normal or papilloma tissues was fractionated on a denaturing sodium dodecyl sulfate-polyacrylamide gel. Separated polypeptides were transferred to a nitrocellulose membrane and probed for the indicated antigens. The amount of actin in the extract is indicated as a loading control. The solid bar stands for relative intensity of the indicated protein in the extract after normalization to actin.

FIG. 3.

Augmented expression of p21^CIP1/WAF1 in papillomas. Forty micrograms of cytoplasmic or nuclear extract prepared from normal or papilloma tissues was electrophoresed on a denaturing sodium dodecyl sulfate-polyacrylamide gel. Separated polypeptides were transferred to a nitrocellulose membrane and probed with an anti-p21^CIP1/WAF1 antibody. Ponceau S staining of the membrane is shown for loading comparison.