Induction of Senescence-like State and Suppression of Telomerase
Activity through Inhibition of HPV E6/E7 Gene Expression
in Cells Immortalized by HPV16 DNA
Chan Jae Lee,*
,1
Eun Jung Suh,* Hyun Tae Kang,* Jun Sub Im,* Soo Jong Um,†
Jong Sup Park,‡ and Eun Seong Hwang*
,2
*Department of Life Science, University of Seoul, 90 Jeonnongdong, Dongdaemungu, Seoul 130-743, Korea; †Department of Bioscience
and Biotechnology, Sejong University, Seoul 143-747, Korea; and ‡Department of Obstetrics/Gynecology,
Catholic University Medical College, Seoul 137-040, Korea
The E6 and E7 oncoproteins of human papillomavi-
rus (HPV) play a major role in the development of
cervical carcinoma. In this study, a recombinant ade-
novirus that expresses the bovine papillomavirus
(BPV) E2, which has been shown to inhibit HPV early
gene expression, was delivered to two HPV-immortal-
ized cell lines as well as CaSki, a cervical carcinoma
cell line. We tested whether the carcinoma and the
immortal cells were equally affected by the expression
of BPV E2. In all cell lines, BPV E2-mediated inhibition
of HPV E6/E7 expression caused a dramatic suppres-
sion of cell growth, being preceded by the activation of
the p53–Rb growth-inhibitory pathway, and a de-
crease in hTERT mRNA expression and telomerase
activity. This suggests that the HPV E6 and E7 pro-
teins are required not only for induction of the prolif-
erative phenotype and telomerase activity, but also for
their maintenance. In both the carcinoma and the im-
mortal lines, the number of cells with enlarged cyto-
plasm and senescence-associated

-galactosidase ac-
tivity, which are markers for cellular senescence, was
significantly increased. These results suggest that a
senescence program exists in cells immortalized by
HPV DNA as well as in cervical carcinoma cells.
© 2002
Elsevier Science (USA)
Key Words: human papillomavirus; cervical carci-
noma; cellular senescence; telomerase; HPV; E6; E7;
BPV E2.
INTRODUCTION
Normal cells, when cultured in vitro, proliferate for a
limited number of population doublings and then enter
a stage called cellular or replicative senescence, where
cells are irreversibly arrested at the G1 phase of the
cell cycle and express certain cellular and biochemical
phenotypes [1–3]. Carcinoma cells must evolve by over-
coming the senescence barrier and becoming immortal.
For most cervical carcinomas, the E6 and E7 oncopro-
teins of human papillomavirus (HPV) are believed to
provide the cells a chance to bypass senescence.
The E6 and E7 genes of high-risk type HPV are
continuously expressed in cervical carcinoma cells
[4–7] and together can immortalize human epithelial
cells and keratinocytes [8–12]. The E6 and E7 proteins
interact and interfere with numerous cellular factors
and activities [13]. Most importantly, E6 and E7 inac-
tivate the p53-mediated cell cycle control function by
targeting p53 and Rb proteins, respectively, for rapid
degradation through ubiquitin-mediated proteolysis
[14, 15]. Furthermore, E6 activates the host telomerase
[16–19] and, in combination with the E7-mediated in-
activation of the Rb function, extends the life span of
primary cells [20]. In addition, induction of chromo-
some instability [21–23] and of abnormal centrosome
synthesis [24, 25] in primary and cancer cells that
ectopically express E6 and E7 indicates that these viral
genes are rather actively involved in carcinogenic
transformation. Taken together, these findings indi-
cate that the E6 and E7 proteins of the high-risk type
HPVs cause uncontrolled cell cycle progression by nul-
lifying the cellular growth-inhibitory pathway and al-
low the cells to become immortal by overcoming the
senescence block induced by the telomere shortening.
At the same time, these proteins likely promote tumor-
igenesis progression by facilitating the accumulation of
oncogenic mutations.
Expression of papillomavirus early genes including
E6 and E7 is regulated by another papillomaviral pro-
tein, E2 [26, 27]. The E2 protein, when overexpressed,
inhibits transcription of the HPV16 P
97
and HPV18 P
105
promoters [28–31] by binding to sites proximal to the
promoters and thereby inhibiting the formation of the
1
Current address: Aminogen Research Institute, Seoul National
University Cancer Research Center Building, 28 Yongondong, Chon-
grogu, Seoul 110-799, Korea.
2
To whom correspondence and reprint requests should be ad
-
dressed. Fax: 082-2-2210-2888. E-mail: eshwang@uos.ac.kr.
0014-4827/02 $35.00
173
© 2002 Elsevier Science (USA)
All rights reserved.
Experimental Cell Research 277, 173–182 (2002)
doi:10.1006/excr.2002.5554