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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 19, 2001 as doi:10.1096/fj.00-0604fje. |
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,
,2
* Institut f. Biomedizinische Alternsforschung der Österreichischen Akademie der Wissenschaften, Rennweg 10, A-6020 Innsbruck, Austria;
Tiroler Krebsforschungsinstitut, Innrain 66, A-6020 Innsbruck, Austria; and
Deutsches Krebsforschungszentrum, Forchungsschewerpunket Angewandte Tumorvirologie, Heidelberg, Germany D-69120
2Correspondence: Tiroler Krebsforschungsinstitut, Innrain 66, A-6020 Innsbruck, Austria. E-mail: werner.zwerschke{at}uklibk.ac.at
SPECIFIC AIMS
In this study, we have addressed the hypothesis that knocking out the function of the HPV-16 E7 oncoprotein in cervical carcinoma cells may prevent proliferation of these cells. By using the peptide aptamer technique, we have screened a random peptide library for artificial peptides that bind to HPV-16 E7 with very high affinity and analyzed the potential of these molecules to interfere with the proliferation of E7-expressing tumor cells.
PRINCIPAL FINDINGS
1. Identification of E7-binding peptide aptamers
Screening a random peptide library of 8.5 x
106 individual peptides in a yeast two-hybrid
screen, 30 peptides have been identified that bind selectively to
HPV-16 E7 with very high affinity. A ß-galactosidase in
situ staining assay was used to quantify the in vivo
binding affinity (Fig. 1A
). We also isolated control peptide aptamers that fail to
bind E7 in the yeast screen. By using extracts from yeast cells in GST
pull-down experiments, we found that the 30-peptide aptamers bound to
GST HPV-16 E7 fusion proteins but not to unfused GST protein, whereas
the control peptide aptamers failed to bind. A representative
experiment is shown in Fig. 1B
for the E7-binding peptide
aptamers #55 (LNFIFDERSDIYVLWLILEG) and #310 (VMQPGVVKGWRRTKVGRYIL), as
well as for a E7-nonbinding peptide aptamer
(KVPVHRTCVACLLVVNSRCM).
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2. E7-binding peptide aptamers suppress proliferation of
E7-expressing but not control cells
We generated E7-expressing sublines from NIH3T3 cells
by stable transfection of an E7-expression vector and used these cells
to determine the influence of E7-binding peptide aptamers on cell
proliferation. By using a colony formation assay, we found that ectopic
expression of peptide aptamer 55 in NIH3T3E7 cells reduced the number
of viable colonies from 89 (±12) to 17 (±2), as compared with the
E7-nonbinding control peptide aptamer; peptide aptamer 310 induced a
similar reduction in the number (to 25 ± 3) of viable colonies.
When the peptide aptamers were expressed in control (non E7-expressing)
cells, similar colony numbers were observed for cells transfected with
vectors for peptide aptamers 55 (46±3), 310 (55±11), and the control
peptide aptamer (41±5), which indicates that the E7-expressing
aptamers do not affect proliferation of control NIH3T3 cells
significantly. These results indicate that E7 is indeed the in
vivo target of the E7-binding peptide aptamers, and that knocking
out the function of E7 reduces the proliferation capacity of NIH3T3E7
cells.
3. E7-binding peptide aptamers suppress cell proliferation
by induction of apoptosis
To clarify the mechanism of growth suppression by E7-binding
peptide aptamers, we determined the rate of apoptosis in E7-expressing
and control NIH3T3 cells after transient expression of the peptide
aptamers 55, 310, or the control peptide aptamer. By using flow
cytometry of propidium iodide stained cells, we found that expression
of the E7-binding peptide aptamer 55 in E7-expressing cells increased
the rate of apoptosis from 2.5% (±1.1%) to 50% (±11%), as
compared with the E7-nonbinding control peptide aptamer; similarly,
expression of peptide aptamer 310 increased the rate of apoptosis to
28% (±6.5%). When the peptide aptamers were expressed in control
(non E7-expressing) cells, a similar rate of apoptosis was observed for
cells transfected with vectors for peptide aptamers 55 (3.0±0.6%),
310 (0.5±0.2%) and the control peptide aptamer (0.83±0.4%), which
indicates that the E7-expressing aptamers do not significantly modulate
the rate of apoptosis in control NIH3T3 cells. These results indicate
that E7-binding peptide aptamers specifically induce apoptosis of
E7-expressing cells. This finding is also supported by our observation
that expression of the E7-binding peptide aptamers resulted in the
appearance of TUNEL-positive cells: In the case of peptide
aptamer 55, the frequency of TUNEL-positive cells increased from 1.7%
(±0.5%) (control aptamer) to 12.7% (±1.9%); whereas in the case of
peptide aptamer 310 the frequency of TUNEL-positive cells increased to
11.9% (±1.2%). Expression of all peptide aptamers in control NIH3T3
cells did not significantly alter the frequency of TUNEL-positive
cells.
4. E7-binding peptide aptamers specifically induce apoptosis in
CaSki cervical carcinoma cells
To determine if E7-binding peptide aptamers can
interfere with the proliferation of naturally occurring HPV-16-induced
tumor cells, peptide aptamers were transiently expressed in CaSki
cervical carcinoma cells by transient transfection. We found that all
peptide aptamers were expressed to similar extent in CaSki cells
(Fig. 2A
). When the rate of apoptosis was assessed by flow
cyotmetry, we found that the control aptamer had no significant effect
on apoptosis; in contrast, expression of peptide aptamers 55 and 310
significantly increased the rate of apoptosis in CaSki cells (Fig. 2B
). This finding indicates that E7-binding peptide
aptamers are useful tools to interfere with the growth of
HPV-positive tumor cells; furthermore, the data reported in this
communication clearly suggest that the function of the E7 protein is
essential for the viability of both E7-expressing experimental cell
lines and HPV-16-positive tumor cells.
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CONCLUSIONS AND SIGNIFICANCE
Human papillomaviruses (HPV) are etiological agents of benign and malignant human tumors. Current therapeutic strategies for HPV-associated tumors include surgical removal of the lesion and nonspecific stimulation of innate immunity. Furthermore, prophylactic and therapeutic vaccination are currently being tested in clinical trials; however, no definitive results are yet available. In an alternative approach, we have explored the possibility of developing new substances that could be used as anti-viral agents, by selecting peptide aptamers that bind with high affinity to the viral protein E7 and thus may have the potential to block the interaction of E7 with cellular proteins. The constrained peptides present in peptide aptamer libraries display an up to 1,000-fold increase in specific binding affinity, when compared with the corresponding free peptides. Due to the high affinity and selectivity inherent to peptide aptamers, these molecules are good candidates for new therapeutic agents with extremely low side effects, in particular when they are directed against viral proteins such as E7, for which no cellular homologue exists. As is described in this communication, we could identify such E7-binding peptide aptamers and present data that some of them fulfil the criteria defined above.
According to the current concept, malignant transformation of mammalian cells requires genetic changes that suppress a cellular suicide program apoptosis that exists in most if not all cells. The apoptotic program is activated by various environmental and genetic insults, and inappropriate cell proliferation is considered as a major apoptosis-inducing signal in mammalian cells; consequently, induction of apoptosis is believed to contribute to tumor suppression. It has long been known that DNA tumor viruses carry genes that can modulate the cellular suicide program. It was shown that the product of the E6 gene of high-risk human papillomaviruses abrogates p53-dependent apoptosis by inducing the proteolytic elimination of p53. However, HPV-16 E6 can also sensitize human keratinocytes to apoptosis and modulate apoptotic pathways in p53-negative cells.
Similar to the findings with E6, it was shown that the E7 protein
of HPV-16 can either induce or prevent apoptotic cell death; hence, the
consequences of E7 expression appear to depend on the genetic
background of the recipient cell. As immortal cell lines can be
generated by the isolated expression of either the E6 or E7 oncogene of
HPV-16 in human cells, both viral genes must be capable of
down-modulating an apoptotic response that results from aberrant cell
proliferation. In this communication, we have used an experimental cell
system in which E7 suppresses apoptosis, and we have found that
expression of E7-binding peptide aptamers in such cells induces the
apoptotic response. That this finding may be relevant for HPV-induced
human tumors is suggested by our finding that targeting E7 by
E7-binding peptide aptamers induces apoptosis in CaSki cells; hence,
CaSki cells appear to require E7 function for survival (Fig. 3
).
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The results presented in this communication suggest that E7-binding peptide aptamers may be useful tools to develop new therapeutic strategies for HPV-associated diseases. Although it is generally assumed that the use of peptides for therapeutic purposes is problematic, the efficient delivery of peptide aptamers may become a realistic option, given the recent progress in virus-based gene therapy systems. Furthermore, the therapeutic use of peptides may benefit from the recent discovery and development of specific oligopeptide sequences that allow the direct penetration of heterologous proteins into mammalian cells and even into a living mouse. In the case of HPV-associated diseases, therapeutic use of peptide aptamers may be facilitated by the fact that topical delivery of therapeutic substances is a reasonable option—due to the nature of HPV-induced lesions which occur exclusively in epithelia or squamous epithelia.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0604fje ; to cite this
article, use FASEB J. (January 19, 2001)
10.1096/fj.00-0604fje 
This article has been cited by other articles:
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A. Cid-Arregui, V. Juarez, and H. z. Hausen A Synthetic E7 Gene of Human Papillomavirus Type 16 That Yields Enhanced Expression of the Protein in Mammalian Cells and Is Useful for DNA Immunization Studies J. Virol., April 15, 2003; 77(8): 4928 - 4937. [Abstract] [Full Text] [PDF]
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