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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 20, 2004 as doi:10.1096/fj.03-1332fje. |
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,1
* Tyrolean Cancer Research Institute at the University of Innsbruck, 6020 Innsbruck, Austria;
Institute for Biomedical Aging Research of the Austrian Academy of Sciences, Innsbruck, Austria; and
Department of Obstetrics and Gynecology, University of Innsbruck, Innsbruck, Austria
1Correspondence: Tyrolean Cancer Research Institute and Institute for Biomedical Aging Research, Innrain 66, 6020 Innsbruck, Austria. E-mail: werner.zwerschke{at}uklibk.ac.at
SPECIFIC AIMS
E7, the major transforming oncogene of HPV-16, codes for an unstable protein whose abundance in cervical biopsies is presently unknown. Using highly specific anti-HPV-16 E7 antibodies, we examined the expression level and subcellular localization of the E7 protein in cervical carcinogenesis in situ and analyzed whether E7-driven degradation of pRb is involved in cervical tumorigenesis in humans.
PRINCIPAL FINDINGS
1. Generation of polyclonal anti-HPV-16 E7 antibodies
To obtain antibodies to HPV-16 E7 suitable for E7 in situ detection, we immunized rabbits with highly purified native HPV-16 E7 oncoprotein. The resulting polyclonal anti-HPV-16 E7 antibodies were affinity purified and recognized the HPV-16 E7 protein as a single band in E7-expressing human U-2 OS cells and in biopsies derived from HPV-16-positive cervical carcinoma patients Western blot; HPV-16 E7 was not detectable in the other cervical tissues. To establish a reliable in situ detection procedure, we first studied by confocal immunofluorescence microscopy the subcellular distribution of E7 in human U-2 OS cells transiently expressing the HPV-16 E7 protein. E7 was predominantly expressed in the nucleus and to a minor extent in the cytoplasm of these cells (Fig. 1
, left panel). These findings were corroborated in the HPV-16-positive cervical carcinoma cell line Ca Ski (Fig. 1
, middle panel). Preimmune serum did not recognize the E7 antigen in Ca Ski cells (Fig. 1
, right panel).
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2. Detection of HPV-16 E7 protein in cervical biopsies in situ
To determine whether HPV-16 E7 protein can be detected in cervical biopsies in situ, we performed immunohistochemistry experiments with anti-HPV-16 E7 antibodies on serial paraffin sections of cervical tissues. The anti-E7 antibodies stained almost all cells in HPV-16 DNA-positive tumor islets but not cells in adjacent connective tissue. Staining by the anti-HPV-16 E7 antibodies could be blocked by preincubating the antibodies with purified HPV-16 E7 antigen. These findings prove the high specificity of the affinity-purified anti-HPV-16 E7 antibodies described here.
3. E7/pRb interaction in LSIL
We used anti-HPV-16 E7 antibodies to evaluate the role E7 plays in cervical carcinogenesis. Normal squamous epithelium obtained from HPV-negative healthy controls and epithelium with apparently normal morphology localized adjacent to an LSIL were not stained by the E7 antibodies. The data suggest that no E7 signal is obtained in HPV-negative tissues and very little, if any, E7 protein is detectable in LSIL adjacent cells. In most of the LSIL analyzed, the E7 protein was hardly detectable (Fig. 2
A).
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pRb is an established molecular high-risk HPV E7 target in cultured cell lines. E7 has been shown to displace pRb from complexes with cellular regulator proteins, such as the transcription factor E2F, and to induce pRb degradation. This results in the activation of many genes essential for cell cycle progression and DNA replication. Since little is known about the molecular pathway by which E7 overrides pRb function in human tumors, we sought to correlate E7 protein levels and pRb levels in cervical biopsies. Staining of normal cervical squamous epithelia from HPV-negative healthy controls with pRb antibodies revealed weak signals in only some cells of the parabasal layers, suggesting that pRb is weakly expressed in such tissues. In morphologically normal epithelium obtained from patients with HPV-16-positive LSIL lesions (LSIL adjacent), surprisingly, high level nuclear staining was reproducibly obtained with the pRb antibody. All cells in the parabasal and intermediate layers stained strongly pRb positive, whereas pRb was not detectable in the basal or upper layers. This observation suggests that at or before the stage of LSIL, a process is initiated that leads to increased abundance of pRb.
Comparing LSIL periphery with the morphologically abnormal sites (Fig. 2D
) of the same section, we found that the pattern of pRb-expressing cells changed drastically. Whereas in the morphologically normal part of the specimen the pRb-positive cells are uniformly localized in the suprabasal layer, in the morphologically abnormal part this pattern has been destroyed. In the latter, pRb-positive and pRb-negative cells are distributed throughout the cellular layer. However, quantification of the pRb-positive cells revealed no major changes in the number of pRb-positive cells. This suggests that the reorganization of the epithelium observed during the outgrowth of an LSIL has led to a redistribution of pRb-positive cells, although no significant decrease in the overall pRb expression level is observed.
4. E7/pRb interaction in HSIL and carcinomas
To obtain further insight into the role of HPV-16 E7 in cervical carcinogenesis, four HSIL biopsies were analyzed. In all HSIL, the E7 protein level significantly increased compared to LSIL. The expression pattern observed for the E7 protein was not always homogeneous, and histologically more advanced lesions expressed higher E7 levels. An example of a histologically more advanced HSIL is shown in Fig. 2
. Here E7 was expressed equally in all cells of the undifferentiated epithelium (Fig. 2B
). In the HSIL analyzed, the level of pRb protein was strongly diminished (Fig. 2E
) relative to earlier stages (Fig. 2D
). Similar to E7, the abundance of pRb and the corresponding expression pattern were not identical in all HSIL analyzed. As analyzed by costaining (pRb red and E7 light brown), in the vast majority of the cells in the HSIL the abundance of pRb and HPV-16 E7 protein was inversely correlated at the level of individual cells. Most cells revealed very low pRb levels in correlation with high E7 expression levels, indicated by the original light-brown E7 staining (Fig. 2H
). A few cells were stained dark brown, suggesting they express significant amounts of pRb in the presence of low amounts of E7 (Fig. 2H
, black arrows). In these cells, either pRb is actually on the way to E7-induced degradation or pRb is stabilized by other means, probably reflecting the inherent heterogeneity of naturally occurring tumors. These results suggest that during precancerous carcinogenesis, the abundance of E7 increases considerably and this increase is correlated with a concomitant decrease in the abundance of pRb, probably reflecting the known ability of HPV-16 E7 to trigger pRb-degradation. Costaining of pRb and E7 in invasive cervical carcinoma (CxCa) sections revealed very low pRb levels along with high E7 expression levels in the vast majority of the cells (Fig. 2I
). These data suggest that the relative abundance of pRb and E7 protein is inversely correlated at the level of individual cells. In only a few cells did we detect a stronger brown tone (black arrows, Fig. 2I
), suggesting that in these cells either pRb is on the way to E7-induced degradation or pRb might have evaded degradation induced by E7. These data suggest that in CxCa, pRb had been eliminated in the vast majority of the cells, probably through proteolytic degradation triggered by E7. In several CxCa analyzed in this study, we found areas of various keratinization (differentiation) grades. The intensity of E7 expression was inversely correlated with the differentiation grade of the tumor area such that the undifferentiated areas contained higher E7 levels. These data suggest that an increase in the E7 expression level could correlate with a decrease in the differentiation level. However, more work is required to address the molecular basis of this difference.
CONCLUSIONS AND SIGNIFICANCE
Experimental evidence derived from many in vitro studies suggests that expression of the E6 and E7 oncogenes is necessary for induction and maintenance of the transformed phenotype. However, the role of the respective oncoproteins in cervical carcinogenesis remains elusive due to a lack of antibodies that would allow their detection in situ. In this study we show that the HPV-16 E7 oncoprotein is expressed in HPV-16-positive cervical biopsies. While the E7 protein was undetectable in most of the LSIL (Fig. 2A
) analyzed, we obtained a significant increase in the E7 protein level during precancerous progression to HSIL (Fig. 2B
). A further increase in the E7 level with malignant progression was found in 70% of the CxCa specimens analyzed (Fig. 2C
). These data suggest an important role for the E7 protein in precancerous and cancerous progression in vivo.
The E7 protein was found predominantly in the nucleus and to a minor extent in the cytoplasm in the cervical cancer cell line Ca Ski and in invasive cervical carcinoma, suggesting that nuclear resident E7 plays a major role in cervical carcinogenesis in humans. In LSIL, our study reveals only weak E7 protein expression in a small subset of specimens. In histologically severe HSIL was the E7 protein distributed across the entire undifferentiated epithelium. The E7 protein was detectable in all CxCa analyzed, but the expression level was very heterogeneous in the various CxCa.
Keratinization has been described as a frequent pathological feature in HPV-16-positive CxCa. In this study we found that high E7 abundance correlates with areas of low keratinization and hence a low degree of differentiation, suggesting an increase in the E7 expression level might correlate with a decrease in the differentiation level.
A striking result reported here is that pRb appears weakly expressed in uninfected normal cervical epithelium but is strongly up-regulated in parabasal layers of histologically normal-looking epithelium adjacent to an LSIL and in LSIL. In more advanced lesions, the pRb expression level decreases and pRb becomes virtually undetectable in CxCa. We were able to show, in situ, that pRb expression is inversely correlated with E7 expression in HSIL whereas pRb staining almost disappeared in carcinomas, concomitant with an increasing E7 level. The inverse correlation of E7 and pRb expression suggests that E7-dependent pRb degradation occurs in vivo and is likely involved mechanistically in cervical carcinogenesis.
The anti-HPV-16 E7 antibodies presented here may now provide an opportunity to study E7 activity and the corresponding protein–protein interactions in the complex environment of human cervical intraepithelial lesions and cancers in vivo. Moreover, the anti-HPV-16 E7 antibodies described might help monitor cervical precancerous and cancerous progression and could be used for diagnostic purposes.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1332fje; doi: 10.1096/fj.03-1332fje
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