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Previous Article | Next Article 
Molecular and Cellular Biology, December 2003, p. 9094-9103, Vol. 23, No. 24
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.24.9094-9103.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin,1 and MIT Cancer Center and Howard Hughes Medical Institute, Cambridge,2 Department of Pathology, Harvard Medical School, Boston,Massachusetts3
Received 4 June 2003/ Returned for modification 21 July 2003/ Accepted 9 September 2003
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ABSTRACT |
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INTRODUCTION |
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HPV-16 E7 is a small nuclear phosphoprotein with potent transforming and oncogenic properties. Coexpression of E6 and E7 is necessary and sufficient to transform primary human keratinocytes (31), and E7 is strongly positive in a number of other in vitro transformation assays (2, 27, 36, 46, 49, 50). In culture, E7-expressing cells exhibit genomic instability (10, 39, 51, 52) and lack normal responses to DNA damage (7, 44). In addition, expression of E7 in primary human keratinocytes results in abnormal centrosome synthesis, with resulting multipolar mitoses and aneuploidy (8-10). Previously, our lab generated mice transgenic for HPV-16 E7 under the control of the keratin 14 (K14) promoter, targeting E7 expression to the basal layer of stratified squamous epithelia such as the skin and cervical epithelium (16). These mice, which express E7 at levels similar to the levels seen in human cervical carcinoma cell lines (I. Frazier, unpublished data), have a broad spectrum of phenotypes. These include epithelial hyperplasia, increased cell cycle progression in all epithelial cell layers, disrupted epithelial differentiation, loss of DNA damage-induced cell cycle arrest, spontaneous skin tumors, and cervical cancers in estrogen-treated mice (16, 40, 45).
The first described function of E7 was binding and inactivation of the retinoblastoma tumor susceptibility gene product, pRb (11). Interaction between E7 and pRb results in proteasomal degradation of pRb in cultured cells (4, 12, 22) and disrupts the ability of pRb to bind to and inactivate the transactivation activity of cellular E2F transcription factors (5, 35). Disruption of the Rb regulatory pathway is frequent in most human cancers and pRb represents a target common to many highly tumorigenic or transforming viruses. E7 has also been reported to bind and inactivate other known cell cycle regulators in vitro, including the pRb family members p107 and p130, and the cyclin-dependent kinase (cdk) inhibitors p21 and p27 (12, 21, 22, 53). Remarkably, at least 14 additional cellular binding partners of the 98-amino-acid E7 protein have now been reported from in vitro studies (6, 30). Thus, E7 is a multifunctional protein in vitro with pleiotropic effects in vivo.
It is unclear which of E7's reported binding interactions is responsible for its phenotypes observed in vivo. pRb inactivation is likely to account for many of E7's effects, since pRb is known to affect cell cycle regulation, differentiation, and DNA damage responses in many cell types in vitro and in vivo (19, 28, 44). Consistent with this, E7 mutants that fail to bind or inactivate pRb fail to induce any phenotypes in vivo (13). These mutants, however, are also deficient for binding multiple other cellular targets, so they do not isolate the importance of the E7-pRb interaction. In addition, the effects of pRb inactivation cannot be studied in adult animals by using traditional knockout technology, since the Rb-/- status is lethal to embryos(19).
In the present study, we analyzed the effect of somatic pRb inactivation in murine skin by using a Cre-lox based system. This system allows comparison of pRb-deleted tissue to E7-expressing tissue in adult mice to determine which effects of E7 expression may be attributable to pRb inactivation. We demonstrate that pRb inactivation recapitulates all known phenotypes associated with E7 expression in murine skin. However, pRb-independent activities of E7 are detectable when E7 is expressed and Rb inactivated in the same tissue. Possible interpretations of these results are discussed.
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MATERIALS AND METHODS |
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Immunohistochemical and immunofluorescence analysis of epidermis. Skin and ear epidermal samples were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and cut into 5-µm sections. Serial sections were used for hematoxylin and eosin staining, immunohistochemical staining for pRb and BrdU, and immunofluorescence staining for K14, K10, and gamma-tubulin.
For immunohistochemical stains, sections were deparaffinized in xylenes and rehydrated through a graded series of ethanol-water solutions. Endogenous peroxidase activity was quenched by treatment in 3% H2O2 in methanol for 10 to 20 min. Slides were washed in phosphate-buffered saline (PBS) and heated in boiling 10 mM sodium citrate (pH 6.0) for 20 min. Further unmasking was achieved with 20 min of immersion in 2 N HCl. Samples were blocked for 30 min at ambient temperature in 5% horse serum in PBS. Primary antibodies were diluted in blocking buffer and applied as follows: 1:50 anti-pRb (clone G3-245, Pharmingen catalog no. 554136) or 1:40 anti-BrdU (Oncogene catalog no. NA20-100UG) for 2.5 h at room temperature. After washes in PBS, biotinylated secondary antibody and streptavidin-peroxidase conjugate were applied according to the Vectastain ABC kit instructions (Vector Labs catalog no. PK-6200). Staining was developed in 3,3'-diaminobenzidine (DAB) solution (Vector Labs catalog no. SK-4100) for 1 to 2 min and then quenched in H2O. Slides were counterstained with hematoxylin, dehydrated through a series of ethanols and xylenes, and covered with a coverslip.
For immunofluorescent stains, sections were deparaffinized and rehydrated as described above. For cytokeratin stains, slides were blocked with 10% horse serum-3% bovine serum albumin for 30 min. A 1:1,000 dilution of anti-mouse K14 (Covance catalog no. PRB-155P) in blocking solution was applied overnight at 4°C. Slides were then washed in PBS, and biotinylated universal secondary antibody (Vector Labs catalog no. PK-6200) at 1:250 was applied for 30 min at room temperature. After washes in PBS, streptavidin-Texas red (Vector Labs catalog no. SA-5006) at 1:150 was applied for 30 min at room temperature, followed by additional washes. Slides were then incubated overnight with fluorescein isothiocyanate-conjugated anti-keratin 10 (Covance catalog no. FITC-159L) at 1:200 in blocking buffer. Slides were rinsed in PBS, mounted in Vectashield mounting medium with DAPI (4',6'-diamidino-2-phenylindole; Vector Labs catalog no. H-1200), and visualized by using a Zeiss Axiophot fluorescence microscope. For centrosome stains, slides were deparaffinized as described above and boiled in 10 mM sodium citrate buffer (pH 6.0) for 30 min, followed by pepsin treatment (Digest-All 3; Zymed, South San Francisco, Calif.) for 10 min at 37°C. Sections were blocked with 10% donkey serum in destilled H2O for 15 min at room temperature, followed by incubation with an anti-gamma-tubulin monoclonal antibody (GTU-88; Sigma, St. Louis, Mo.) at a 1:1,000 dilution in PBS overnight at 4°C. After a wash in PBS, cells were incubated with a rhodamine red-conjugated donkey anti-mouse secondary antibody (Jackson Immunoresearch, West Grove, Pa.) at a 1:100 dilution in PBS for at least 2 h at 37°C. After a final wash in PBS, sections were counterstained with DAPI and then analyzed by using a Leica DMLB epifluorescence microscope.
Quantitation of pRb loss and cell proliferation. To quantify the loss of pRb expression, sections of ear epidermis were stained for pRb and counted. Similarly, BrdU incorporation into newly synthesized DNA was used as a measure of keratinocyte proliferation by counting BrdU-stained skin and ear sections. All keratinocytes in 10 visual fields were scored as either positive (brown) or negative (blue) for pRb expression or BrdU incorporation in both basal and suprabasal layers of the epidermis. For counting purposes, even slightly brown cells were counted as positive. Three to six mice were counted per genotype. Statistical analyses of results were performed by using the two-sided Wilcoxon rank sum test.
Quantitation of centrosome abnormalities. On each tissue section, cells showing a dot-like staining for gamma-tubulin were identified, and the number of centrosomes was determined. Since normal cells contain up to two centrosomes, cells with more than two centrosomes were considered abnormal. Due to tissue sectioning, centrosomes were detectable only in a proportion of cells on each section. On average, 164 cells were assessed per section, and the mean ± standard error of the percentage of cells with more than two centrosomes was calculated. Statistical significance was assessed by using Student two-tailed t test for independent samples.
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RESULTS |
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To assess the efficiency of Cre-induced inhibition of pRb expression, ear and dorsal skin epidermal sections from mice of different ages were stained for pRb. To minimize variations in staining intensity, all slides were stained simultaneously using the same reagents and developed in DAB for identical times. When the animals were 21 days old, pRb staining was drastically reduced in K14CreRbf/f ear and skin epidermis but still strongly evident in Cre-negative Rbf/f epidermis (Fig. 1B). Since Cre-mediated recombination could occur independently in each keratinocyte, inactivation of pRb was quantified on a per-cell basis by counting cells as positive or negative for pRb staining. This revealed a 96% decrease in the fraction of cells expressing detectable pRb in K14CreRbf/f mice compared to Cre-negative Rbf/f controls, with the remaining positive cells in K14CreRbf/f epidermis staining very weakly (Fig. 1C). Rb inactivation was only slightly less efficient in dorsal skin, where a 91% reduction in pRb-positive cells was seen. Thus, pRb expression is inhibited in the vast majority of keratinocytes in K14CreRbf/f mice by 21 days of age. Interestingly, pRb staining was detected in most keratinocytes in K14E7 mice, although staining intensity in individual cells was often diminished compared to Cre-negative Rbf/f epidermis. This observation supports the hypothesis that E7 not only binds but also degrades pRb in vivo (4, 12, 22). Since nearly complete loss of detectable pRb was achieved in K14CreRbf/f mice by 21 days of age, all further analyses were performed on 21-day-old mice.
Hyperproliferation in
Rb-deleted and E7-expressing epidermis.
Pronounced hyperplasia was evident in
the ear epidermis of both K14E7 and
K14CreRbf/f mice (Fig.
1B and
2A; see also Fig.
5). To compare quantitatively the hyperproliferative effects of E7
expression versus loss of pRb, mice were injected with the
deoxynucleotide analog BrdU 1 h prior to sacrifice. Sections
were stained with anti-BrdU antibody to detect DNA synthesized during
the hour prior to sacrifice (Fig.
2A). Counting of these
sections revealed several phenotypes in Rb-deleted epidermis.
These phenotypes included an 
2-fold increase in DNA synthesis
in the basal layer of keratinocytes, as well as inappropriate DNA
synthesis in normally quiescent suprabasal keratinocytes (Fig.
2B). These phenotypes are
strikingly similar to those seen in K14E7 mice (Fig.
2). Indeed, the fraction
of cells incorporating BrdU in K14CreRbf/f mice was
indistinguishable from that in K14E7 mice in both the basal
and suprabasal epidermal layers. Since the severity of E7-associated
phenotypes is known to vary with the expression level of E7
(16), it is important to
note here that the line of K14E7 mice used in these studies
expresses E7 in ear epidermis at a level similar to the E7 expression
levels in human cervical carcinoma cell lines, as measured by an
enzyme-linked immunosorbent capture assay (Frazier,
unpublished).
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pRb loss inhibits radiation-induced cell cycle arrest. Having seen similarities between the effects of loss of pRb and the expression of E7 on epithelial cell proliferation, we conducted experiments to determine whether other acute effects of E7 expression in epithelium were reproduced in Rb-deleted tissues.
Expression of E7 in stratified squamous epithelia in mice inhibits the ability of these cells to suppress DNA synthesis in response to DNA damage (45). To determine whether loss of pRb also abolishes the normal DNA damage response of epithelia, K14CreRbf/f and K14E7 mice were exposed to 0 or 5 Gy of ionizing radiation at 21 days of age, BrdU was injected 24 h later, animals were sacrificed 1 h after that, and ear epidermal sections were taken. Histological sections were stained for BrdU, and the percentage of epidermal cells staining positively for BrdU in tissues from irradiated versus unirradiated mice was determined (Fig. 3). As expected, the epidermis from control mice (Rbf/f and K14CreRbf/wt mice) displayed a drastic decrease in BrdU incorporation after irradiation compared to that from the unirradiated control mice; in contrast, the epidermis from K14E7 mice failed to display any significant decrease in the frequency of cells supporting DNA synthesis after irradiation. Epidermis of K14CreRbf/f mice also failed to display any decrease in the frequency of cells supporting DNA synthesis after irradiation. Thus, the loss of pRb function is sufficient to inhibit DNA damage-induced responses in the mouse epidermis.
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Importantly, the dysplasia and hyperplasia seen in K14E7K14CreRbf/f mice is not due to any effect of E7 on the timing or efficiency of Cre-mediated Rb deletion, since the extent of Rb inactivation is the same in K14E7K14CreRbf/f and K14CreRbf/f mice (data not shown). Similarly, since E7-mediated pRb inactivation does not affect the K14Cre transgene in K14E7K14CreRbf/f mice, it is unlikely that Cre-mediated Rb inactivation has any effect on expression of the K14E7 transgene in these mice. Thus, the detection of E7-induced phenotypes in Rb-deleted tissue indicates that E7 posesses pRb-independent effects that lead to either increased dysplasia or increased hyperplasia and dysplasia, depending on the epithelial tissue.
Centrosome
abnormalities in K14E7 and K14CreRbf/f
mice.
E7 expression has been
shown to induce aberrant centrosome duplication and chromosomal
instability in keratinocytes in monolayer and organotypic cultures
(8-10).
To determine whether pRb inactivation is sufficient to deregulate
centrosome synthesis, epidermal sections from the ears of mice were
stained for the centrosome marker gamma-tubulin, counterstained with
DAPI, and the proportion of cells with abnormal centrosome numbers was
enumerated (Fig. 6). We
detected a statistically significant (P 
0.05)
6.1-fold increase of the proportion of cells with more than two
centrosomes when we compared K14CreRbf/f
(4.3% ± 1.5%) to Rbf/f
controls (0.7% ± 0.6%). Similarly, statistically
significant 6- and 6.9-fold increases of cells with numerical
centrosome abnormalities were found when we compared K14E7
mice (4.2% ± 0.4%) and
K14E7K14CreRbf/f mice (4.8% ±
1.2%), respectively, to controls (P 0.01). No
significant differences were observed between K14E7,
K14CreRbf/f, and
K14E7K14CreRbf/f (P > 0.05). These
data indicate that E7 deregulates centrosome synthesis in vivo and that
inactivation of pRb is sufficient to deregulate centrosome
synthesis.
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DISCUSSION |
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TopAbstractIntroductionMaterials and
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Consequences of somatic inactivation of pRb in murine epithelia. We achieved nearly complete (>90%) inhibition of pRb expression in murine stratified squamous epithelia by 21 days of age (Fig. 1). Loss of pRb expression in keratinocytes resulted in multiple phenotypes, including epithelial hyperplasia with increased and deregulated cell cycle progression (Fig. 2). This was accompanied by a wrinkled skin phenotype with visible thickening of the ears and a scruffy appearance. These results were anticipated given the well-established role of pRb in cell cycle regulation (33). Interestingly, however, the extent of hyperplasia induced by Rb deletion varied between different epidermal sites, with pronounced hyperplasia in ear epidermis and only mild hyperplasia in dorsal skin (Fig. 5). This observation indicates different roles for pRb in epidermal tissue from different anatomic locations. A similar difference has long been noted with our K14E7 mice (16) (Fig. 5). There, however, we could not discriminate between a difference in the level of expression of E7 versus a difference in the biology of the different tissues. Inactivation of pRb also disrupted the normal differentiation program of the stratified squamous epitheium, demonstrating that pRb plays an important role in the differentiation of this tissue in vivo (Fig. 4). We observed delayed differentiation in Rb-deleted epidermis, with an expansion of the poorly differentiated, K14-positive cell compartment, and a delay in the onset of expression of the differentiation marker K10. These observations are consistent with studies in which a role for pRb in the differentiation of other cell types has been discerned in vitro and in vivo (28).
We found that the loss of pRb was sufficient to inhibit DNA damage responses in murine epidermis (Fig. 3). Prior studies have argued that E7's binding to pRb was necessary but not sufficient for E7 to inhibit radiation responses in human keratinocytes in tissue culture (14, 15, 44). In that system, E7's inactivation of p21 was also found to contribute to the ability of E7 to inhibit DNA damage responses. A likely explanation is that inactivation of pRb is sufficient to disrupt DNA damage responses both in vitro and in vivo, but E7 does not inactivate pRb solely by binding and degrading pRb. Rather, E7 must also promote pRb phosphorylation by suppressing the activity of cdk inhibitors such as p21. This is of particular importance in the context of DNA-damaging agents, which lead to the induction of p21.
Loss of pRb also deregulated centrosome synthesis in murine keratinocytes (Fig. 6). Although the molecular mechanism of this effect is not clear, centrosome duplication is normally coupled to the onset of DNA replication at the G1/S transition (17, 18, 23) and is dependent on cyclinE-cdk2 activity in many systems (18). Thus, since cyclin E is an E2F-responsive gene (43), loss of pRb could result in inappropriate cyclin E expression and cdk2 activation, triggering abnormal centrosome synthesis.
Centrosome abnormalities result in multipolar mitoses, with consequent genomic instability and aneuploidy (25, 29, 38). Therefore, the demonstration of a direct role for pRb in the regulation of proper centrosome synthesis indicates yet another important tumor suppressor function of pRb. This finding may have broad implications for tumor biology, since the pRb pathway is disrupted in nearly all human tumors (33). Importantly, however, dysregulation of the pRb pathway in primary human epithelial cells by loss of p16INK4A function and/or ectopic expression of the pRb kinase cdk4 does not cause centrosome abnormalities (37). Thus, the effect of pRb pathway disruption on centrosome control may vary depending on precisely how the pathway is disrupted and/or the affected cell type. Further studies will more clearly elucidate the relationship between pRb function and centrosome regulation.
Both HPV-16 E7 and the adenovirus E1A oncoprotein induce abnormal centrosome synthesis (6, 8-10). This activity of E1A is reportedly mediated by an interaction between E1A and Ran-GTPase (6). In our study, loss of pRb function deregulated centrosome synthesis to the same extent as E7 expression, and E7 expression in pRb-deficient cells had no additional effect on centrosome abnormalities. Thus, our data suggest that E7 deregulates centrosome synthesis primarily via a pRb-dependent mechanism rather than a Ran-dependent mechanism. Importantly, cervical epithelium from K14E7 mice expresses increased levels of cyclin E (T. Brake, J. Connor, D. Petereit, and P. F. Lambert, unpublished data), supporting the possibility that E7 deregulates centrosome synthesis by the pRb-dependent activation of cdk2, as described above. Given the variability inherent in counting centrosomes in histological slides, however, we cannot rule out the possibility that interactions between E7 and other binding partners could have subtle effects on centrosome synthesis that were not detected here.
Evidence for additional targets of E7 in mediating its phenotypes. There are two important implications to the observation that the K14E7K14CreRbf/f mice display increased hyperplasia and dysplasia compared to the K14E7 and K14CreRbf/f mice. First, other activities besides E7's inactivation of pRb must contribute to E7's biological properties in vivo (since, if inactivation of pRb is the only relevant activity, we would not see a difference in the phenotypes between K14CreRbf/f and K14E7K14CreRbf/f mice). It is possible that E7 targets p107, p130, or other cellular factors more efficiently in pRb-null tissue than in pRb-sufficient tissue. However, because E7 does not appear to fully inactivate pRb (see below) and since multiple in vitro studies suggest that non-pRb interactions are important to E7's function (14, 15, 24), we conclude that the pRb-independent activities observed in the K14E7K14CreRbf/f mice are physiologically relevant and not simply a reflection of E7 altering its targets in the absence of pRb.
It is also interesting that the more severe phenotypes bestowed by the combination of E7 expression and pRb deficiency (compared to the consequences of either effect alone) differed between epidermal sites. In the torso epidermis we saw both increased hyperplasia and onset of dysplasia in the K14E7K14CreRbf/f mice, but in the ear epidermis we only observed onset of gross dysplasia without increased hyperplasia in the K14E7K14CreRbf/f mice. Although this difference may simply reflect different efficiencies with which E7 affects the same cellular factors, it could reflect, alternatively, E7 targeting different cellular proteins in these tissues.
The second implication of our findings is that E7 does not completely inactivate pRb in this mouse model (since, if E7 were to completely inactivate pRb, there would be no difference in the phenotype of K14E7 and K14E7K14CreRbf/f mice). This conclusion is in agreement with a previous study in which pRb-E2F complexes were detectable in a HPV-18-positive human cervical carcinoma cell line, suggesting that E7's inactivation of pRb is similarly incomplete in human cervical carcinogenesis (48). It is interesting, then, that the K14E7 mice display a phenotype similar to that of the K14CreRbf/f mice. Thus, in addition to partially inactivating pRb, E7 must be targeting other cellular factors, the consequence of which mimics the biological consequences that result from the complete inactivation of pRb. It is unclear which cellular targets of E7 besides pRb contribute to E7's phenotypes in vivo; however, as discussed below, p107 and p130 are the most suspect candidates.
What are the other
relevant cellular targets for E7?
E7 is known to bind to at least 18
different cellular proteins in addition to pRb
(30). The cellular
proteins most likely mediating E7's phenotypes in vivo in addition
to pRb are the other pocket proteins, p107 and p130, that are targeted
by E7. They, like pRb, bind to and modulate the function of overlapping
subsets of E2F family members, transcription factors known to regulate
expression of cell cycle regulatory factors and DNA synthesis machinery
(28). Interestingly, the
combined inactivation of p107 and pRb in the epidermis does lead to a
more hyperplastic mouse epidermis (J. Sage and T. Jacks, unpublished
data; A. Berns, unpublished data). Also, a mutant version of E7,
E7
DLYC, encoding a protein defective in binding all
three pocket proteins (pRb, p107, and 130) fails to confer any
phenotype when directed in its expression to stratified squamous
epithelia in mice (13).
Thus, p107 and/or p130 likely represent relevant targets for E7 in
vivo.
Another potentially important set of cellular factors are
the cdk inhibitors targeted by E7, p21 and p27, given their role in
cell cycle regulation. Further studies are necessary to discriminate
between relevant cellular factors, however, since mutations in E7 often
are not sufficiently informative. Particularly, the
E7DLYC mutant protein used in the above-cited study
(13) is not only
defective for binding the pocket proteins
(1,
32) but more recently has
been found to be defective for binding p21
(21). The experimental
mouse shall continue to provide us a unique basis for carrying out
these studies by providing the means to disrupt the expression or alter
the function of potentially relevant cellular targets in the context of
the natural host tissue.
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ACKNOWLEDGMENTS |
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This study was supported by grants CA22443 (P.F.L.), CA84227 (P.F.L.), and CA66980 (K.M.) from the National Institutes of Health. S.B. was supported by training grant T32 GM07215) and the American Cancer Society.
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FOOTNOTES |
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