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Cell Growth & Differentiation Vol. 10, 101-111, February 1999
© 1999 American Association for Cancer Research

Concordant Induction of Cyclin E and p21cip1 in Differentiated Keratinocytes by the Human Papillomavirus E7 Protein Inhibits Cellular and Viral DNA Synthesis1

Yichun Jian, Brian A. Van Tine, Wei-Ming Chien, George M. Shaw, Thomas R. Broker and Louise T. Chow2

Departments of Biochemistry and Molecular Genetics [Y. J., W-M. C., T. R. B., L. T. C.], Pathology [B. A. V. T.], and Medicine [B. A. V. T., G. M. S.], The Howard Hughes Medical Institute [G. M. S.], University of Alabama at Birmingham, Birmingham, Alabama 35294-0005


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Productive infections by human papillomaviruses (HPVs) occur only in differentiated keratinocytes in squamous epithelia in which the HPV E7 protein reactivates the host DNA replication machinery to support viral DNA replication. In a fraction of the differentiated keratinocytes, E7 also posttranscriptionally induces p21cip1, which is distributed in a mutually exclusive manner with unscheduled cellular DNA synthesis. In this study, double immunofluorescence labeling unexpectedly revealed that E7 caused a concordant accumulation of both cyclin E and p21cip1 to high levels in patient papillomas and in organotypic cultures of primary human keratinocytes. The induction of cyclin E is mutually exclusive with unscheduled cellular DNA synthesis or abundant viral DNA. These novel virus-host interactions in differentiated keratinocytes are in contrast to previous observations made in submerged proliferating cultures, in which HPV E7 induces cyclin E and overcomes p21cip1 inhibition of S-phase entry. We propose that an appropriately timed induction of cyclin E/cyclin-dependent kinase 2 by HPV E7 in postmitotic cells enables S-phase reentry and HPV DNA amplification, whereas prematurely induced cyclin E stabilizes p21cip1 protein, which then inhibits cyclin E/cyclin-dependent kinase 2. Consequently, cyclin E and p21cip1 both fail to turn over, and DNA synthesis does not occur.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The large family of HPVs3 is tropic for cutaneous and mucosal squamous epithelial cells. HPVs induce warty lesions in which viral mRNA transcription, DNA amplification, and progeny virion production are dependent on squamous differentiation (reviewed in Ref. 1 ). Because HPV replication requires host DNA replication machinery (2) , which is no longer present in postmitotic, differentiated cells (3 , 4) , the virus reactivates all of the cellular genes necessary to support DNA synthesis. Using organotypic (raft) cultures of PHKs grown at the media-air interface on a dermal equivalent raft, we have shown previously that HPV-18 E7 driven by the native differentiation-dependent viral enhancer-promoter accomplishes this task. We demonstrated an induction of unscheduled host DNA synthesis in postmitotic, differentiated keratinocytes in raft cultures (5 , 6) . HPV-18 and HPV-16 are considered to be high-risk viral types because infections can occasionally progress to cancers. Infections by low-risk types such as HPV-6 and HPV-11 virtually never progress to higher-grade lesions, yet unscheduled host DNA synthesis does take place in a fraction of the postmitotic, differentiated cells (5 , 6) . The distinct pathological properties of these HPV types are attributed to differences in the interactions between the viral E6 and E7 proteins and the tumor suppressor proteins, p53 and the retinoblastoma susceptibility protein (pRB) family of proteins, respectively. In vitro, viral oncoproteins E7 and E6 from high-risk HPVs can immortalize PHKs (see reviews in Refs. 1 and 7 ).

Cell cycle progression is controlled by cyclins and Cdks (8) . The unphosphorylated form of pRB binds to a family of E2F/DP transcription factor complexes and represses the E2F-responsive genes necessary for S-phase entry and progression (for reviews, see Refs. 9 , 10 ; Fig. 7). The D-type cyclins and Cdk4 and Cdk6 phosphorylate and inactivate pRB in response to mitogenic signals. Cyclin E/Cdk2 activity is present briefly in the late G1 and early S phases and is critical for S-phase entry. Transfection of a cyclin E expression vector is able to speed up entry into the S phase 11, 12, 13, 14, 15, 16 . In tumor cells in which cyclin D/Cdk4 or Cdk6 is functionally inactivated by the over-expression of inhibitor p16INK4a, over-expression of cyclin E/Cdk2 results in the phosphorylation of pRB (17) . Furthermore, ectopic expression of cyclin E/Cdk2 can drive cells into S phase, despite the presence of a mutated pRB that can no longer be phosphorylated (Refs. 16 , 18 and 19 ; reviewed in Ref. 20 ). Conversely, depletion of Cdk2 or addition of the p21cip1 protein inhibits chromosomal DNA replication in vitro (21, 22, 23, 24, 25, 26) . p21cip1 is a universal inhibitor of cyclin/Cdk, which sequesters cyclin E/Cdk2 into a quaternary complex along with PCNA, a processivity factor of DNA polymerase {delta} (8) . Thus, cyclin E is a limiting factor downstream of the pRB protein at the level of initiation of chromosomal DNA replication.

By binding and inactivating the unphosphorylated form of pRB, HPV-16 E7 and the adenovirus counterpart, the E1A protein, can reactivate DNA replication in differentiated cells (5 , 27) . Both viral oncoproteins activate cyclin E in proliferating cells in culture because the promoter of the cyclin E gene contains E2F binding sites (28, 29, 30, 31, 32, 33) . Moreover, a weak association between E7 and p21cip1 has been demonstrated in vitro, and high levels of E7 can overcome p21cip1 inhibition of cell-free replication from the SV40 origin (34 , 35) . These E7 functions are thought to play important roles in overcoming the inhibition of S-phase entry under conditions in which p21cip1 protein expression is highly induced in vitro and in the course of HPV-associated carcinogenesis in patient lesions (34, 35, 36, 37) .

We have recently reported that p21cip1 protein is induced posttranscriptionally in a fraction of the spinous cells in papillomas, condylomas, and low-grade cervical intraepithelial neoplasias caused by both the low-risk and the high-risk HPVs (38) . Using organotypic raft cultures of PHKs, we have recapitulated this induction by expressing HPV-18 E7 in postmitotic cells from the differentiation-dependent HPV-18 enhancer-promoter contained in the URR (6) . In that study, double IF studies of both papillomas and epithelial raft cultures have revealed that p21cip1 protein induction occurred in a fraction of differentiated PHKs in which PCNA was reactivated by E7. Furthermore, unscheduled host DNA synthesis and p21cip1 protein induction took place in separate populations of PCNA-positive spinous cells, suggesting that p21cip1 protein inhibited host DNA synthesis. Regardless of the HPV type present in benign clinical specimens, there is an inverse relationship between p21cip1 protein induction and the abundance of HPV DNA and RNA in serial sections of patient specimens (38) . We infer from these observations that p21cip1 protein also inhibits viral DNA amplification. Until now, there has been no data to substantiate this hypothesis. The mechanism of this posttranscriptional induction of p21cip1 protein is not known either.

In the present study, we asked the following questions. Is cyclin E induced in benign warts? If so, where is it induced in the stratified epithelium? Is E7 responsible for such an induction? Does unscheduled cellular DNA synthesis occur in differentiated cells in which cyclin E is induced? Does viral DNA amplification have the same relationship to cyclin E and p21cip1 induction as the unscheduled cellular DNA synthesis? We have found completely concordant results in benign patient specimens and in PHK raft cultures transduced with HPV-18 URR E7. We present evidence that cyclin E was below the threshold of detection by in situ methods in proliferating basal/parabasal cells, regardless of the presence or absence of HPVs, but it was highly induced in a fraction of the differentiated keratinocytes in the presence of E7. However, contrary to the expectation from observations made in proliferating cells in submerged cultures, both cyclin E and p21cip1 were invariably induced in the same nuclei in a subpopulation of PCNA-positive, differentiated cells. Furthermore, the induction of cyclin E protein occurred in different cells from those in which unscheduled cellular DNA synthesis or viral DNA amplification took place. Interpretations and implications regarding the control of cellular DNA replication will be presented.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Induction of Cyclin E in Differentiated Keratinocytes in Laryngeal Papillomas and in Organotypic Raft Cultures Transduced by HPV-18 URR E7.
Sections (4 µm) of formalin-fixed, paraffin-embedded laryngeal papillomas infected with low-risk HPV-11 or HPV-6 were examined by immunohistochemistry for the presence of cyclin E. It was detected in a fraction of the spinous cells, but not in the proliferating basal and parabasal cells (data not shown; see Citation Figs. 2–5Citation ). Citation Citation Citation To ascertain whether cyclin E induction in spinous cells is attributable to HPV E7, as reported for proliferating cells in cultures, we examined cyclin E expression in raft cultures of PHKs transduced with the recombinant retroviruses described previously (5) . These vectors express HPV-18 E7, E6, or both, each under the control of the 1.1-kb differentiation-dependent HPV-18 URR E6 promoter (5 , 39) . The raft cultures were well differentiated, consisting of a proliferating basal layer, several layers each of differentiated spinous and granular cells, and multiple superficial layers of squames (Fig. 1, a and b)Citation . Differentiation stage-specific keratins and profilaggrin were appropriately expressed (data not shown; Ref. 5 ).



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Fig. 1. Detection of cyclin E protein in epithelial raft cultures and foreskin by immunohistochemistry. The paraffin-embedded sections of epithelial raft cultures (a and b) or foreskin (c) were reacted with anti-cyclin E monoclonal antibody after antigen retrieval. PHK rafts were infected with recombinant retroviruses containing (a) HPV-18 URR E7 or (b) HPV-18 URR E6, respectively. Arrowheads point to the basal layer.

 


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Fig. 2. Cyclin E protein was induced in a fraction of PCNA-positive postmitotic cells in laryngeal papillomas and in E7-expressing epithelial raft cultures. Double IF staining was performed as described in "Materials and Methods." Each section was photographed with a Texas red filter to detect cyclin E protein (left column) and with a fluorescein filter (green) to detect PCNA (right column) and merged electronically (middle column) to detect both. a–c, an HPV-11-infected laryngeal papilloma. d–f, PHK raft cultures transduced with HPV-18 URR E6-E7; g–i, uninfected PHK raft cultures; j–l, PHK raft cultures transduced with HPV-18 URR E7 and stained in parallel, except that the anti-PCNA antibody was omitted. A negative control for cyclin E is shown in Fig. 4Citation . Arrowheads point to the basal layer.

 


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Fig. 3. Cyclin E protein induction and host DNA replication occurred in separate cells in laryngeal papillomas and in epithelial raft cultures. Exposure of raft cultures (30 h) and laryngeal papillomas to BrdUrd was described in "Materials and Methods." Double IF staining was performed using antibodies against cyclin E, as revealed by Texas red (left column), or against BrdUrd, as revealed by fluorescein (right column). Individual images were merged electronically to show both signals (middle column). a–c, an HPV-11-infected laryngeal papilloma. d–f, PHK raft cultures transduced with HPV-18 URR E6-E7; g–i, uninfected PHK raft cultures; j–l, PHK raft cultures transduced with HPV-18 URR E7 but not exposed to BrdUrd. Arrowheads point to the basal layer.

 


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Fig. 4. Coinduction of cyclin E and p21cip1 in laryngeal papillomas and in E7-expressing epithelial raft cultures. Double IF staining was performed as described in "Materials and Methods." Each section was photographed with a Texas red filter to detect p21cip1 protein (left column) and with a fluorescein filter to detect cyclin E (right column) and merged electronically (middle column) to detect both. a–c, an HPV-11-infected laryngeal papilloma. d–f, PHK raft cultures transduced with HPV-18 URR E6-E7; g–i, PHK raft cultures transduced with HPV-18 URR E6*-E7 and stained in parallel, except that the anti-cyclin E antibody was omitted; j–l, PHK raft cultures transduced with HPV-18 URR E6*-E7 and stained in parallel, except that the anti-p21 antibody was omitted. Arrowheads point to the basal layer.

 


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Fig. 5. Super-induction of cyclin E and p21cip1 proteins is mutually exclusive with HPV DNA amplification in a vaginal condyloma (A–D) and laryngeal papillomas (E–H), all of which were caused by HPV-11. All sections were stained with 4`,6-diamidino-2-phenylindole to reveal nuclei. For A–F, the sections were treated with RNAase before in situ hybridization to reveal HPV DNA amplification shown by fluorescein (green) that saturates to yellow when the signals are very high. Cyanine-3 (orange) revealed cyclin E (C and D) or p21cip1 (A, B, E, and F). Images A–F were captured directly with a digital color camera using a triple-pass filter. In G and H, the sections were not pretreated with RNAase; viral nucleic acids were revealed by cyanine-3 (orange), and cyclin E was revealed by fluorescein (green). Images G and H were captured with a digital camera with single-pass filters and merged electronically. There was essentially no white nucleus to indicate colocalization of all three colored probes.

 
Cyclin E was consistently induced in a fraction of the differentiated keratinocytes transduced with an intact HPV-18 E7 gene (the genomic E6-E7, E7 only, or the spliced E6*-E7 cDNA) (Fig. 1aCitation ; data not shown; see Figs. 2Citation , 3Citation and 4Citation ). It was not detected in native foreskin (Fig. 1c)Citation , in raft cultures transduced with the HPV-18 E6 gene or with the empty vector, nor in the uninfected raft cultures (Fig. 2bCitation ; data not shown; but see Figs. 2Citation and 4Citation ). Thus, HPV-18 E7 was responsible for the induction of cyclin E. HPV-18 E6*-E7(T), which expresses an E7 protein truncated of the COOH-terminal zinc binding domain, also induced cyclin E, but the signal was extremely weak (data not shown). We conclude that cyclin E is normally below the sensitivity of this detection method in cycling cells, but it is induced to a high level by HPV infection, specifically by the E7 gene product, but only in differentiated cells.

Double IF Studies in Papillomas and in Raft Cultures.
To determine the possible relationships between cyclin E induction and unscheduled DNA synthesis in the differentiated keratinocytes, we performed double IF studies. We localized nuclei in which cyclin E, PCNA, or p21cip1 was induced by antibody reactivity and nuclei in which unscheduled cellular DNA synthesis took place by monitoring antibody reactivity to BrdUrd incorporated into chromosomal DNA (Figs. 2Citation 3Citation 4)Citation . These investigations were conducted in parallel with surgically removed HPV-infected laryngeal papillomas and with the HPV-18 E7-expressing raft cultures just described. Digitally captured microscope images are shown for individual antigens (left and right columns) and electronically merged (center columns) to reveal their relative distributions (Fig. 2Citation 3Citation 4)Citation . All signals were strictly nuclear. Uninfected PHK raft cultures were processed in parallel, and the absence of signals in the differentiated cells ruled out nonspecific reactivity of antibodies and reagents. In addition, we also assessed HPV DNA abundance in benign patient lesions by in situ hybridization and determined its relationship with p21cip1 and cyclin E induction (Fig. 5)Citation . The results are presented below.

Cyclin E Induction and Unscheduled Host DNA Synthesis Are Mutually Exclusive in Differentiated Cells in Vivo and in Vitro.
Texas red was used to reveal cyclin E, and fluorescein marked PCNA. In agreement with the immunohistochemical results, we detected cyclin E induction only in the differentiated cells in laryngeal papillomas (Fig. 2a)Citation . Notably, the induction of cyclin E occurred in only a fraction of the PCNA-positive, spinous cells (Fig. 2, b and c)Citation . Similar results were obtained with HPV-18 E7-expressing raft cultures (Fig. 2, d–f)Citation . Again, cyclin E was not detected in the proliferating basal and parabasal cells in any of the sections. There was only a background signal when primary antibodies were omitted or when uninfected PHK raft cultures were examined (Fig. 2, g–lCitation ; data not shown).

Only some of the PCNA-expressing differentiated keratinocytes in papillomas and in E7-transduced raft cultures were able to support unscheduled cellular DNA synthesis as revealed by [3H]thymidine or BrdUrd incorporation (5 , 6) . To determine whether these BrdUrd-positive cells contained high levels of cyclin E and whether BrdUrd-positive cells were cycling cells that never left the cell cycle, the HPV-18 E7-expressing raft cultures were exposed to BrdUrd for 18 or 30 h before formalin fixation. We reasoned that, over an 18-h exposure, we might not be able to detect cells positive for both BrdUrd and cyclin E, had the cells been cycling. This is because cells positive for cyclin E should be in the late G1 and early S phases, and there might not be sufficient time to incorporate BrdUrd to a detectable level. However, the 30-h exposure to BrdUrd, which is much longer than one full cell cycle, should allow us to detect cells that were in late G1 or early S phase and were thus high in cyclin E but had incorporated BrdUrd in the previous S phase. Thus, the distribution patterns of cyclin E and BrdUrd should be different at these two time points, if the cells had been cycling. Laryngeal papillomas were exposed to BrdUrd for 8 h, the maximal duration before extensive tissue disintegration occurred.

Texas red reveals cyclin E signals, and fluorescein signifies the BrdUrd incorporation. As reported previously, BrdUrd incorporation was observed in many of the proliferating basal and parabasal cells. It was also detected in some of the differentiated spinous cells of laryngeal papillomas (Fig. 3c)Citation and of E7-expressing raft cultures (Fig. 3fCitation ; data not shown). The BrdUrd signals were specific because they were absent in the HPV-18 E7-transduced raft cultures that were not incubated with BrdUrd (Fig. 3, k and l)Citation . Contrary to our expectation, a great majority of the cyclin E-positive spinous cells did not synthesize DNA; conversely, very few spinous cells that incorporated BrdUrd expressed a detectable level of cyclin E, regardless of the duration of BrdUrd exposure (Fig. 3, a, b, d, and eCitation ; data not shown). As just described, cyclin E was not observed in basal and parabasal cells, regardless of whether these cells were positive for BrdUrd.

In uninfected PHK raft cultures, BrdUrd was detected primarily in basal and a few parabasal cycling cells in cultures exposed to BrdUrd for 18 h (Ref. 6 ; data not shown). Over the course of a 30-h exposure, most of the basal and parabasal cells of uninfected PHKs replicated once or twice. Together with upward cell migration, there were two, three, and occasionally four layers of BrdUrd-positive nuclei in the lower strata (Fig. 3i)Citation . At the time of tissue fixation, some of these basal and parabasal cells should have been in late G1 or early S phase and thus should have been positive for both BrdUrd and cyclin E. However, cyclin E was not observed in any of the strata.

Collectively, these data demonstrate that: (a) normal proliferating basal cells in uninfected raft cultures and basal or suprabasal keratinocytes transduced with HPV-18 URR E7 cells in which cellular DNA synthesis took place had a level of cyclin E below the threshold of detection; (b) the inability to detect cyclin E in BrdUrd-positive nuclei cannot be explained by chance tissue fixation at inappropriate phases of the cell cycle; (c) the suprabasal cells that incorporated BrdUrd were not cycling cells that never left the cell cycle; rather, they were differentiated cells that were stimulated by E7 to reenter S phase; and (d) super-induction of cyclin E by E7 in differentiated keratinocytes was not able to promote unscheduled cellular DNA synthesis, as originally anticipated.

Cyclin E Colocalizes with p21cip1 in Differentiated Cells in Vivo and in Vitro.
The mutually exclusive patterns of cyclin E induction and BrdUrd incorporation in differentiated cells are reminiscent of the relationship between the E7-induced p21cip1 protein and unscheduled host DNA synthesis (6) . Furthermore, because p21cip1-positive cells do not synthesize cellular DNA, the observations above suggest that cyclin E might be present in the same cells as the p21cip1 protein but was not able to overcome the inhibitory effect of p21cip1 on S-phase entry. We tested this hypothesis by double IF staining for cyclin E and p21cip1 proteins. This was indeed the case in laryngeal papillomas (Fig. 4, a–c)Citation and in HPV-18 E7-transduced PHK raft cultures (Fig. 4, d–f)Citation . Fluorescein reveals the nuclei containing elevated cyclin E antigen, and Texas red denotes those positive for p21cip1. The two antigens colocalized precisely in the differentiated cells. The omission of primary antibodies in the staining process with the HPV-18 E7-transduced raft cultures revealed no specific signals, only a low background fluorescence (Fig. 4, g–l)Citation .

Super-Induction of Cyclin E and p21cip1 in Spinous Cells Is Mutually Exclusive with HPV DNA Amplification in Benign Papillomas and a Condyloma.
Because the HPV genome is 1.3 x 10-6 the size of the human diploid genome, most if not all of the BrdUrd incorporation detected by the anti-BrdUrd antibody in patient specimens was likely to represent cellular DNA synthesis (6) . To investigate whether HPV DNA amplification was also regulated by the same mechanism and was thus inhibited by the super-induction of cyclin E and p21cip1, we examined the relative distribution of p21cip1 protein and cyclin E and the abundance of HPV DNA in several laryngeal papillomas and in a vaginal condyloma.

p21cip1 antibody reactivity is represented by cyanine-3 tyramide (orange), HPV DNA is detected by in situ hybridization with HPV DNA probes and revealed with fluorescein after tyramide signal enhancement, and nuclei are stained with 4',6-diamidino-2-phenylindole (Fig. 5, A, B, E, and F)Citation . In cells in which high levels of p21cip1 accumulated, little or no HPV DNA was detected. Conversely, p21cip1 protein was not detected in cells that had a high copy number of HPV DNA. Analogous double IF probing revealed that high levels of cyclin E (cyanine-3) and abundant HPV DNA (fluorescein) were also mutually exclusive (Fig. 5, C and D)Citation . When the tissue sections were not pretreated with RNAase, the double-stranded DNA probe hybridized to viral DNA and possibly viral mRNA (cyanine-3). Cytoplasmic RNA was clearly lost after antibody staining. The mutually exclusive pattern of cyclin E induction (fluorescein) and high levels of viral nucleic acids (cyanine-3) remained (Fig. 5, G and H)Citation . Because the two fluorophores used in Fig. 6, G and HCitation , were reversed from those in Fig. 6, C and DCitation , these results also demonstrated that the outcome of the experiment is independent of the signal strengths of the fluorophores. We thus conclude that super-induction of cyclin E did not overcome the inhibitory effects of p21cip1 on viral DNA amplification. At any one epithelial stratum, the distribution of these three subpopulations of cells appeared to be stochastic in the several patient specimens examined, but nuclei containing high copies of viral DNA were often located more superficially than those containing cyclin E and p21cip1 protein, consistent with a previous report (38) . This spatial distribution of cells containing abundant viral DNA also agrees with numerous reports in which viral DNA was detected by in situ hybridization using conventional radiolabeled riboprobes or nonisotopic detection methods (reviewed in Ref. 1 ).



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Fig. 6. A model for the relationship between cyclin E and p21cip1 induction and cellular and viral DNA replication in postmitotic, differentiated keratinocytes in benign lesions and in E7-expressing epithelial raft cultures. Left side, the prior induction of all replication genes followed by cyclin E/Cdk2 allows the cells to initiate DNA synthesis, leading to cyclin E degradation and no costabilization with p21cip1 protein. Right side, an untimely induction of cyclin E/Cdk2 can inhibit DNA replication either by premature phosphorylation of the initiation complex or via sequestration of cyclin E/Cdk2 into an inactive complex with p21cip1 along with PCNA. The net result is a concordant accumulation of both cyclin E and p21cip1 proteins in which neither unscheduled cellular DNA nor viral DNA amplification can take place.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The interactions between viral oncoproteins and host tumor suppressor proteins have traditionally been examined in cycling cells. These investigations have yielded critical information on cell cycle regulation and mechanisms of cell transformation. We have taken a distinctly different approach and examined the virus-host interactions in the natural setting of productively infected patient specimens and in an experimental model system that closely simulates such infected tissues. These studies revealed unexpected virus-host interactions and produced new insights into the relationships between cell cycle-regulatory proteins and S-phase entry.

Using immunohistochemical staining and IF detection, we demonstrated that cyclin E, which is critical for S-phase entry, accumulated to high levels in differentiated keratinocytes in papillomas and condyloma caused by HPV-6 or HPV-11. In contrast, there was no detectable cyclin E in the proliferating basal and parabasal cells, regardless of HPV infection. We have recapitulated this pattern of induction in PHK raft cultures transduced by HPV-18 URR E7 (Figs. 1Citation 2Citation 3Citation 4)Citation . In vivo and in vitro, cyclin E accumulation was only observed in a fraction of the postmitotic cells that were positive for PCNA (Fig. 2)Citation , the induction of which is also dependent on E7 (5) . The CR1 and CR2 of E7 are sufficient for this induction because a truncated E7 mutation of the COOH-terminal CR3 zinc-binding domain was able to induce cyclin E, albeit much less effectively (data not shown), in agreement with a previous report (30) .

Our double IF studies clearly demonstrated that the levels of cyclin E (Fig. 3)Citation and p21cip1 proteins (6) were below the sensitivity of detection in patient specimens and in HPV-18 E7-expressing epithelial raft cultures in cells in which cellular DNA synthesis took place, regardless of their location in the stratified epithelium. In contrast, cyclin E invariably accumulated simultaneously with high levels of the p21cip1 protein in a fraction of PCNA-positive, postmitotic differentiated keratinocytes in which unscheduled cellular DNA synthesis did not occur (Figs. 2Citation 3Citation 4)Citation . These results demonstrated that, in the great majority of the differentiated keratinocytes, neither the expression of E7 nor a high level of cyclin E was able to overcome the inhibitory effect of the p21cip1 protein, contrary to previous observations in cycling cells and in a cell-free replication system (34, 35, 36, 37) . One key difference between these two systems is that, in cycling cells, p21cip1 protein expression is regulated at the transcription level by p53 (40) , whereas, in the differentiated keratinocytes, its transcription regulation is independent of p53, and protein accumulation is mediated by posttranscriptional mechanisms (6 , 38) .

We have demonstrated that super-induction of cyclin E or p21cip1 was also incompatible with HPV-11 viral DNA amplification (Fig. 5)Citation . Because viral DNA abundance is independent of the phase of the cell cycle at the moment of tissue fixation, these results support our previous interpretation that p21cip1 induction inhibited cellular DNA synthesis (6) . Thus, both viral and cellular DNA synthesis respond comparably to regulations by cyclin E and p21cip1. We note that a fraction of the spinous cells that did not contain high levels of cyclin E or p21cip1 did not amplify viral DNA either. Evidently, in these cells, other conditions for viral DNA amplification were not met.

In support of the notion that cyclin E also regulates papillomavirus DNA replication, a recent report showed that DNA replication initiator protein E1 of bovine papillomavirus type 1 associated with Xenopus cyclin E and was a substrate of cyclin E/Cdk2 in vitro. This interaction enhanced bovine papillomavirus type 1 origin plasmid replication in Xenopus egg extracts (41) . HPV E1 proteins also directly associated with several human cyclins, including cyclins A, B, E, and F. In particular, HPV-11 E1 is a substrate of Cdk2, and mutations in HPV-11 E1 that reduced or abolished phosphorylation by Cdk2 significantly impaired HPV origin replication in transfected cells and in a cell-free system (42) .

What might be the mechanisms that lead to the mutually exclusive patterns of unscheduled cellular DNA synthesis and viral DNA amplification and the concordant super-induction of cyclin E and p21cip1 proteins in postmitotic cells, despite the critical roles played by cyclin E/Cdk2 in both cellular and viral DNA synthesis? One possibility is that E7 might interfere with the ubiquitin pathways that normally mediate the turnover of cyclin E and p21cip1 by analogy to the ability of adenovirus E1A to stabilize E2F factors (43 , 44) ; this could then lead to an inability to enter into S phase when cyclin E/Cdk2 was inhibited by p21cip1. This mechanism may indeed contribute to the stability of these two proteins, but it cannot explain why they did not accumulate in all cells in which E7 was expressed, as inferred by PCNA-positive suprabasal cells. We prefer another interpretation.

We propose that the p21cip1 protein is normally short-lived in differentiated cells in squamous epithelium and has thus escaped detection by in situ methods, but it is stabilized by forming a complex containing cyclin E when cyclin E is induced in an untimely fashion in differentiated cells. We also suggest that the high levels of cyclin E result from both transcription activation by E7 mediated via E2F, as reported previously, and by costabilization with the p21cip1 protein. We believe that, in differentiated cells, the induction by E7 of the S-phase genes such as cyclin E/Cdk2, DNA replication enzymes, and enzymes that synthesize deoxyribonucleoside triphosphates is stochastic rather than orderly (as it is in normal cycling cells). This randomness then sets up two possible outcomes, based on current knowledge concerning G1-S progression and the data presented in this investigation.

If cyclin E/Cdk2 complex is induced before one or more of the other host genes necessary to support DNA replication, DNA synthesis cannot occur (Fig. 6Citation , right side). Prematurely induced cyclin E/Cdk2 would then sequester the short-lived p21cip1 protein into a stable quaternary complex with PCNA (8) because p21cip1 protein can simultaneously interact with cyclin E (45 , 46) , Cdk2, and PCNA through different domains (47, 48, 49) . p21cip1, in turn, would inhibit cyclin E/Cdk2. Inhibition of cylin E/Cdk2 prevents not only the initiation of DNA synthesis (22 , 23) but also the autophosphorylation of cyclin E. Cyclin E phosphorylation is required for its degradation via the ubiquitin-mediated pathway (50 , 51) , leading to the costabilization of both cyclin E and p21cip1 (Fig. 6Citation , right side). Once this occurs, the initiation of DNA synthesis might remain inhibited even when the missing replication components are eventually activated by E7, as suggested by the inability of cyclin E/ p21cip1/PCNA-positive nuclei to incorporate BrdUrd even after a 30-h exposure to this nucleoside analogue (Fig. 4)Citation . Alternatively, prematurely induced cyclin E/Cdk2 might inhibit the assembly of the preinitiation complex on the origin and thus block chromosomal DNA replication, as reported for the DNA replication system in Xenopus egg extracts (52) . When DNA replication fails to occur, cyclin E might not turn over and would then sequester p21cip1 protein into stable complexes. Either scenario would result in the concordant accumulation of these two proteins in PCNA-positive postmitotic cells (Figs. 2Citation and 4)Citation in which DNA synthesis did not occur (Figs. 4Citation and 6)Citation . This model also explains the posttranscriptional induction of p21cip1 protein 6 , 38 . No Cdk2 antibody suitable for paraffin-embedded tissues is currently available for further confirmation. However, we note that elevated levels of cyclin E and p21cip1 proteins were previously detected in cultured cells arrested by irradiation (53) .

In contrast, a properly timed induction of cyclin E by HPV E7 in postmitotic cells would lead to S-phase entry and permit unscheduled cellular synthesis and viral DNA amplification to occur. S-phase entry is followed by prompt cyclin E autophosphorylation by Cdk2, triggering its degradation and resulting in no accumulation of either cyclin E or p21cip1 protein (Fig. 6Citation , left side), events that normally occur in cycling cells. Thus, we suggest that the state of replication readiness is a critical condition that normal cycling cells must meet to pass through the restriction point before the activation of cyclin E/Cdk2 for S-phase entry.

In summary, our studies of HPVs in squamous epithelia have provided the first example in which high levels of endogenous p21cip1 protein induced by conditions other than DNA damage and p53 up-regulation are incompatible with DNA synthesis in tissues (6 , 38) . We have also demonstrated for the first time in patient specimens and in an epithelial model system that high levels of cyclin E fail to overcome p21cip1 and to promote S-phase entry. Rather, cyclin E may have contributed to the posttranscriptional induction of p21cip1 by protein costabilization. Thus, in these differentiated keratinocytes, cyclin E/Cdk2 can be a double-edged sword in regulating the initiation of DNA synthesis. This property of cyclin E/Cdk2 is also likely to be true for cycling cells because it would then fit or explain the timing when cyclin E is normally synthesized late in G1 after the restriction point to activate the catalytic subunit Cdk2, whereas D-type cyclins/Cdk4 or Cdk6 are activated before the restriction point to inactivate pRB. Our experiments and interpretation also provide for the first time a molecular explanation to the long-standing observation that seemingly identical, differentiated keratinocytes are heterogeneous in their ability to support viral DNA amplification and high levels of viral mRNA transcription (1) . These novel virus-host interactions could not have been predicted from experiments performed in proliferating cells in submerged cultures and further demonstrate the importance of the examination of native tissue specimens and in vitro model systems that faithfully simulate the native tissues in vivo. We also note that this premature induction of cyclin E/p21cip1 fortuitously becomes a host defense against viral propagation. It will now be most interesting to investigate whether it is possible to suppress HPV productive infections further by manipulating the timing or extent of p21cip1 or cyclin E/Cdk2 induction in the differentiated keratinocytes.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Tissue Specimens.
Neonatal foreskins were collected from Cooper Green Hospital (Birmingham, AL). Juvenile laryngeal papillomas were collected from The Children’s Hospital of Alabama (Birmingham, AL) as part of routine therapies. Some tissues were incubated with 50 µg/ml BrdUrd in DMEM with 10% fetal bovine serum for 8 h before being fixed in 10% buffered formalin (Sigma) and embedded in paraffin. Additional specimens were fixed and paraffin-embedded directly. A vaginal condyloma was obtained through University of Alabama at Birmingham Comprehensive Cancer Center Tissue Procurement Service. The HPV types in the patient specimens were determined previously by in situ hybridization with whole genomic HPV antisense riboprobes to detect total viral transcripts.

Retrovirus-mediated Gene Transfer.
Moloney murine leukemia virus-based retroviral pLJd vectors containing either genomic E6, E7, E6-E7, the spliced E6*-E7 cDNA, or a cDNA with a truncated E7 have been described previously (5) . Briefly, all vectors express the bacterial neomycin resistance gene from the SV40 promoter. The expression of the HPV-18 gene product is under the control of the 1.1-kb HPV-18 URR enhancer-E6 promoter. Recombinant retroviruses were produced as described previously. High-titer amphotropic viruses were then used to infect early-passage PHKs recovered from neonatal foreskin. After a 2-day selection with 400 µmg/ml G418, the majority of the cells survived, whereas all uninfected cells died. The selected cells were used immediately without further expansion for developing raft cultures.

Epithelial Raft Cultures.
PHKs recovered from neonatal foreskins were grown in serum-free medium (Life Technologies, Inc.) as described previously (54) . Passage 2 cells were used to develop epithelial raft cultures as described previously (5) . After 9 days at the liquid:air interface, the cultures were fixed in 10% buffered formalin, paraffin-embedded, and cut into 4-µm sections. BrdUrd (50 µg/ml) was added to some raft cultures for 18 or 30 h before harvesting on day 9.

Antigen Detection and in Situ Hybridization.
Immunohistochemical staining was carried out with the anti-cyclin E monoclonal antibody (Novocastra Laboratories) at a dilution of 1:50 using a modification of antigen retrieval (5) . 3-Amino-9-ethylcarbazole was used as the chromogen, and the slides were counterstained lightly with hematoxylin. Images were captured with a digital SPOTTM (Diagnostic Instruments) camera on an Olympus BH2 microscope. For double IF staining of cyclin E and PCNA or BrdUrd, cyclin E antibody reactivity was revealed by biotinylated secondary antibodies and Texas red-labeled streptavidin (Vector Laboratories). The sections were then probed with anti-PCNA antibody (DAKO), goat antimouse IgG2a, and then with fluorescein-antigoat IgG or fluorescein-labeled anti-BrdUrd monoclonal antibody (Boehringer Mannheim). For double IF staining of cyclin E and p21cip1, the sections were first probed with anti-p21cip1 antibody (Oncogene), biotinylated anti-mouse IgG, and then streptavidin-conjugated Texas red. The sections were then reprobed with anti-cyclin E antibody, goat anti-mouse IgG2a, and then fluorescein anti-goat IgG. The images were individually captured using an Olympus IX70 inverted fluorescence microscope equipped with a Photometrics Sensys Digital Camera and a Ludl filter wheel and processed by an IP Lab Spectrum image program.

For the double IF studies presented in Fig. 5, A–FCitation , the sections were first stained with anti-p21 or anti-cyclin E as described, except that horseradish peroxidase was used to label the anti-mouse secondary IgG antibody, and cyanine-3 tyramide was used as the substrate. The sections were treated with RNase for 1 h at 37°C and with proteinase K for 30 min before hybridization with DNA probes. HPV DNA probes were prepared by nick translation in the presence of biotin-N6-dUTP (New England Nuclear Life Sciences Products), precipitated after the addition of cot-1 DNA and salmon sperm DNA, and resuspended in Hybrisol VII (Oncor). Sections were denatured in 50% formamide and 2x SSC at 72°C. After an overnight hybridization at 37°C with the probe and extensive washes, fluorescein tyramide (NEN Life Sciences Products) was added to detect the biotinylated DNA probes. IF images were captured with a CoolCam digital camera on a Nikon E-800 microscope using a triple-pass filter. For Fig. 5, G and HCitation , the fluorescence labels were reversed between nucleic acids and cyclin E, and images were captured with single-pass filters and then merged electronically.


    Acknowledgments
 
We appreciate the help of the Digital Imaging Facility managers Albert Tousson and Shawn Williams. We thank Brian J. Wiatrak, M.D. (Children’s Hospital of Alabama) for laryngeal papillomas specimens, and the nurses of The Cooper Green Hospital for collecting foreskins. We thank Ge Jin for tissue embedding and sectioning and Yafen Niu of the University of Alabama at Birmingham Center for AIDS Research for helping with in situ hybridization.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by USPHS Grants CA36200 and DE/CA11910. Y. J. is a recipient of USPHS Training Grant T32 CA09467 in Molecular and Viral Oncology. Partial support for the Digital Imaging Microscopy Facility was provided by a grant from the University of Alabama at Birmingham Health Services Foundation (to T. R. B.) and USPHS P30 AI27767 (to Eric Hunter). Back

2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Genetics, 1918 University Boulevard, McCallum Basic Science Research Building, Room 510, University of Alabama at Birmingham, Birmingham, AL 35294-0005. Phone: (205) 975-8300; Fax: (205) 975-6075; E-mail: ltchow{at}uab.edu Back

3 The abbreviations used are: HPV, human papillomavirus; Cdk, cyclin-dependent kinase; PHK, primary human keratinocyte; PCNA, proliferating cell nuclear antigen; URR, upstream regulatory region; IF, immunofluorescence; BrdUrd, bromodeoxyuridine; CR, conserved region. Back

Received for publication 11/20/98. Revision received 1/ 6/99. Accepted for publication 1/ 6/99.


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