p21cip1 Degradation in Differentiated Keratinocytes Is Abrogated by Costabilization with Cyclin E Induced by Human Papillomavirus E7

ABSTRACT The human papillomavirus (HPV) E7 protein promotes S-phase reentry in a fraction of postmitotic, differentiated keratinocytes. Here we report that these cells contain an inherent mechanism that opposes E7-induced DNA replication. In organotypic raft cultures of primary human keratinocytes, neither cyclin E nor p21cip1 is detectable in situ. However, E7-transduced differentiated cells not in S phase accumulate abundant cyclin E and p21cip1. We show that normally p21cip1 protein is rapidly degraded by proteasomes. In the presence of E7 or E6/E7, p21cip1, cyclin E, and cyclin E2 proteins were all up-regulated. The accumulation of p21cip1 protein is a posttranscriptional event, and ectopic cyclin E expression was sufficient to trigger it. In constract, cdk2 and p27kip1 were abundant in normal differentiated cells and were not significantly affected by E7. Cyclin E, cdk2, and p21cip1 or p27kip1 formed complexes, and relatively little kinase activity was found associated with cyclin E or cdk2. In patient papillomas and E7 raft cultures, all p27kip1-positive cells were negative for bromodeoxyuridine (BrdU) incorporation, but only some also contained cyclin E and p21cip1. In contrast, all cyclin E-positive cells also contained p27kip1. When the expression of p21cip1 was reduced by rottlerin, a PKC δ inhibitor, p27kip1- and BrdU-positive cells remained unchanged. These observations show that high levels of endogenous p27kip1 can prevent E7-induced S-phase reentry. This inhibition then leads to the stabilization of cyclin E and p21cip1. Since efficient initiation of viral DNA replication requires cyclin E and cdk2, its inhibition accounts for heterogeneous viral activities in productively infected lesions.

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Alternative Fates of Keratinocytes Transduced by Human Papillomavirus Type 18 E7 during Squamous Differentiation

Journal of Virology ◽

10.1128/jvi.76.6.2964-2972.2002 ◽

2002 ◽

Vol 76 (6) ◽

pp. 2964-2972 ◽

Cited By ~ 19

Author(s):

Wei-Ming Chien ◽

Francisco Noya ◽

Heather M. Benedict-Hamilton ◽

Thomas R. Broker ◽

Louise T. Chow

Keyword(s):

Human Papillomavirus ◽

Cyclin E ◽

Dna Amplification ◽

Human Keratinocytes ◽

S Phase ◽

Primary Human Keratinocytes ◽

P27kip1 Protein ◽

Differentiated Cells ◽

Hpv 18

ABSTRACT The human papillomavirus type 18 (HPV-18) E7 protein promotes S-phase reentry in postmitotic, differentiated keratinocytes in squamous epithelium to facilitate vegetative viral DNA amplification. To examine the nature and fate of the differentiated cells that reenter S phase, organotypic cultures of primary human keratinocytes transduced with HPV-18 E7 were pulse-chase-pulse-labeled with 3H-thymidine (3H-TdR) and bromodeoxyuridine (BrdU). The kinetics of the appearance of doubly labeled suprabasal cells demonstrate that E7 expression did not promote prolonged S phase. Rather, there was a considerable lag before a small percentage of the cells reentered another round of S phase. Fluorescence in situ hybridization analysis, indeed, revealed a small fraction of the cells with more than 4n chromosomes in the differentiated strata. Differentiated cells positive for 3H-TdR, BrdU, or both often had enlarged nuclei or were binucleated. These results suggest that S phase is not followed by cell division, although nuclear division may occur. Interestingly, a significant fraction of differentiated cells that entered S phase subsequently accumulated p27kip1 protein with a kinetics preceding the accumulation of cyclin E. We conclude that E7-transduced, differentiated keratinocytes that enter S phase have two alternative fates: (i) a low percentage of cells undergoes endoreduplication, achieving higher than 4n ploidy, and (ii) a high percentage of cells accumulates the p27kip1, cyclin E, and p21cip1 proteins, resulting in arrest and preventing further S-phase reentry.

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Mutations of the Drosophila dDP,dE2F, and cyclin E Genes Reveal Distinct Roles for the E2F-DP Transcription Factor and Cyclin E during the G1-S Transition

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.141 ◽

1998 ◽

Vol 18 (1) ◽

pp. 141-151 ◽

Cited By ~ 52

Author(s):

Robert J. Duronio ◽

Peter C. Bonnette ◽

Patrick H. O’Farrell

Keyword(s):

Dna Replication ◽

Transcriptional Activation ◽

Cyclin E ◽

S Phase ◽

Brdu Incorporation ◽

Gene Products ◽

Hypomorphic Allele ◽

Transition Mutation ◽

Replication Factors ◽

Phase Entry

ABSTRACT Activation of heterodimeric E2F-DP transcription factors can drive the G1-S transition. Mutation of the Drosophila melanogaster dE2F gene eliminates transcriptional activation of several replication factors at the G1-S transition and compromises DNA replication. Here we describe a mutation in theDrosophila dDP gene. As expected for a defect in the dE2F partner, this mutation blocks G1-S transcription ofDmRNR2 and cyclin E as previously described for mutations of dE2F. Mutation of dDP also causes an incomplete block of DNA replication. When S phase is compromised by reducing the activity of dE2F-dDP by either a dE2F ordDP mutation, the first phenotype detected is a reduction in the intensity of BrdU incorporation and a prolongation of the labeling. Notably, in many cells, there was no detected delay in entry into this compromised S phase. In contrast, when cyclin E function was reduced by a hypomorphic allele combination, BrdU incorporation was robust but the timing of S-phase entry was delayed. We suggest that dE2F-dDP contributes to the expression of two classes of gene products: replication factors, whose abundance has a graded effect on replication, and cyclin E, which triggers an all-or-nothing transition from G1 to S phase.

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The human papillomavirus (HPV) E7 protein antagonises an Imiquimod-induced inflammatory pathway in primary human keratinocytes

Scientific Reports ◽

10.1038/srep12922 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 15

Author(s):

Kathryn H. Richards ◽

Christopher W. Wasson ◽

Oliver Watherston ◽

Rosella Doble ◽

G. Eric Blair ◽

...

Keyword(s):

Human Papillomavirus ◽

Human Keratinocytes ◽

Primary Human Keratinocytes ◽

Inflammatory Pathway ◽

Hpv E7

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Sp100 Provides Intrinsic Immunity against Human Papillomavirus Infection

mBio ◽

10.1128/mbio.00845-13 ◽

2013 ◽

Vol 4 (6) ◽

Cited By ~ 41

Author(s):

Wesley H. Stepp ◽

Jordan M. Meyers ◽

Alison A. McBride

Keyword(s):

Human Papillomavirus ◽

Dna Replication ◽

Human Keratinocytes ◽

Hpv Infection ◽

Nuclear Bodies ◽

Host Cells ◽

Viral Transcription ◽

Primary Human Keratinocytes ◽

Dna Viruses ◽

Hpv Dna

ABSTRACTMost DNA viruses associate with, and reorganize, nuclear domain 10 (ND10) bodies upon entry into the host nucleus. In this study, we examine the roles of the ND10 components PML, Sp100, and Daxx in the establishment of human papillomavirus type 18 (HPV18) infection of primary human keratinocytes. HPV18 DNA or HPV18 quasivirus was introduced into primary human keratinocytes depleted of each ND10 protein by small interfering RNA technology, and genome establishment was determined by using a quantitative immortalization assay and measurements of viral transcription and DNA replication. Keratinocyte depletion of Sp100 resulted in a substantial increase in the number of HPV18-immortalized colonies and a corresponding increase in viral transcription and DNA replication. However, Sp100 repressed viral transcription and replication only during the initial stages of viral establishment, suggesting that Sp100 acts as a repressor of incoming HPV DNA.IMPORTANCEThe intrinsic immune system provides a first-line defense against invading pathogens. Host cells contain nuclear bodies (ND10) that are important for antiviral defense, yet many DNA viruses localize here upon cell entry. However, viruses also disrupt, reorganize, and modify individual components of the bodies. In this study, we show that one of the ND10 components, Sp100, limits the infection of human skin cells by human papillomavirus (HPV). HPVs are important pathogens that cause many types of infection of the cutaneous and mucosal epithelium and are the causative agents of several human cancers. Understanding how host cells counteract HPV infection could provide insight into antimicrobial therapies that could limit initial infection.

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Genetic Analysis of High-Risk E6 in Episomal Maintenance of Human Papillomavirus Genomes in Primary Human Keratinocytes

Journal of Virology ◽

10.1128/jvi.76.22.11359-11364.2002 ◽

2002 ◽

Vol 76 (22) ◽

pp. 11359-11364 ◽

Cited By ~ 56

Author(s):

Regina B. Park ◽

Elliot J. Androphy

Keyword(s):

Human Papillomavirus ◽

Dna Replication ◽

High Risk ◽

Viral Replication ◽

Human Keratinocytes ◽

Binding Motif ◽

Primary Human Keratinocytes ◽

Hpv16 E6 ◽

E6 And E7 ◽

Episomal Maintenance

ABSTRACT Papillomaviruses possess small DNA genomes that encode five early (E) proteins. Transient DNA replication requires activities of the E1 and E2 proteins and a DNA segment containing their binding sites. The E6 and E7 proteins of cancer-associated human papillomavirus (HPV) transform cells in culture. Recent reports have shown that E6 and E7 are necessary for episomal maintenance of HPV in primary keratinocytes. The functions of E6 necessary for viral replication have not been determined, and to address this question we used a recently developed transfection system based on HPV31. To utilize a series of HPV16 E6 mutations, HPV31 E6 was replaced by its HPV16 counterpart. This chimeric genome was competent for both transient and stable replication in keratinocytes. Four HPV16 E6 mutations that do not stimulate p53 degradation were unable to support stable viral replication, suggesting this activity may be necessary for episomal maintenance. E7 has also been shown to be essential for episomal maintenance of the HPV31 genome. A point mutation in the Rb binding motif of HPV E7 has been reported to render HPV31 unable to stably replicate. Interestingly, HPV31 genomes harboring two of the three p53 degradation-defective E6 mutations combined with this E7 mutation were maintained as replicating episomes. These findings imply that the balance between E6 and E7 functions in infected cells is critical for episomal maintenance of high-risk HPV genomes. This model will be useful to dissect the activities of E6 and E7 necessary for viral DNA replication.

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PTPN14 degradation by high-risk human papillomavirus E7 limits keratinocyte differentiation and contributes to HPV-mediated oncogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819534116 ◽

2019 ◽

Vol 116 (14) ◽

pp. 7033-7042 ◽

Cited By ~ 20

Author(s):

Joshua Hatterschide ◽

Amelia E. Bohidar ◽

Miranda Grace ◽

Tara J. Nulton ◽

Hee Won Kim ◽

...

Keyword(s):

Gene Expression ◽

Human Papillomavirus ◽

High Risk ◽

Gene Expression Signature ◽

Human Keratinocytes ◽

Keratinocyte Differentiation ◽

Oncogenic Transformation ◽

Hpv E7 ◽

Oncogenic Activity ◽

High Risk Hpv

High-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate retinoblastoma tumor suppressor (RB1) but cannot degrade PTPN14, we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion ofPTPN14from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation andPTPN14deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV+but not HPV−cancers exhibit a gene-expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.

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A Role for Cdk2 Kinase in Negatively Regulating DNA Replication during S Phase of the Cell Cycle

The Journal of Cell Biology ◽

10.1083/jcb.137.1.183 ◽

1997 ◽

Vol 137 (1) ◽

pp. 183-192 ◽

Cited By ~ 98

Author(s):

Xuequn Helen Hua ◽

Hong Yan ◽

John Newport

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Cyclin E ◽

Embryonic Cell ◽

S Phase ◽

Negative Regulation ◽

Sperm Chromatin ◽

High Concentration ◽

Low Concentrations ◽

Embryonic Cell Cycle

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.

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Premature Expression of the Winged Helix Transcription Factor HFH-11B in Regenerating Mouse Liver Accelerates Hepatocyte Entry into S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8570 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8570-8580 ◽

Cited By ~ 140

Author(s):

Honggang Ye ◽

Ai Xuan Holterman ◽

Kyung W. Yoo ◽

Roberta R. Franks ◽

Robert H. Costa

Keyword(s):

Dna Replication ◽

Liver Regeneration ◽

Nuclear Translocation ◽

Cyclin B1 ◽

S Phase ◽

Hepatocyte Proliferation ◽

Liver Remnant ◽

Winged Helix ◽

Fork Head ◽

Differentiated Cells

ABSTRACT Two-thirds partial hepatectomy (PH) induces differentiated cells in the liver remnant to proliferate and regenerate to its original size. The proliferation-specific HNF-3/fork head homolog-11B protein (HFH-11B; also known as Trident and Win) is a family member of the winged helix/fork head transcription factors and in regenerating liver its expression is reactivated prior to hepatocyte entry into DNA replication (S phase). To examine whether HFH-11B regulates hepatocyte proliferation during liver regeneration, we used the −3-kb transthyretin (TTR) promoter to create transgenic mice that displayed ectopic hepatocyte expression of HFH-11B. Liver regeneration studies with the TTR–HFH-11B mice demonstrate that its premature expression resulted in an 8-h acceleration in the onset of hepatocyte DNA replication and mitosis. This liver regeneration phenotype is associated with protracted expression of cyclin D1 and C/EBPβ, which are involved in stimulating DNA replication and premature expression of M phase promoting cyclin B1 and cdc2. Consistent with the early hepatocyte entry into S phase, regenerating transgenic livers exhibited earlier expression of DNA repair genes (XRCC1, mHR21spA, and mHR23B). Furthermore, in nonregenerating transgenic livers, ectopic HFH-11B expression did not elicit abnormal hepatocyte proliferation, a finding consistent with the retention of the HFH-11B transgene protein in the cytoplasm. We found that nuclear translocation of the HFH-11B transgene protein requires mitogenic signalling induced by PH and that its premature availability in regenerating transgenic liver allowed nuclear translocation to occur 8 h earlier than in wild type.

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