Overriding of cyclin-dependent kinase inhibitors by high and low risk human papillomavirus types: evidence for an in vivo role in cervical lesions

Zehbe, Ingeborg; Rätsch, Andreas; Alunni-Fabbroni, Marianna; Burzlaff, Annett; Bakos, Evi; Wilander, Erik; Tommasino, Massimo

doi:10.1038/sj.onc.1202549

Cited by 42 publications

(42 citation statements)

References 36 publications

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“…In HPV-induced papillomas, condylomas, and low-grade dysplasia, some koilocytes, which may contain high numbers of HPV DNA copies, were also found to be positive for cyclin E (38) or p21cip1 (44). However, our experiments with raft cultures or patient specimens clearly demonstrate that cells with active DNA replication at the time of tissue fixation did not simultaneously have detectable levels of cyclin E, p21cip1, or p27kip1 protein (19,30).…”

Section: Discussionmentioning

confidence: 51%

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

Chien

Noya

Benedict-Hamilton

et al. 2002

J Virol

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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 3 H-thymidine ( 3 H-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 3 H-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.Human papillomaviruses (HPVs) cause benign proliferative lesions in cutaneous or mucosal epithelia. Infections by certain virus types, such as HPV type 16 (HPV-16) and HPV-18, can also lead to anogenital cancers. Despite the difference in viral pathogenesis, viral DNA amplification and progeny virion production invariably depend on squamous differentiation and occur only in benign warty growths (7). Because viral DNA replication requires the host replication machinery, HPVs induce S-phase reentry in a subset of postmitotic, differentiated keratinocytes (5), which we called unscheduled DNA synthesis. Organotypic cultures of primary human keratinocytes (PHKs) grown at the air-medium interface (abbreviated as raft cultures) resemble closely the native squamous epithelia from which the PHKs were derived (8). As in native skin, only the basal and parabasal cells maintain the ability to divide, whereas cells above are postmitotic and become progressively differentiated by histological and molecular criteria. In these cultures, expression of the HPV-18 E7 gene from its differentiationdependent promoter located in the upstream regulatory region (URR) recapitulates the unscheduled cellular DNA synthesis observed in benign papillomas caused by the nononcogenic HPV-6 or HPV-11 (5).The G 1 -to-S-phase transition during the cell cycle is normally controlled by the retinoblastoma susceptibility protein (pRb), the cyclin-dependent kinases (cdks), and cdk inhibitors. pRb bin...

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Section: Discussionmentioning

confidence: 51%

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

Chien

Noya

Benedict-Hamilton

et al. 2002

J Virol

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“…Waf1 and p27 Kip1 have been found in cervical samples containing HPV16 (Zehbe et al, 1999). While our study does not examine the effect of E7 on p21 Waf1 , it is interesting that in HFKE6/E7 cells p27 Kip1 is the most likely cause of G1 arrest upon quercetin exposure.…”

Section: Waf1mentioning

confidence: 79%

Quercetin elevates p27Kip1 and arrests both primary and HPV16 E6/E7 transformed human keratinocytes in G1

Beniston

Campo

2003

Oncogene

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“…Immunofluorescence on raft culture sections was performed using the appropriate primary antibodies-a rabbit anti-K5 polyclonal antibody (#24647, Abcam, Cambridge, MA, USA), a mouse anti-K10 monoclonal antibody or a mouse anti-Ki67 (both from DAKO)-followed by secondary antibodiesAlexaFluor 488 donkey anti-rabbit or AlexaFluor 594 donkey anti-mouse-as described (Zehbe et al, 2009). Ki67 was detected using a peroxidase-based reporter molecule system as described (Zehbe et al, 1999).…”

Section: Flow Cytometrymentioning

confidence: 99%

The immortalizing and transforming ability of two common human papillomavirus 16 E6 variants with different prevalences in cervical cancer

et al. 2010

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Persistent infection with high-risk human papillomaviruses (HPVs), especially type 16 has been undeniably linked to cervical cancer. The Asian-American (AA) variant of HPV16 is more common in the Americas than the prototype in cervical cancer. The different prevalence is based on three amino acid changes within the E6 protein denoted Q14H/H78Y/L83V. To investigate the mechanism(s) behind this observation, both E6 proteins, in the presence of E7, were evaluated for their ability to extend the life span of and transform primary human foreskin keratinocytes (PHFKs). Longterm cell culture studies resulted in death at passage 9 of vector-transduced PHFKs (negative control), but survival of both E6 PHFKs to passage 65 (and beyond). Compared with E6/E7 PHFKs, AA/E7 PHFKs were significantly faster dividing, developed larger cells in monolayer cultures, showed double the epithelial thickness and expressed cytokeratin 10 when grown as organotypic raft cultures. Telomerase activation and p53 inactivation, two hallmarks of immortalization, were not significantly different between the two populations. Both were resistant to anoikis at later passages, but only AA/E7 PHFKs acquired the capacity for in vitro transformation. Proteomic analysis revealed markedly different protein patterns between E6/E7 and AA/E7, particularly with respect to key cellular metabolic enzymes. Our results provide new insights into the reasons underlying the greater prevalence of the AA variant in cervical cancer as evidenced by characteristics associated with higher oncogenic potential.

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Overriding of cyclin-dependent kinase inhibitors by high and low risk human papillomavirus types: evidence for an in vivo role in cervical lesions

Cited by 42 publications

References 36 publications

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

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

Quercetin elevates p27Kip1 and arrests both primary and HPV16 E6/E7 transformed human keratinocytes in G1

The immortalizing and transforming ability of two common human papillomavirus 16 E6 variants with different prevalences in cervical cancer

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