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

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 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...

Human Papillomavirus (HPV) E7 Induces Prolonged G2 following S Phase Reentry in Differentiated Human Keratinocytes

“…Indeed, we observe very few mitotic figures in the HPV-18 E7 raft cultures. Moreover, many of the differentiated cells in H18 E7 raft cultures had an enlarged nucleus or appeared multinucleated, consistent with an aberrant G 2 -M transition (8).…”

“…Plasmids-The retroviral constructs expressing wild type HPV-18 E7 (18E7) and E7 mutations (C27S, ⌬DLLC, E35Q,E36Q,E37Q, and S32Q,S33Q) have been described (3,6,8,9). In these vectors, E7 is expressed from the HPV-18 enhancer-promoter located in the upstream regulatory region, whereas the neomycin resistance gene is driven by the SV40 promoter.…”

Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes

“…We considered a number of possible explanations: (1) Cyclin B1 was degraded as cells transitioned through mitosis. This is unlikely because the enlarged nuclei in these cells are consistent with a greater than 2n DNA content (Chien et al 2002). (2) Cyclin B was degraded to allow cellular DNA rereplication.…”

“…For trans-complementation experiments, the empty vector-only retrovirus pLC and pLJ HPV-18 URR-E6 or URR-E6/E7 retroviruses were used. Each expresses the Neomycin resistance gene (Cheng et al 1995;Chien et al 2002). All plasmids were purified by banding in CsCl-ethidium bromide equilibrium density gradients.…”

HPV E7 expression in skeletal muscle cells distinguishes initiation of the postmitotic state from its maintenance

“…Whether the virus reactivates S phase in a terminally differentiated, postmitotic cell or keeps active the cell cycle of a differentiating cell is unclear. Indeed, the phenomenon is variably described as S phase reentry in postmitotic, differentiated cells (Jian et al, 1998;Chien et al, 2002), maintenance of cells in a proliferative state in the course of differentiation (Kyo et al, 1997;Nguyen et al, 2002) or, perhaps most appropriately, left undecided (Jones et al, 1997).…”