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.

FIG. 1.

HPV-18 URR-E7 does not prolong S phase in differentiated keratinocytes of raft cultures. (A) Schematic diagram of pulse-chase-pulse experiments with two TdR analogues. Cultures were pulsed with ³H-TdR for 6 or 12 h as indicated, chased for different durations, and then exposed to BrdU for the last 6 h or longer before harvest (downward arrow). The values at the top indicate hours before harvest, whereas those below indicate the ages of raft cultures in days after they were raised to the air-medium interface. The incorporation of BrdU and ³H-TdR was detected by immunohistochemistry (in red) and autoradiography (black dots), respectively. The percentages and numbers (in parenthesis) of doubly labeled cells among all ³H-TdR-positive cells in the differentiated strata are shown on the right. Positive cells along the entire section were scored. (B) Immunostaining of keratin 1 and autoradiography of ³H-TdR in T6/C0/B6 raft cultures. (C) Example of cells positive for ³H-TdR, BrdU, or both in T6/C0/B6 and T6/C42/B6 cultures.

FIG. 2.

E7 induces multinucleated cells in the suprabasal strata of raft cultures. Examples of binucleated and trinucleated cells in E7-transduced cultures. All cells were positive for ³H-TdR or BrdU, except those in panel b, which were positive for both. In panel a, one of the two binucleated cells did not incorporate any nucleoside analogue. Insets are enlarged views of selected nuclei (boxed). Arrows point to the basal stratum.

FIG. 3.

HPV-18 E7 induces endoreduplication in differentiated keratinocytes. Fluorescent images were taken from the differentiated strata in E7-transduced cultures. The labeling scheme is similar to that depicted in Fig 1A (top rows), except that the order of exposure to TdR analogues was reversed. FISH for panels A and B was conducted with cy3-conjugated, pericentromeric probes specific for the X chromosome (red), while BrdU was revealed with an FITC-conjugated anti-BrdU antibody (green) and nuclei were revealed with DAPI (in blue) in a 5-μm section. (A) B6/C42/T6 culture images were captured with a triple-pass filter (left) or with dual filters for the X chromosome and BrdU (middle) or for the X chromosome and the nucleus (right). Also note that the boxed nucleus is significantly larger than the nuclei of surrounding cells. (B) Percentages of differentiated cells containing 0, 1, or 2 or more X chromosome copies in BrdU-positive cells were scored in three E7-transduced cultures, B6/C42/T6, B6/C36/T6, and B6/C0/T6. (C) A FISH assay was conducted with fluorescein-conjugated pericentromeric probes of chromosome 17. Examples of six dots in a BrdU-negative nucleus from the B6/C36/T6 culture are shown. Weaker signal dots were not in focus because they were situated at different focal planes than the strong-signal dots. Arrowheads point to the pericentromeric signals.

FIG. 4.

Delayed accumulation of abundant p27kip1, cyclin E, and p21cip1 proteins in HPV-18 URR-E7-transduced, differentiated keratinocytes that have reentered S phase prevents further rounds of S phase. The cultures were those described in Fig. 1A (top), except that BrdU was not assayed in these experiments. Sections were immunostained for the protein of interest (in red) and then autoradiographed to reveal ³H-TdR incorporation. (A) ³H-TdR labeling scheme and the percentage of cells positive for both ³H-TdR and cyclin E or p27kip1 among ³H-TdR-positive cells in the differentiated strata. (B) Cyclin E immunostaining in control and E7-transduced raft cultures. Immunostaining of p21cip1 (C) and p27kip1 (D) in control and E7-transduced cultures. The insets in panels B and D are enlarged images of selected (boxed) binucleated, doubly positive cells. The inset in panel C is an enlarged view of the selected (boxed), doubly positive cell with an enlarged nucleus. (E) Images of control and E7-transduced raft cultures that had been exposed to BrdU continuously for 96 h. BrdU incorporation was revealed by FITC-conjugated anti-BrdU (green), and nuclei were stained by DAPI (blue). Arrows in panels B through E point to the basal stratum.

FIG. 5.

Alternative cell fates of HPV-18 URR-E7-transduced keratinocytes during squamous differentiation. (A) Kinetic profiles of percentages of differentiated keratinocytes doubly positive for ³H-TdR and BrdU (open triangles), for ³H-TdR and cyclin E (filled squares), or for ³H-TdR and p27kip1 (open circles) in E7-transduced cultures. The data were taken from Fig. 1A, and 4A. (B) Proposed model of alternative paths induced by HPV-18 E7 in differentiated keratinocytes. Lightly shaded ovals represent nuclei capable of entering S phase, whereas filled ovals represent nuclei accumulating abundant p27kip1 or cyclin E/p21cip1 as well. The size of the nucleus reflects its DNA content. Nuclear division without cytokinesis generates binucleated cells. Attrition of cells to the lower path leads to low and decreasing percentages of cells capable of entering into one or more rounds of S phase.