FANCD2 foci in normal cervical tissue and cervical SCCs. Immunofluorescence analysis of normal cervical tissue and a cervical SCC for FANCD2 demonstrates a small punctate nuclear staining in the control (top panels) in contrast to large nuclear FANCD2 foci in cervical cancer cells (bottom panels). The white line in the top right panel denotes the basement membrane. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm.

HPV-16 E7 stimulates recruitment of monoubiquitinated FANCD2 to chromatin. (A to D) Immunoblot analyses of subcellular fractions S3 (soluble nuclear proteins) and P3 (chromatin-enriched) (19) obtained from hTERT-immortalized primary human keratinocytes stably expressing empty vector (control) or HPV-16 E7 (A), HPV-16 E6 (B), HPV-16 E6 and E7 (C), or low-risk HPV-6 E6 or E7 (D) (same cell populations as used in Fig. 2C to F). Antibodies used for immunoblotting were directed against FANCD2 (Genetex) or FANCD1/BRCA2 (Calbiochem, San Diego, CA). Note the differences in the monoubiquitinated form of FANCD2 (long form, L) in comparison to the nonubiquitinated form (short form, S). Immunoblot for ORC2 (BD Biosciences, San Diego, CA) is shown to demonstrate chromatin enrichment of P3 fractions. Ponceau S (Pon S) staining is shown to visualize total protein loading.

Expression of HPV-16 E7 in FANCD2 fibroblasts accelerates chromosomal instability. (A) Metaphase spreads from stably transfected empty vector (control) or HPV-16 E7-expressing immortalized FANCD2 fibroblasts. Chromosomes were stained with DAPI. Inserts depict chromatid breaks (top right and bottom right) or chromosome fusion (bottom left). (B and C) Quantification of the percentage of metaphase spreads with chromosome aberrations from stably transfected empty vector (control) or HPV-16 E7-expressing immortalized human keratinocytes (B) or immortalized FANCD2-deficient fibroblasts stably expressing low-risk HPV-6 E6 or E7 or high-risk HPV-16 E6 and/or E7 (C). Populations expressing HPV-16 E6 and E7 and the corresponding control (HPV-16 E7/neo) underwent two rounds of transfection and antibiotic selection. Each bar represents the mean and standard error for at least triplicate quantifications of a minimum of 25 metaphases. Asterisks indicate statistically significant differences in comparison to controls (Student's t test for independent samples).

Induction of large nuclear FANCD2 foci involves HPV-16 E7 and occurs independently of viral integration. (A) Immunofluorescence microscopic analysis of organotypical cultures of primary human keratinocytes that were either untransfected (control) or stably transfected with full-length HPV-16 episomes. Note the small punctate staining in a control cell in comparison to the large FANCD2 focus in a cell containing HPV-16. Nuclei were stained with DAPI. Scale bar, 10 μm. (B) Quantification of the percentage of cells showing large nuclear FANCD2 foci in organotypical raft cultures that either were untransfected (control) or contained HPV-16 episomes. In addition, raft cultures obtained from keratinocyte populations transduced with E7-deficient HPV-16 episomes with a TTL inserted at nucleotide 711 of the E7 gene were analyzed. Each bar indicates the mean and standard error for at least two independent immunofluorescence experiments, with at least 100 cells counted per experiment. Statistical significance was calculated using Student's t test for independent samples. (C) Immunofluorescence microscopic analysis of hTERT-immortalized primary human keratinocytes either stably expressing pCMVneo-based empty vector (control) or HPV-16 E7. Note the small punctate staining in a control cell in comparison to the large nuclear FANCD2 foci in a cell expressing HPV-16 E7. Nuclei stained with DAPI. Scale bar, 10 μm. (D to F) Quantification of the proportion of cells with large FANCD2 foci in immortalized human keratinocyte populations stably expressing high-risk HPV-16 E6 or E7 (D), coexpressing HPV-16 E6 and E7 (E), or of low-risk HPV-6 E6 or E7 (F). In all experiments, the expression of HPV genes was confirmed by PCR. Each bar represents the mean and standard error for triplicate quantifications of at least 100 cells. Asterisks indicate statistically significant differences in comparison to controls (Student's t test for independent samples).