Apoptotic cells are engulfed by fibroblasts.

<p>(A) Co-culture of HPFs and apoptotic HeLa cells. (B) HPFs co-cultured with apoptotic Ca Ski cells (upper left), apoptotic C-33 A cells (upper right), alone (bottom left) and the supernatant of apoptotic HeLa cells (bottom right). (C) HPFs were cultured with apoptotic HeLa cells for different lengths of time: 1 h (Ci), 2 h (Cii) and 3 h (Ciii). The pictures show actin recruitment and membrane expansion formation (arrows). (D) HPFs, Ca Ski cells and HPFs plus apoptotic Ca Ski or HeLa cells were stained with anti-vimentin (TRITC) and anti-cytokeratin (Cy2) antibodies. Microscope observations were performed with a confocal microscope (scale bar: 1 µm). All results are representative of four independent experiments. (E) HPFs were incubated with apoptotic HeLa, Ca Ski or C-33 A cells for 48 h and apoptotic cell internalization was quantified by flow cytometry.</p

Downregulation of p53 in transformed fibroblasts expressing E6 HPV transcripts.

<p>(A) E6 HPV18 and E6 HPV16 RNA quantification from HPFs, parental cells and fibroblasts transformed by apoptotic HeLa cells or apoptotic Ca Ski cells (named FH and FC respectively) was performed by real-time quantitative PCR following reverse transcription. The graphs represent the mean of three independent experiments (+/− SD). (B) Immunoblotting analyses of p53 and p21 expression in cancer cell lines, HPFs and FH and FC transformed fibroblasts, using mouse monoclonal antibodies against p53 and p21. Blots were also probed with a β-actin antibody.</p

HPV oncogenes are transferred through apoptotic cells to transformed fibroblasts.

<p>(A) The images illustrate the DNA transfer from the apoptotic cells to the fibroblast recipients (arrows). Microscopic observations were performed with a confocal microscope (scale bar: 1 µm). (B) High-risk HPV DNA was detected by <i>in situ</i> hybridization in parental and apoptotic HeLa and Ca Ski cells, FH and FC but not in HPFs. Cells were counterstained with eosin (scale bar: 1 µm). (C) Agarose gel electrophoresis of amplified human albumin and E6 HPV18 and E6 HPV16 DNA (MW: molecular weight). The images are representative of three independent experiments.</p

Transformed fibroblasts acquire tumorigenic characteristics.

<p>(A and B) HPFs and HPFs transformed by apoptotic HeLa (FH) or apoptotic Ca Ski (FC) cells were grown for the indicated lengths of time. The cell proliferation was monitored each day by counting the total numbers of cells (A) and by MTT assays (B). The graphs present the mean (+/− SD) of three independent experiments. (C) HPFs, FH and FC at passages 5 (P5) and 15 (P15) were stained (10⁶ cells) with propidium iodide solution, and analyzed by flow cytometry. (D) The MFI of the G0/G1 peak (mean of three independent experiments +/− SD) displays the ploidy of HPF, FH and FC at P5 and FH and FC at P15.</p

Cytokeratin expression and DNA profiles of transformed fibroblasts differ from the parental cancer cells.

<p>(A) HPFs, Ca Ski, HeLa, FC and FH cells were analyzed by immunocytofluorescence for cytokeratine expression (scale bar: 1 µm). (B) DNA from Ca Ski and FC cells was amplified by fluorescent PCR using 20 microsatellites. Four representative microsatellite amplifications are shown. Different types of allele are observed using four markers: D18S61 TP53, D8S264 and D17S250.</p

Apoptotic cells infected by HPV transform fibroblasts.

<p>(A) HPFs and HeLa, Ca Ski and C-33 A cervical cancer cells (upper line); HPFs after 48 h of exposure to apoptotic cells (apo HPF, apo HeLa, apo Ca Ski, apo C-33 A) (middle line) were cultured in soft agar for 21 days. Apoptotic cells alone were also cultured as a control (lower line). Photographs were taken with a 20× magnification lens. (B and C) HPFs, HeLa cells, Ca Ski cells and transformed fibroblasts by apoptotic HeLa (FH) and apoptotic Ca Ski (FC) cells (after selection on soft-agar), were grown at a limit-dilution for 21 days. (B) The colonies stained with purple crystal were counted, and the plating efficiency (PE, percentage of cells able to form colonies; SD, standard deviation) was calculated. (C) Colony magnifications were photographed with a 40× magnification lens.</p