Depletion of the cdk Inhibitor p16INK4a Differentially Affects Proliferation of Established Cervical Carcinoma Cells

  1. Pidder Jansen-Dürra,b
  1. aInstitute for Biomedical Aging Research of Innsbruck University, Innsbruck, Austria
  2. bTyrolean Cancer Research Institute at the Medical University of Innsbruck, Innsbruck, Austria
  3. cClinic for Gynecology, CCM/CBF, Charité-Universitätsmedizin Berlin, Berlin, Germany
  1. M. J. Imperiale, Editor
  1. FIG 1
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    FIG 1

    Knockdown of p16INK4A induces senescence of HeLa cells. (A) HeLa cells were transfected with p16INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7). p16INK4A knockdown was verified by Western blotting (inset). Cells were counted, and a growth curve was established. Shown are the results (mean ± standard deviation [SD]) from three independent experiments. cPDL, cumulated population doublings. (B) At day 12, cells were assessed for presence of SA-β-gal activity. Bars represent the relative percentage of SA-β-gal-positive cells (mean ± SD) observed within three visual fields from three independent experiments. Two representative micrographs are also shown. (C) HeLa cells were treated as described for panel A and harvested at day 10 posttransfection. Left panel, extraction of total RNA was followed by subsequent cDNA synthesis, and mRNA levels of B2M and p21WAF-1/Cip-1 were assessed by qRT-PCR. p21WAF-1/Cip-1 mRNA levels were normalized to B2M mRNA. Shown are the results (mean ± SD) of three independent experiments. Right panel, cell extracts were subjected to SDS-PAGE and analyzed by Western blotting using antibodies for the detection of p21WAF-1/Cip-1, p53, and tubulin, as indicated. Extracts of cisplatin-treated human fibroblasts were added as a positive control for the expression of p21WAF-1/Cip-1 and p53; as a further control, extracts of untreated HeLa cells were used. Three independent experiments were performed, and results of one representative experiment are shown. (D) Left panel, HeLa cells were treated as for panel A, stained with annexin V, and analyzed by flow cytometry, and the relative percentage of annexin V-positive cells was determined. Shown are the results (mean ± SD) of three independent experiments. Right panel, HeLa cells were treated as for panel A, and caspase-3/7 activity was determined and displayed as relative luminescence units (RLU). Staurosporine-treated cells were used as a positive control for the assay. Shown are the results (mean ± SD) of three independent experiments. n.s., not significant.

  2. FIG 2
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    FIG 2

    Knockdown of p16INK4A leads to reduced expression of the E7 oncogene in HeLa cells (A) Left panel, HeLa cells were transfected with p16INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7) and harvested at different time points after initial transfection (24 h, 6 days, and 8 days). Extraction of total RNA was followed by subsequent cDNA synthesis, and HPV-18 E7 mRNA levels were assessed by qRT-PCR. Shown are the results (mean ± SD) of three independent experiments. Right panel, HeLa cells were transfected with p16INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7) and harvested at different time points after initial transfection (24 h, 48 h, 6 days, and 8 days). Cell extracts were subjected to SDS-PAGE and analyzed by Western blotting using antibodies for the detection of p16INK4A, HPV-18 E7 protein, and alpha-tubulin, as indicated. Three independent experiments were performed, and results from one representative experiment are shown. (B) HeLa cells were stably transfected with pLSXN and pLSXN-HPV-18 E6/E7, as indicated. p16INK4A was silenced by siRNA as for panel A, and the percentage of senescent cells was determined after SA-β-gal staining. Shown are the results (mean ± SD) of three independent experiments. (C) Left panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-18 E7 or the empty vector pCMV. HPV-18 E7 mRNA was analyzed by qRT-PCR in both cases; the fold difference of expression is shown on a logarithmic scale. Right panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-18 E7 and cotransfected by p16INK4A siRNA or control siRNA, as indicated. HPV-18 E7 mRNA was analyzed by qRT-PCR in both cases; shown are the results (mean ± SD) of three independent experiments. (D) Left panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for p16INK4A or the empty vector pCMV. p16INK4A mRNA was analyzed by qRT-PCR in both cases; the fold difference of expression is shown on a logarithmic scale. Right panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for p16INK4A and cotransfected by p16INK4A siRNA or control siRNA, as indicated. p16INK4A mRNA was analyzed by qRT-PCR in both cases; shown are the results (mean ± SD) of three independent experiments.

  3. FIG 3
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    FIG 3

    p16INK4A knockdown reduces E7 levels in CaSki and MS-751 cells. (A) CaSki cells were transfected with p16INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day, 3 and day 6). Left panel, at day 8, RNA was prepared and the mRNA levels for p16INK4A and HPV-16 E7 were analyzed by qRT-PCR. Shown are the results (mean ± SD) of three independent experiments. Right panel, at day 8, cells were lysed and protein levels of p16INK4Aand HPV-16 E7 were analyzed by Western blotting. One representative Western blot is shown. (B) Left panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-16 E7 or the empty vector pCMV. HPV-16 E7 mRNA was analyzed by qRT-PCR in both cases; the fold difference of expression is shown on a logarithmic scale. Right panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-16 E7 and cotransfected by p16INK4A siRNA or control siRNA, as indicated. HPV-16 E7 mRNA was analyzed by qRT-PCR in both cases; shown are the results (mean ± SD) of three independent experiments. (C) MS-751 cells were transfected with p16INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 8). Left panel, at day 8 RNA was prepared and the mRNA levels for p16INK4A and HPV-45 E7 were analyzed by qRT-PCR. Shown are the results (mean ± SD) of three independent experiments. Right panel, at day 10, cells were lysed and protein levels of p16INK4A and HPV-45 E7 were analyzed by Western blotting. One representative Western blot is shown. (D) Left panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-45 E7 or the empty vector pCMV. HPV-45 E7 mRNA was analyzed by qRT-PCR in both cases; the fold difference of expression is shown on a logarithmic scale. Right panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-45 E7 and cotransfected by p16INK4A siRNA or control siRNA, as indicated. HPV-45 E7 mRNA was analyzed by qRT-PCR in both cases; Shown are the results (mean ± SD) of three independent experiments.

  4. FIG 4
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    FIG 4

    p16INK4A knockdown does not affect cell proliferation in CaSki and MS-751 cells. (A) CaSki cells were transfected with p16INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 3, and day 6). Upper panel, cells were counted, and a growth curve was established. Shown are the results (mean ± SD) of three independent experiments. Lower panel, at day 9, cells were assessed for presence of SA-β-gal activity. Two representative micrographs are shown. (B) MS-751 cells were transfected with p16INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 8). Upper panel, cells were counted, and a growth curve was established. Shown are the results (mean ± SD) of three independent experiments. Lower panel, at day 11, cells were assessed for presence of SA-β-gal activity. Two representative micrographs are shown.

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  1. Accepted manuscript posted online 5 March 2014, doi: 10.1128/JVI.03817-13 J. Virol. vol. 88 no. 10 5256-5262
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