Restoration of p53 expression in human cancer cell lines upregulates the expression of Notch1: implications for cancer cell fate determination after genotoxic stress

Abstract

Following genotoxic stress, transcriptional activation of target genes by p53 tumor suppressor is critical in cell fate determination. Here we report that the restoration of p53 function in human cancer cell lines that are deficient in p53 function upregulated the expression of Notch1. Interestingly, the expression of wild-type p53 in human prostate and breast cancer cell lines correlated well with increased expression of Notch1. Furthermore, knockdown of p53 expression in cancer cells that express wild-type p53 resulted in reduced expression of Notch1. Importantly, genotoxic stress to cancer cells that resulted in activation of p53 also upregulated the expression of Notch1. Moreover, p53-mediated induction of Notch1 expression was associated with stimulation of the activity of Notch-responsive reporters. Notably, p53 differentially regulated the expression of Notch family members: expression of Notch2 and Notch4 was not induced by p53. Significantly, treatment of cells with gamma secretase inhibitor, an inhibitor of Notch signaling, increased susceptibility to apoptosis in response to genotoxic stress. Together, our observations suggest that p53-mediated upregulation of Notch1 expression in human cancer cell lines contributes to cell fate determination after genotoxic stress.

Figure 1

Restoration of p53 expression in the human Saos-2 osteosarcoma cell line upregulates the expression of Notch1. (A) Subconfluent cultures of Saos^Arg72 cells were either incubated at 39°C (lane 1), incubated at 32°C for 16 hours (lane 2), or incubated at 32°C for 24 hours (lane 3). After incubation, total cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins. (B) Saos-2 cells were nucleofected with either an empty vector pCMV (lane 1) or the plasmid pCMV-p53 encoding wild-type p53 (lane 2). Sixty hours after the nucleofection of cells, cells were lysed, and cell lysates containing an equal amount of proteins were processed for immunoblotting using antibodies specific to the indicated proteins. (C) Subconfluent cultures of Saos^Arg72 cells were incubated either at 39°C (lane 1) or at 32°C (lane 2). Twenty-four hours after incubation, total RNA was isolated, and steady-state levels of Notch1, p21, and actin mRNA were analyzed by semiquantitative reverse transcription-polymerase chain reaction. (D and E) Two sets of Saos^Arg72 cell cultures (in 60-mm plates) were transfected with p21-luc, CBF1-luc, or Hes1-luc reporter plasmid (1.8 µg) along with pRL-TK plasmid (0.2 ag; plasmids in a 9:1 ratio) using FuGene6 transfection reagent. One set of plates for each reporter was incubated at 39°C, and the other sets of plates were incubated at 32°C. Forty-four hours after incubation, cells were processed for dual luciferase reporter activity assays, as described in Materials and Methods. Firefly luciferase reporter activity was normalized to Renilla luciferase activity to control for variations in nucleofection efficiencies. Luciferase activity for p21-luc (C) and for CBF1-luc and Hes1-luc (D) in control cells is shown.

Figure 2

The functional status of p53 and its expression levels in human cancer cell lines correlate with expression levels of Notch1 protein. (A) Total cell lysates from human prostate cancer cell lines LNCaP (lane 1), DU-145 (lane 2), or PC-3 (lane 3) were analyzed by immunoblotting using antibodies specific to the indicated proteins. (B) Total cell lysates from the human breast cancer cell line MCF-7 (lane 1) or MDA-468 (lane 2) were analyzed by immunoblotting using antibodies specific to the indicated proteins. (C) PC-3 cells were nucleofected with either pCMV vector (2 µg; lanes 1 and 2) or pCMV-p53 (wild-type) plasmid (2 µg; lanes 3 and 4), as described in Materials and Methods. Twenty-four hours after nucleofection, cells were either left untreated (lanes 1 and 3) or treated with etoposide (45 M) for 24 hours. Forty-eight hours after nucleofection, total cell extracts were analyzed by immunoblotting using antibodies specific to the indicated proteins. (D) LNCaP cells were transfected with either control siRNA (lane 1) or a pool of p53 siRNA (lane 2), as described in Materials and Methods, using Lipofectamine transfection reagent. Sixty hours after the transfection of cells, total cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins.

Figure 3

Etoposide treatment-induced genotoxic stress in p53-positive human cancer cell lines upregulates Notch1 expression. (A) Total cell lysates from the human prostate cancer cell line LNCaP, treated with either DMSO alone (lane 1), 45 µM etoposide (final concentration; lane 2), or 90 µM etoposide (final concentration; lane 3) for 15 hours, were analyzed by immunoblotting using antibodies specific to the indicated proteins. (B) Total cell lysates from the human breast cancer cell line MCF-7, either treated with DMSO alone (lane 1) or treated with 45 µM etoposide (final concentration) for the indicated time (hours), were analyzed by immunoblotting using antibodies specific to the indicated proteins. (C and D) Total cell lysates from the human prostate cancer cell line PC-3 (C) or DU-145 (D), either treated with DMSO alone (lane 1) or treated with indicated concentrations of etoposide for 24 hours, were analyzed by immunoblotting using antibodies specific to the indicated proteins.

Figure 4

p53 differentially regulates the expression of Notch receptor family members. Subconfluent cultures of Saos^Arg72 cells were incubated at either 39°C (lane 1) or 32°C (lane 2) for 24 hours. After incubation, total cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins.

Figure 5

Inhibition of Notch1 activity and genotoxic stress increase susceptibility to apoptosis. (A) Subconfluent cultures of MCF-7 cell line were either left untreated (lane 1) or treated with GSI (lane 2; 25 µM), etoposide (lane 3; 45 µM), or both GSI and etoposide (lane 4). Cells were incubated for 20 hours, and total cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins. Two arrows indicate two forms of PARP protein: the precursor form (113 kDa) and the cleaved form (85 kDa). (B) Subconfluent cultures of the MCF-7 cell line were either left untreated (control; top left panel) or treated with GSI (25 µM; top right panel), etoposide (45 µM; bottom left panel), or both GSI and etoposide (bottom right panel). After incubation of cells for 20 hours, floating and attached cells were collected and processed for propidium iodide staining followed by flow cytometry. The percentage of cells in sub-G₀ phase is indicated in each panel.