Wild-type p53 induces LMα1 expression in epithelial cells via binding to the <i>LAMA1</i> promoter.

<p>(<b>A</b>) HCT116 cells were seeded on LM-111 or LM-511-enriched matrix. (Left) Endogenous p53 mRNA was quantified by RT-qPCR (normalized to <i>GAPDH</i>) and expressed as ratio relative to the control (plastic dishes). The values are given as a mean +/− SEM of 9 independent experiments which were pooled (n = 3 at 2 days; n = 3 at 3 days; n = 3 at 4 days). (Right) Representative immunoblot of p53 and actin from HCT116 cells in the different conditions. Data show that LM substrata do not activate endogenous expression of p53. (<b>B</b>) Relative mRNA expression of LM chains in HCT116 cells upon transfection with a <i>TP53</i> vector assessed at different time points. Transcript levels were determined by RT-qPCR and normalized to <i>GAPDH</i> and are presented relative to the control vector (n = 3 to 4 experiments, except for α1 at 6 h n = 6 and at 48 h n = 8). Data show that wild-type (wt) p53 induces selectively and progressively LMα1 mRNA levels. (<b>C</b>) Semiquantitative analysis of intracellular LMα1 after immunofluorescence staining of HCT116 cells (48 h of irinotecan treatment). Note a 2.3-fold intracellular deposition of LMα1 in irinotecan-treated cells as compared to untreated cells (n = 15). (<b>D</b>) Expression of LMα1 mRNA from two independent stable sh-<i>TP53</i> HCT116 cell lines and in a sh-RNA control cell line, upon treatment with irinotecan. After 48 h, relative mRNA expression of LMα1 was assessed by RT-qPCR and normalized to <i>GAPDH;</i> values are given as ratios relative to those found in the corresponding untreated cells (n = 3). In p53-deprived cells, irinotecan was unable to stimulate LMα1 expression. (<b>E</b>) Relative <i>LAMA1</i> mRNA expression in HCT116 cells following transfection with wt or mutants p53 (ratios calculated as stated above; n = 6). LMα1 mRNA were only activated by wt p53. (<b>F</b>) Diagram showing the location of putative p53 binding motifs (sites 1 to 6) in the <i>LAMA1</i> promoter 7 kb upstream of the transcription site and in the 5 kb region of intron 1 (i). (<b>G</b>) Chromatin immunoprecipitation experiments. Chromatin was prepared from HCT116 cells transfected with either the control (pCMV) or <i>TP53</i> vector (p53). Cross-linked p53-DNA complexes were immunoprecipitated by either IgG (negative control) or anti-p53 antibodies (DO1 or 1801) followed by PCR amplification using primers that flank the putative p53 binding sites. Input represents chromatin before immunoprecipitation. Note that p53 binds to 7 candidate p53 binding sites. Bars represent mean +/−SEM; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.