FIG. 3.

Expression of mutant p53 up-regulates genes in H1299 cells. (A) mRNA was extracted from exponentially growing plates of the indicated cell lines, and cDNA was prepared. The cDNA was analyzed by QPCR using gene-specific primers for various genes identified by microarray analysis. The degree of expression was quantitated using a relative standard curve and normalized to an internal control (brome mosaic virus) corresponding to the cDNA batch as described previously (69, 73). The normalized mRNA levels in the HC-5 control cell line were arbitrarily set to 1, and the relative differences (n-fold) were calculated. Additional mutations at amino acids 22 and 23 decrease up-regulation by the mutant p53 protein. Expression levels of NF-κB2, estrogen receptor binding site-associated antigen 9 (EBAG9), integrin α6 (ITGA6), the transcription factor E2F-5, minichromosome maintenance protein 6 (MCM6), and the proto-oncogene c-Syn have been tested. (B) On the left, results from an analysis of cDNA from mutant p53-expressing H1299 cells by QPCR using gene-specific primers for NF-κB1 and NF-κB2 expression are shown. NF-κB2 but not NF-κB1 is up-regulated by mutant p53. Shown at the right is a Western blot demonstrating up-regulation of NF-κB2 in mutant p53-expressing H1299 cell lines and in a murine cell line [10(3)] stably transfected to express vector alone (V4) or mutant p53-D281G. Cell extracts were prepared using reporter lysis buffer (Promega). NF-κB2 was detected using a specific antibody as specified in Materials and Methods.