Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines

Abstract

A comprehensive analysis of the TP53 gene and its protein status was carried out on a panel of 56 colorectal cancer cell lines. This analysis was based on a combination of denaturing HPLC mutation screening of all exons of the p53 gene, sequencing the cDNA, and assessing the function of the p53 protein by assaying the induced expression of phosphorylated p53 and p21 after exposing cells to gamma-rays. In a few cases where there was no production of p53 message nor evidence of functional p53 protein, all of the p53 exons were sequenced directly. Thirteen of the 56 cell lines had functional p53, 21 lines had missense mutations (one of which made no detectable protein), 4 lines produced no p53 transcripts, and the remaining 18 lines carried truncating TP53 mutations. Thus, our results showed a relatively high frequency of TP53 mutations (76.8%) in our cell lines, with almost half of the mutations being truncating mutations. This is a rather higher frequency of such mutations than usually reported. Of the 18 cell lines with truncating mutations, 12 had detectable truncated protein based on Western blot analysis, whereas no protein was detected in the remaining 6 cell lines. Our data provide a valuable source of TP 53 mutations for further studies and raise the question of the extent to which truncating mutations may have dominant negative effects, even when no truncated protein can be detected by standard methods.

Fig. 1.

P53 status in CRC cell lines after γ-radiation. Cell lines were irradiated with 6 Gy and harvested after 6 and 24 h. The corresponding cell extracts were then analyzed by Western blotting with the anti-p53 antibodies DO-1 and Ser15P as described in Materials and Methods. An increase in Ser15P-detected activity shows that P53 was stabilized in cell lines with WT p53: HCT116, LS180, SW48, and NCI-747. There was either no change in reactivity after irradiation, or lack of reactivity with either antibody in cell lines with mutant p53: LS411, NCI-H716, LS123, and VACO5. LS411 is a mutant line with truncated p53 that cannot be detected, NCI-H716 has a missense mutation whose mutant p53 protein product cannot be detected, and LS123 and VACO5 both have a mutant p53 that is stabilized without stimulation by DNA damage.

Fig. 2.

P21 expression detected by p21 antibody in CRC cell lines after DNA damage. Cells from each line were irradiated with a dose of 6 Gy γ-ray and harvested after 6 and 24 h. Cell extracts were then analyzed by Western blotting with an anti-p21 antibody as described in Materials and Methods. P21 expression was increased after radiation in cell lines HCT116, LS180, SW48, C32, and LOVO, but was not detected at all in NCI-747. None of these lines carried TP53 mutations.

Fig. 3.

Detection of truncated p53 in CRC cell lines with anti-p53 antibody DO-1. Cell extracts were analyzed by Western blotting by using DO-1 as described in Materials and Methods. Samples from 1–10 are: C70, C84, COLO678, COLO741, Gp2d, HCA7, LS513, SW948, VACO4A, and VACO429. Cell line LS513 (position 7) is a positive control from a line with normal TP53. C70 (position 1) and HCA7 (position 6) each showed two different products and SW948 (position 8) lacked evidence of any product, whereas the remaining 6 lines each showed a single product.

Fig. 4.

Comparisons of the distributions of types of TP53 mutations in cell lines and in the IARC database. (Left) Overall TP53 mutation effects in 43 CRC cell lines (Upper Left) compared with that in colon carcinomas in the IARC database updated in July 2005 (Lower Left). (Right) Detailed distribution of TP53 mutational events observed in 43 CRC cell lines (Upper Right) compared with that in CRCs in the IARC database updated in July 2005 (Lower Right).