Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Sep 14;96(19):10764-9.
doi: 10.1073/pnas.96.19.10764.

Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents

M J Hickman  1 , L D Samson
Affiliations

Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents

M J Hickman et al. Proc Natl Acad Sci U S A. .

Abstract

All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed, O(6)-alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that O(6)-methylguanine (O(6)MeG) and O(6)-chloroethylguanine (O(6)CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutSalpha branch of the DNA mismatch repair pathway (but not the MutSbeta branch) is absolutely required for signaling the initiation of apoptosis in response to O(6)MeGs and is partially required for signaling apoptosis in response to O(6)CEGs. Further, O(6)MeG lesions signal the stabilization of the p53 tumor suppressor, and such signaling is also MutSalpha-dependent. Despite this, MutSalpha-dependent apoptosis can be executed in a p53-independent manner. DNA mismatch repair status did not influence the response of cells to other inducers of p53 and apoptosis. Thus, it appears that mismatch repair status, rather than p53 status, is a strong indicator of the susceptibility of cells to alkylation-induced apoptosis. This experimental system will allow dissection of the signal transduction events that couple a specific type of DNA base lesion with the final outcome of apoptotic cell death.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Northern and band-shift analyses of DC3F and A3 Chinese hamster lung fibroblasts. (A) Northern blot of total RNA probed with mMsh3 cDNA probe. The middle panels show an overexposure of the top panels. The positions of the 28S and 18S rRNA bands are shown. The size of the msh3 transcript is in agreement with a previous study (39). The lower panels represent total RNA probed with human β-actin cDNA, to verify equal loading. (B) Band-shift assays in which extract proteins were incubated with labeled DNA oligonucleotide duplexes containing a mismatch (G⋅T), three unpaired nucleotides (TTT), or no mismatch (A⋅T). The reaction mixtures were electrophoresed in 6% polyacrylamide gels; the positions of the DNA duplexes in complex with MutSα (α) and MutSβ (β) are shown, and other unlabeled bands represent nonspecific protein⋅DNA complexes. The specificity of MutSα and MutSβ binding was verified by competition assays using a 40-fold molar excess of unlabeled duplex (data not shown), and results were consistent with a previous study (45).
Figure 2
Figure 2
Cell survival (A, D, and G) and apoptosis (B, C, E, F, H, and I) after treatment of DC3F (□) and A3 (●) cells with MNNG (A, B, and C), IR (D, E, and F) and BCNU (G, H, and I). Representative survival curves are shown. Apoptosis was measured by nuclear morphology (B, E, and H) and fluorescence-activated cell sorting analysis (C, F, and I). Each point represents the mean ± SD of at least two independent determinations. Note the different y-axis scales.
Figure 3
Figure 3
Cell survival (A and B) and apoptosis (C and D) after treatment of human lymphoblastoid TK6 (□) and MT1 (●) cells with MNNG (A and C) and BCNU (B and D), as described in Fig. 2. Apoptosis was quantitated by nuclear morphology.
Figure 4
Figure 4
Apoptosis and cell growth after treatment of human lymphoblastoid cells. Apoptosis was quantitated by nuclear morphology; for A–C, TK6 (□), MT1 (●), TK6 containing control vector, TK6-P1 (▵), TK6 expressing MGMT, TK6-M12 (▿). Me-Lex, methyl-lexitropsin. Cell density was monitored for TK6-P1 (D), TK6-M12 (E), and MT1 (F) after exposure to MNNG and IR.
Figure 5
Figure 5
Western blot analysis of p53 expression after 0.02 μg/ml MNNG (A and C) or 1,000 cGy of IR (B). p53 expression was measured 2 or 24 h after IR or MNNG treatment, respectively. Equal amounts of protein (determined by Bradford assay) were loaded onto the gels.
Figure 6
Figure 6
Apoptosis in TK6-E6 (□) and TK6-E6C (●) cells after 200 cGy of IR (Top), 0.02 μg/ml MNNG (Middle), and serum withdrawal (Bottom), as described in Fig. 2. Apoptosis was quantitated by nuclear morphology (Left) and cell sorting analysis (Right).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hollstein M, Sidransky D, Vogelstein B, Harris C C. Science. 1991;253:49–53. - PubMed
    1. Clarke A R, Purdie C A, Harrison D J, Morris R G, Bird C C, Hooper M L, Wyllie A H. Nature (London) 1993;362:849–852. - PubMed
    1. Dou Q P, An B, Will P L. Proc Natl Acad Sci USA. 1995;92:9019–9023. - PMC - PubMed
    1. Sheikh M S, Burns T F, Huang Y, Wu G S, Amundson S, Brooks K S, Fornace A J, Jr, el-Deiry W S. Cancer Res. 1998;58:1593–1598. - PubMed
    1. Kastan M B, Onyekwere O, Sidransky D, Vogelstein B, Craig R W. Cancer Res. 1991;51:6304–6311. - PubMed

Publication types

MeSH terms