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
. 2003 Apr 29;100(9):5205-10.
doi: 10.1073/pnas.0935978100. Epub 2003 Apr 11.

Acidic pH induces topoisomerase II-mediated DNA damage

Hai Xiao  1 , Tsai-Kun Li, Jin-Ming Yang, Leroy F Liu
Affiliations

Acidic pH induces topoisomerase II-mediated DNA damage

Hai Xiao et al. Proc Natl Acad Sci U S A. .

Abstract

Acidic pH plays an important role in various pathophysiological states and has been demonstrated to be carcinogenic in animal models. Recent studies have also implicated acidic pH in the development of preneoplastic Barrett's esophagus in human. However, little is known about the molecular mechanism underlying acidic pH-induced carcinogenesis. In the current study, we show that acidic pH, like the topoisomerase II (TOP2) poison VP-16 (demethylepipodophyllotoxin ethylidene-beta-D-glucoside), induces tumors in 9,10-dimethyl-1,2-benzanthracene(DMBA)-initiated mice. The following studies in tissue culture models have suggested that acidic pH acts like a TOP2 poison to induce TOP2-mediated DNA damage: (i) acidic pH induces TOP2-dependent DNA damage signals as evidenced by up-regulation of p53 and Ser-139 phosphorylation of H2AX [a substrate for ataxia telangiectasia mutated (ATM)ATM and Rad3-related (ATR) kinases]; (ii) acidic pH-induced cytotoxicity in tumor cells is reduced in TOP2-deficient cells; (iii) acidic pH increases the mutation frequency of the hypoxanthine phosphoribosyl transferase (HPRT) gene in a TOP2-dependent manner; and (iv) acidic pH induces reversible TOP2-mediated DNA strand breaks in vitro. We discuss the possibility that TOP2-mediated DNA damage may contribute to acidic pH-induced carcinogenesis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Acidic pH and VP-16 induce tumors in the mouse skin carcinogenesis model. (A) Acidic pH and VP-16 induce papillomas in DMBA-initiated mice. Groups of five mice were treated with different drugs (in DMSO) or pH 2.5 medium as described in Materials and Methods. DMBA (600 nmol in 100 μl of DMSO) was applied only once during the first week. TPA (10 nmol in 100 μl of DMSO), VP-16 (5 μmol in 100 μl of DMSO), and pH 2.5 medium (250 mM citrate phosphate-buffered RPMI medium 1640) were applied twice every week for 16 wk. The total number of papillomas appearing on the mouse skin in each group was measured. (B) DMBA induces papillomas in VP-16-initated mice. A group of five mice were treated with VP-16 (5 μmol in 100 μl of DMSO) twice weekly for 10 wk. At the end of the 10th week, a single application of DMBA (600 nmol in 100 μl of DMSO) was performed. Tumor formation was measured at the end of the 16th week.
Figure 2
Figure 2
Acidic pH induces up-regulation of p53 and phosphorylation of H2AX. (A) Acidic pH induces up-regulation of p53. Breast cancer ZR75-1 cells were treated with VP-16 (25 μM), CPT (25 μM), or RPMI medium 1640 (pH 6.0) for 1 h. Treated cells were lysed directly with SDS sample buffer and prepared for immunoblotting by using anti-p53. (B) Acidic pH induces phosphorylation of H2AX. Mouse embryo fibroblast (MEF) cells were treated with VM-26 (10 μM), CPT (10 μM), and different pH media for 1 h. Cells were then lysed and processed for detection of the phosphorylated H2AX by immunoblotting using antibodies against the phosphorylated H2AX epitope.
Figure 3
Figure 3
Acidic pH-induced H2AX phosphorylation is defective in a TOP2 mutant cell line. (A) HL-60 and HL-60/MX2 cells were treated with VM-26 (10 μM), CPT (10 μM), and different pH media for 1 h. Cells were then lysed and processed for detection of the phosphorylated H2AX. (B) HL-60 and HL-60/MX2 cells were lysed and processed for detection of TOP2α and TOP2β.
Figure 4
Figure 4
Acidic pH cytotoxicity is reduced in TOP2-deficient cells. Cytotoxicity was measured by using the clonogenic assay as described in Materials and Methods. (A) Acidic pH cytotoxicity is reduced in TOP2-deficient MDA-MB-231/3000 cells. MDA-MB-231 and TOP2-deficient MDA-MB-231/3000 (attached cells) were treated with 50 μM VP-16, 1 μM CPT, pH 4.5 medium, or pH 7.5 medium for 30 min. Cells were then replenished with drug-free neutral medium and incubated for ≈2 wk. Clonogenic survival was measured by counting colonies on plates. (B) Acidic pH cytotoxicity is reduced in TOP2-deficient T47D/VP1 cells. T47D and TOP2-deficient T47D/VP1 cells (attached cells) were treated with 50 μM VP-16 or pH 4.5 medium for 30 min. Cells were then incubated in drug-free neutral medium for 2 wk. Clonogenic survival was also measured as described in A. (C) Acidic pH cytotoxicity is reduced in TOP2-deficient HL-60/MX2 cells. HL-60 and TOP2-deficient HL-60/MX2 cells (suspension cells) were treated with 2 μM VM-26 or pH 4.5 medium for 30 min. Cells were then resuspended in soft agar (in drug-free neutral medium), and clonogenic survival in soft agar was measured after ≈2 wk as described in Materials and Methods.
Figure 5
Figure 5
Acidic pH increases 6-TG resistance. MDA-MB-231 cells were treated with 0.5 μM VM-26, 0.5 μM CPT, pH 6.0 medium, or pH 7.4 medium (control) for 1 h in the presence or absence of ICRF-193 (25 μM), followed by recovery in RPMI medium 1640 (pH 7.4) for 7 days. Selection with 6-TG was performed as described in Materials and Methods.
Figure 6
Figure 6
Acidic pH induces TOP2-mediated DNA breaks in vitro. (A) Acidic pH induces TOP2-mediated DNA cleavage in vitro. 3′-end 32P-labeled linearized YEpG DNA and hTOP2α were incubated in reactions with different pHs (lanes 4–13) for 30 min at 37°C as described in Materials and Methods. Reactions were terminated with SDS/proteinase K. VM-26 (0, 0.5, and 5 μM) was used as control in a standard cleavage reaction at pH 7.5 (lanes 1–3). (B) Acidic pH-induced DNA cleavage is reversible. DNA cleavage in the presence of hTOP2α was performed as described in A at pH 6.0 (lanes 8–14), pH 7.5 (control), or pH 7.5 with 5 μM VM-26 (lanes 2–7). Before termination, the reactions were subjected to a second incubation under different reversal conditions (indicated on top of each lane) for 30 min. The reversal conditions for the second incubation include different temperatures (lanes 3–6 and 9–12), neutralization of the reactions to pH 7.5 (lane 14), and excess EDTA (10 mM; lanes 7 and 13). (C) Acidic pH induces both single- and double-stranded DNA breaks. Supercoiled pBluescript SK(+) DNA was used to react with hTOP2α in various pH conditions as described in A. The reactions were terminated by SDS/proteinase K and subjected to electrophoresis in 1.0% agarose gel containing 0.5 μg/ml ethidium bromide. N, nicked DNA; L, linear DNA; SC, supercoiled DNA.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kubasiak L A, Hernandez O M, Bishopric N H, Webster K A. Proc Natl Acad Sci USA. 2002;99:12825–12830. - PMC - PubMed
    1. Williams A C, Collard T J, Paraskeva C. Oncogene. 1999;18:3199–3204. - PubMed
    1. Yamamoto D, Kiyozuka Y, Uemura Y, Yamamoto C, Takemoto H, Hirata H, Tanaka K, Hioki K, Tsubura A. J Cancer Res Clin Oncol. 2000;126:191–197. - PubMed
    1. Chevfec G, Schnell T, Sontag S. Am J Clin Pathol. 1992;98:5–7. - PubMed
    1. Shaheen N, Ransohoff D F. J Am Med Assoc. 2002;287:1972–1981. - PubMed

Publication types

MeSH terms