Mutagenic potential of hypoxanthine in live human cells

Abstract

Hypoxanthine (Hx) is a major DNA lesion generated by deamination of adenine during chronic inflammatory conditions, which is an underlying cause of various diseases including cancer of colon, liver, pancreas, bladder and stomach. There is evidence that deamination of DNA bases induces mutations, but no study has directly linked Hx accumulation to mutagenesis and strand-specific mutations yet in human cells. Using a site-specific mutagenesis approach, we report the first direct evidence of mutation potential and pattern of Hx in live human cells. We investigated Hx-induced mutations in human nonmalignant HEK293 and cancer HCT116 cell lines and found that Hx is mutagenic in both HEK293 and HCT116 cell lines. There is a strand bias for Hx-mediated mutations in both the cell lines; the Hx in lagging strand is more mutagenic than in leading strand. There is also some difference in cell types regarding the strand bias for mutation types; HEK293 cells showed largely deletion (>80%) mutations in both leading and lagging strand and the rest were insertions and A:T→G:C transition mutations in leading and lagging strands, respectively, whereas in HCT116 cells we observed 60% A:T→G:C transition mutations in the leading strand and 100% deletions in the lagging strand. Overall, Hx is a highly mutagenic lesion capable of generating A:T→G:C transitions and large deletions with a significant variation in leading and lagging strands in human cells. In recent meta-analysis study A→G (T→C) mutations were found to be a prominent signature in a variety of cancers, including a majority types that are induced by inflammation. The deletions are known to be a major cause of copy-number variations or CNVs, which is a major underlying cause of many human diseases including mental illness, developmental disorders and cancer. Thus, Hx, a major DNA lesion induced by different deamination mechanisms, has potential to initiate inflammation-driven carcinogenesis in addition to various human pathophysiological consequences.

Keywords: Base excision repair; Cancer; DNA damage; Deamination damage; Hypoxanthine; Inflammation; Inosine; MPG; Mutation; OGG1; Reactive oxygen and nitrogen species; Site-specific mutagenesis.

Fig. 1. Strategy for testing Hx-induced mutations on the leading or lagging strand DNA template of a duplex plasmid

A) Structures of deoxyadenosine (dA) and deoxyinosine (dI). B) The SV40-containing replicating shuttle vector used for the construction of plasmid DNA containing a site-specific Hx lesion. C) The target sequence containing Hx lesion at EcoRI site in the leading strand (pBSII(KS⁺)-SV40) and the lagging strand (pBSII(KS⁻)-SV40) are shown with respect to SV40 origin.

Fig. 2. 8-oxo-G-induced mutations in HCT116 cells

A) OGG1 and APE1 digestion of undamaged and 8-oxo-G containing pBSII-sv40ori plasmids. Upon treatment with OGG1 and APE1, 8-oxo-G-containing CCC DNA is converted to nicked DNA. See Materials and methods for details. B) Replication efficiency of undamaged and 8-oxo-G-containing plasmid DNA. C) 8-oxo-G-induced mutation frequency. D) 8-oxo-G-induced mutation distributions. CCC: covalently closed circular DNA; 8-oxo-G: 7,8-Dihydro-8-oxoguanine. *p=0.00342.

Fig. 3. Hx-induced mutations in HEK 293 cells

A) MPG and APE1 digestion of undamaged and Hx-containing pBSII-sv40ori plasmids. Upon treatment with MPG and APE1, Hx-containing CCC DNA is converted to nicked DNA. See Materials and methods for details. B) Replication efficiency of undamaged and Hx-containing plasmid DNA. C) Hx-induced mutation frequency. D) Hx-induced mutation distributions. CCC: covalently closed circular DNA. *p≤0.02, ****p<0.0001.

Fig. 4. Hx-induced mutations in HCT116 cells

A) Replication efficiency of undamaged and Hx-containing plasmid DNA. B) Hx-induced mutation frequency. C) Hx-induced mutation distributions.*p=0.02, ***p≤0.0007.