Targeting of mutant hogg1 in mammalian mitochondria and nucleus: effect on cellular survival upon oxidative stress

Abstract

Background: Oxidative damage to mitochondrial DNA has been implicated as a causative factor in a wide variety of degenerative diseases, aging and cancer. The modified guanine, 7,8-dihydro-8-oxoguanine (also known as 8-hydroxyguanine) is one of the major oxidized bases generated in DNA by reactive oxygen species and has gained most of the attention in recent years as a marker of oxidative DNA injury and its suspected role in the initiation of carcinogenesis. 8-hydroxyguanine is removed by hOgg1, a DNA glycosylase/AP lyase involved in the base excision repair pathway.

Methods: We over-expressed wild type and R229Q mutant hOGG1 in the nucleus and mitochondria of cells lacking mitochondrial hOGG1 expression through an expression vector containing nuclear and mitochondrial targeting sequence respectively. We used quantitative real time PCR to analyze mtDNA integrity after exposure to oxidative damaging agents, in cells transfected with or without mitochondrially-targeted mutant hogg1.

Result: Over-expression of wild type hOgg1 in both nucleus and mitochondria resulted in increased cellular survival when compared to vector or mutant over-expression of hOGG1. Interestingly, mitochondrially-targeted mutant hogg1 resulted in more cell death than nuclear targeted mutant hogg1 upon exposure of cells to oxidative damage. Additional we examined mitochondrial DNA integrity after oxidative damage exposure using real-time quantitative PCR. The presence of mutant hogg1 in the mitochondria resulted in reduced mitochondrial DNA integrity when compared to the wild type. Our work indicates that the R229Q hOGG1 mutation failed to protect cells from oxidative damage and that such mutations in cancer may be more detrimental to cellular survival when present in the mitochondria than in the nucleus.

Conclusion: These findings suggest that deficiencies in hOGG1, especially in the mitochondria may lead to reduced mitochondrial DNA integrity, consequently resulting in decreased cell viability.

Figure 1

Sequence of hogg1 Mutant. hOGG1 (wild type) and mutant-hogg1 (mutant) were transfected in HeLa cells. 72 h post transfection, total RNA was isolated and RT-PCR was performed using Superscript II (Invitrogen, Carlsbad, CA) as per manufacturer's instructions. Automated DNA Sequencing results indicated that cells transfected with mutant-hogg1 harbored mutant hogg1, where CGA is changed to CAA at codon 229. Arrow indicates mutated position (G to A).

Figure 2

Expression of hOGG1 targeted to mitochondria. HeLa cells were transfected with empty vector, MTS-hOGG1, MTS-mutant-hogg1, Nuc-hOGG1, and Nuc-mutant-hogg1. Mitochondrial, nuclear and total cellular extracts were isolated and analyzed by Western blot analysis using anti-OGG1 antiserum. Twenty microgram of mitochondrial, nuclear and total cellular extract for each indicated transfection was loaded into each lane (Figure 2A. & B.). Protein extracts in each lane are as indicated. Immunodetection of Lamin B and COX II was done to assure that the transfected proteins were in nucleus and mitochondria respectively.

Figure 3

Cell Survival of hOGG1 Regulated Cells. HeLa cells transfected with indicated plasmids were grown in regular growth media, for 72 h post transfection. The cells were plated at a density such that they reached a 70% confluence on the day of the treatment. The cells were then treated with 400 μM H₂O₂ (A) for 2 h and for 1 h with 4NQO (B) in serum free media, and allowed to recover in normal growth medium for 16 h. Cellular survival was assessed using the MTT cell proliferation assay kit. Error bars represent standard deviation of four points. Nuc-hOGG1 (nuclear targeted hOGG1); MTS-hOGG1 (mitochondrially-targeted hOGG1), Nuc-mutant-hogg1 (nuclear targeted mutant hogg1), MTS-mutant-hogg1 (mitochondrially-targeted mutant hogg1), Vector (empty vector). p < 0.05 when the data from Nuc-hOGG1, MTS-hOGG1 and Nuc-Mutant-hogg1 transfectants was compared with vector-only cells using Student's t test.

Figure 4

Real Time PCR Amplification Curves. Representative real-time PCR amplification curves generated for nuclear β-actin gene (Fig. 4A.), mitochondrial D-Loop (Fig. 4B.) and Cox I (Fig. 4C.) with and without wild type MTS-hOGG1 after treatment with 400 μM H₂O₂. Each experiment was performed in triplicate and is shown by overlapping amplification curves. ΔRn = (Rn⁺) - (Rn^-), where Rn⁺ is the fluorescence emission intensity of reporter/emission intensity of quencher at any time point, and Rn^- is the initial emission intensity of reporter/emission intensity of quencher in the same reaction vessel before PCR amplification is initiated.

Figure 5

mtDNA integrity after oxidative damage exposure. MtDNA integrity of indicated mitochondrial regions in HeLa cells transfected with empty vector, MTS-hOGG1 and MTS-Mutant hogg1 and treated with (A) 400 μM of H₂O₂ for 2 h; (B) 28 μM of Adriamycin for 2 h and (C) 50 μM of 4-NQO for 1 h were analyzed by using quantitative real-time PCR amplification. The extent of decrease in mtDNA integrity was analyzed by calculating the mtDNA/nuclear DNA ratio, and normalizing to the untreated control set at 100%. The error bars represent standard deviation of each experiment done twice in triplicates. An asterisk indicates a significant difference (0.004 ≥ p ≥ 0.0001), when compared to the vector using Student's t test.