Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag−/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.National Institutes of Health (U.S.) (NIH grant R01-CA075576)National Institutes of Health (U.S.) (NIH grant R01-CA055042)National Institutes of Health (U.S.) (NIH grant R01-CA149261)National Institutes of Health (U.S.) (NIH grant P30-ES00002)National Institutes of Health (U.S.) (NIH grant P30-ES02109)National Center for Research Resources (U.S.) (grant number M01RR-01066)National Center for Research Resources (U.S.) (grant number UL1 RR025758, Harvard Clinical and Translational Science Center

The interstitial lung disease multidisciplinary meeting: A position statement from the Thoracic Society of Australia and New Zealand and the Lung Foundation Australia

Interstitial lung diseases (ILD) are a diverse group of pulmonary diseases for which accurate diagnosis is critical for optimal treatment outcomes. Diagnosis of ILD can be challenging and a multidisciplinary approach is recommended in international guidelines. The purpose of this position paper is to review the evidence for the use of the multidisciplinary meeting (MDM) in ILD and suggest an approach to its governance and constitution, in an attempt to provide a standard methodology that could be applied across Australia and New Zealand. This position paper is endorsed by the Thoracic Society of Australia and New Zealand (TSANZ) and the Lung Foundation Australia (LFA)

<i>AagTg</i> mice are more susceptible to MMS-induced toxicity.

<p>(A) Body weight (BW) of WT (n = 14), <i>Aag</i>^−/− (n = 12) and <i>AagTg</i> (n = 12) mice 24 h following MMS treatment (75 mg/kg). Representative data (mean ± standard deviation) from 3 independent experiments are shown. (B) BW is illustrated for WT (n = 14), <i>Aag</i>^−/− (n = 12) and <i>AagTg</i> (n = 9) mice following MMS treatment (75 mg/kg). Data represent mean ± standard deviation. (C) Tissue weights of spleen and thymus are illustrated for n>6 per genotype. Striped bars represent untreated tissue weights and solid bars represent tissue weights 24 h following MMS treatment (75 mg/kg). Percent decrease in tissue weight observed following MMS treatment is shown above bars. Data represent mean ± SEM. (D) <i>Ex vivo</i> bone marrow (BM) clonogenic survival assays were performed using BM isolated from WT (n = 3), <i>Aag</i>^−/− (n = 3) and <i>AagTg</i> (n = 3). Data represent mean ± SEM. All the mice used in this figure are males on a pure C57BL/6 background.</p

Approximate MMS LD₅₀ for <i>Aag</i> transgenic mice.

*<p>Indicates that the <i>Aag</i> transgene is expressed in an <i>Aag^−/−</i> background.</p

Parp1 deficiency protects against Aag-dependent, MMS-induced motor dysfunction.

<p>(A) H&E stained cerebellar sections are shown from WT, <i>Aag</i>^−/−, <i>Parp1</i>^−/−, <i>AagTg</i>, and <i>AagTg</i>/<i>Parp1</i>^−/− under untreated conditions or 24 h following MMS treatment (150 mg/kg). Scale bar is 15 µm. Representative images for n = 5 mice/genotype are shown. (B) Quantitation of cerebellar phenotype is shown. Three or more images/cerebella were quantitated per mouse, and 3 mice per genotype analyzed for quantitation; the average sum of object area (edema) per image is presented. (C) Performance during the rotarod challenge in WT (n = 15), <i>AagTg</i> (n = 18), and <i>AagTg</i>/<i>Parp1</i>^−/− (n = 9) is illustrated under untreated conditions and following MMS treatment (60 mg/kg). (D) Performance for the rotarod challenge is shown for WT (n = 8), <i>Aag</i>^−/− (n = 11), and <i>Parp1</i>^−/− (n = 15) mice four hours following MMS treatment (140 mg/kg). All the mice used in this figure are mixed C57BL/6:129S background. All data represent mean ± SEM.</p

MMS induces severe cerebellar lesions <i>AagTg</i> mice.

<p>(A) H&E stained image of cerebellar granule cells from WT, <i>Aag</i>^−/− and <i>AagTg</i> mice either in untreated conditions or 24 h following MMS treatment (150 mg/kg). Representative images are shown of n>6 experiments. Yellow arrows indicate pyknotic nuclei. Scale bar is 100 µm on low magnification images (black bar) and 15 µm on high-magnification images (white bar). Insets contain magnified images of area in dashed boxes. (B) Quantitation of cerebellar phenotype was performed on images from WT (n = 4), <i>Aag</i>^−/− (n = 3) and <i>AagTg</i> mice (n = 4). Representative images with identified objects (edema) colorized for visualization. Greater than 3 images/cerebella were quantitated per mouse, and the average sum of object area per image is presented. Data represent mean ± SEM. All the mice in this figure are on a pure C57BL/6 background.</p

Approximate LD₅₀ of <i>Aag</i>^−/− and <i>Aag</i> transgenic mice to various genotoxic agents.

<p>MMS, Methyl methanesulfonate; MNU, N-methyl-N-nitrosourea; AOM, Azoxymethane; MMC, Mitomycin C; CAA, Chloroacetaldehyde.</p>*<p>Indicates that the <i>Aag</i> transgene is expressed in an <i>Aag^−/−</i> background.</p

Human peripheral blood mononuclear cells (PBMCs) exhibit a wide range in AAG activity.

<p>An <i>in vitro</i> glycosylase assay determined AAG activity in PBMCs isolated from 80 healthy individuals.</p

MMS induces an Aag-dependent decrease in motor function.

<p>(A) Representations of gait are shown for WT (n = 3), <i>Aag</i>^−/− (n = 3) and <i>AagTg</i> (n = 3) mice three hours following MMS treatment (90 mg/kg). (B) Rotarod performance is shown for WT (n = 18), <i>Aag</i>^−/− (n = 17) and <i>AagTg</i> (n = 25) mice under untreated conditions and following MMS treatment (60 mg/kg). Data represent mean ± SEM. (C) Performance for the rotarod challenge is shown for WT (n = 17) and <i>Aag</i>^−/− (n = 22), 3 and 4 h following MMS treatment (140 mg/kg). Data represent mean ± SEM. All the mice in this figure are on a pure C57BL/6 background.</p

Parp1 deficiency protects against Aag-dependent, MMS-induced toxicity in retina photoreceptors.

<p>H&E stained retinal sections for WT, <i>Aag</i>^−/−, <i>Parp1</i>^−/−, <i>AagTg</i>, and <i>AagTg</i>/<i>Parp1</i>^−/− under untreated conditions or 7 d following MMS treatment (75 mg/kg). Scale bar is 15 µm. Representative images for n = 5 mice/genotype are shown. All the mice used in this figure are mixed C57BL/6:129S background. ONL, Outer nuclear layer; INL, inner nuclear layer.</p