DNA Adducts in Aldehyde Dehydrogenase-Positive Lung Stem Cells

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DNA Adducts in Aldehyde Dehydrogenase-Positive Lung Stem Cells of A/J Mice Treated with the Tobacco Specific Lung Carcinogen 4‑(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) Silvia Balbo,* Pramod Upadhyaya, Peter W. Villalta, Xuemin Qian, and Fekadu Kassie The Masonic Cancer Center, University of Minnesota, MMC 806, 420 Delaware Street Southeast, Minneapolis, Minnesota 55455, United States S Supporting Information *

ABSTRACT: Lung cancer is the leading cause of cancer death in the world. Evidence suggests that lung cancer could originate from mutations accumulating in a subpopulation of self-renewing cells, lung stem cells. Aldehyde dehydrogenase (ALDH) is a marker of stem cells. To investigate the presence of DNA modifications in these cells, we isolated ALDHpositive lung cells from A/J mice exposed to the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Using LC−NSI-HRMS/MS−PRM, O6-methyl-G, 7-POB-G, and O2-POB-dT were positively identified in ALDH-positive cell DNA. This is the first example of detection of carcinogen-DNA adducts in lung stem cells, supporting the hypothesis of their role in lung carcinogenesis.

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NNK in ALDH-positive stem cells. Female A/J mice were treated with NNK, and formation of NNK-induced DNA adducts, precursors of mutations, was quantified in ALDHpositive mouse lung cells. Sixteen 6-week-old female A/J mice were exposed to NNK by ip injection, once a week for 6 weeks, at a dose of 50 mg/kg of body weight. This dose produces lung tumors with 100% incidence in A/J mice.6 One week after the last dose of NNK, the mice were euthanized, and the lungs were harvested. Fifteen mouse lungs were pooled to isolate ALDH-positive pulmonary epithelial cells from which DNA was extracted, whereas one mouse lung was kept to extract DNA from the whole organ. To isolate the ALDH-positive lung stem cells, a single cell suspension of pulmonary epithelial cells was obtained using an ALDEFLUOR kit (Stemcell Technologies, Vancouver, BC).7 ALDH-positive lung stem cells were sorted using a FACSAria flow cytometer (DB Biosciences). Cells in the ALDH-positive area were collected, and DNA was isolated. The DNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA). A total of 6.3 μg of DNA was obtained from the isolated ALDH-positive lung stem cells. Figure 1 shows how ALDH-positive pulmonary cells (P10 area) were separated, by flow cytometry, from ALDH-negative pulmonary cells (P7 area) or a mixed population of pulmonary cells (P4 area). Pyridyloxobutyl (POB)-DNA adducts and methyl-DNA adducts are among the major DNA modifications occurring after reaction of NNK metabolites with DNA.5 Among these adducts, O6-methyl-G, O4-methyl-T, O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (O6-POB-dG), and O2-[4-(3-pyr-

ung cancer is the leading cause of cancer death in the world, causing more deaths than the next three most common cancers combined (colon, breast, and prostate). Lung cancer prognosis still remains very poor, with a five-year survival rate of 16.3%, which is lower than the survival rates of many other leading cancers.1 These figures clearly show the need for a better understanding of lung cancer formation and ultimately for the identification of better therapeutic targets. Recent evidence suggests that human lung cancer, like other cancers, may originate from a small subset of self-renewing tumor cells known as cancer stem cells.2 These cells originate from normal stem cells that acquire mutations leading to loss of natural growth control mechanisms and expansion into a selfrenewing pool of cancer stem cells, ultimately resulting in proliferation of aberrantly differentiated cancer cells. Aldehyde dehydrogenase (ALDH), a detoxifying enzyme responsible for the oxidation of intracellular aldehydes, is a stem cell marker in several tissue types and tumors, including lung tumors.3,4 Flow cytometric analysis of several lung cancer cell lines and patient tumors has shown that most non-small cell lung cancer cells contain a subpopulation of cells with elevated ALDH activity. Additionally, isolated ALDH-positive lung cancer cells were observed to be highly tumorigenic and clonogenic and capable of self-renewal compared to ALDHnegative cells.2 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen in rodents. This tobacco specific nitrosamine undergoes cytochrome P450-mediated metabolism, resulting in the formation of species that react with DNA forming adducts, which are believed to play a fundamental role in chemically induced carcinogenesis.5 To explore a potential role of this carcinogenic pathway in the context of the cancer stem cell theory in lung cancer, we investigated the effect of © XXXX American Chemical Society

Received: February 11, 2013

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co-eluting with the respective internal standard peaks, as shown in Figure S1 in the Supporting Information. This highresolution accurate mass approach has a dramatic effect on decreasing chemical noise, providing extremely clean traces and leading to low limits of detection and quantitation in complex samples. Additionally, the method is more selective because of the high-resolution accurate mass measurement (5 ppm) of the fragment ions, which also provides sufficient mass accuracy to allow for confirmation of their molecular formula. Figure 2

Figure 1. Aldefluor 488 E-A vs FSC-A dot plot of mouse lung cancer stem cells stained with ALDEFLUOR. The cells exposed to ALDEFLUOR are the test samples, while a cell sample exposed to ALDEFLUOR in the presence of the ALDH inhibitor, DEAB, was used as a negative control. P7, created with negative control sample data, is drawn to encompass all nucleated cells and gate RBCs and debris. P10 is drawn to include all ALDH-positive cells. P4 is drawn to collect the cells in the area between the ALDH-positive and ALDHnegative areas. Figure 2. Levels of POB-DNA adducts and O6-methyl-G (femtomoles per milligram of DNA) measured in ALDH-positive lung stem cell DNA (■) and whole lung DNA (□) from A/J female mice treated with NNK (50 mg/kg of body weight once a week for 4 weeks).

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idyl)-4-oxobut-1-yl]thymidine (O -POB-dT) are known to have miscoding properties. In earlier studies, these adducts were identified and quantified in lung DNA of rats and mice exposed to NNK.11−13 In this work, we focused on the identification and quantitation of O6-methyl-G, O6-POB-dG, 7-[4-(3-pyridyl)-4oxobut-1-yl]-2′-deoxyguanosine (7-POB-dG), analyzed as the corresponding base 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7POB-G), and O2-POB-dT in the DNA isolated from the ALDH-positive lung stem cells and compared the results with the levels found in the DNA isolated form the whole lung. A stable-isotope liquid chromatography−nanoelectrospray highresolution tandem mass spectrometry−parallel reaction monitoring (LC−NSI-HRMS/MS−PRM) method was used to analyze the DNA samples. Deuterated analogues of the analytes were used as internal standards for quantitation. Five femtomoles of [CD3]-O6-methyl-G and 50 fmol of [pyridineD4]-O2-POB-dT, [pyridine-D4]-7-POB-G, and [pyridine-D4]O6-POB-dG were added to the DNA samples before hydrolysis. DNA hydrolysis and sample enrichment and purification were performed following the methods reported in the literature (summarized in the Supporting Information).11,13 Because of the small amounts of DNA available for the analysis, higher sensitivity was achieved using a nanoelectrospray source coupled to an LTQ-Orbitrap Velos (Thermo Scientific) instrument. O6-Methyl-G was analyzed starting from 1.3 μg of ALDHpositive lung stem lung cell DNA and 1.3 μg of whole lung DNA. POB adducts were measured starting from 2.5 μg of ALDH-positive lung stem cell DNA and 2.7 μg of whole lung DNA. Two calf thymus DNA samples were used as negative controls, and two buffer blanks with no DNA were included in the analysis. No adduct signal was measured in the negative control samples or buffer blanks, confirming zero detectable contamination or carryover in the analytical method used. ALDH-positive lung stem cells showed clear peaks corresponding to O6-methyl-G, 7-POB-G, and O2-POB-dT,

summarizes the results obtained upon analysis of the DNA samples for the NNK-derived DNA adducts. Levels of the adducts measured in ALDH-positive lung stem cell DNA were as follows: 50 fmol of 7-POB-G/mg of DNA, 2300 fmol of O2POB-dT/mg of DNA, and 1100 fmol of O6-methyl-G/mg of DNA. To the best of our knowledge, this is the first report of detection of any carcinogen-DNA adduct in stem cells. The same DNA adducts were detected in the DNA isolated from the whole lung. Levels of the adducts were comparable to the levels measured in previous studies.11−13 However, the levels measured in the whole lung were significantly higher for all three DNA adducts than the levels measured in ALDHpositive stem cells. Levels of 7-POB-G and O2-POB-dT in whole lung DNA were 700 and 39000 fmol/mg of DNA, respectively, around 15-fold higher than the levels detected in ALDH-positive lung stem cell DNA. The level of O6-methyl-G was 29000 fmol/mg in whole lung DNA, more than 20-fold higher than what was measured in ALDH-positive lung stem cell DNA. Carcinogen-induced DNA adduct levels, measured at any point in time, reflect the balance between the extent of carcinogen activation and detoxification by phase 2 enzymes, DNA adduct formation and removal (DNA repair), and other events. Therefore, the marked differences in NNK-induced DNA adduct levels between ALDH-positive lung stem lung cells and whole lung tissue could be attributed, at least in part, to the low degree of NNK activation, high level of detoxification and/or a rapid repair of DNA adducts in the stem cell population, which could be responsible for the high genetic stability and lower mutation frequencies in stem cells compared to somatic cells. Indeed, in a recent study hepatic stem cells expressed several P450 isoforms at low levels relative to total hepatic cell populations but had a higher capacity for glutathione S-transferase conjugation reactions through mu and B

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pi class isoforms.14 Moreover, intrapulmonary stem cells that express Clara cell secretory protein (CCSP) showed weak expression of cytochrome P450-2F2 compared to Clara cells, which results in a high resistance to the Clara cell specific toxicant naphthalene.15 Additionally, both normal stem cells and cancer stem cells are also known for their high rate of DNA repair capacity.16−18 Altogether, these observations could explain the higher levels of adducts observed in whole lung DNA as compared to those measured in ALDH-positive lung stem cells. The comparative levels of the three adducts were similar in the ALDH-positive cell preparation and whole lung DNA, with the level of O2-POB-dT being the highest and the level of 7POB-G the lowest among the three adducts measured. These findings are in accordance with previous studies of NNKtreated rats and mice showing O2-POB-dT to be the most abundant adduct and the most persistent over time compared to the more rapidly repaired 7-POB-G and O6-methyl-G. No traces of O6-POB-dG were detected in our samples. This POB adduct is generally present in smaller amounts. The levels in our samples may have been below our limit of detection. One of the limitations of this work is the relatively small amount of DNA available for the DNA adduct analysis. This may have limited our ability to detect O6-POB-dG and has limited the possibility of performing multiple measurements on each sample. For this reason, our results should be considered as an indication of the presence and relative amounts of the DNA adducts in lung ALDH-positive stem cells. The absolute amounts of these adducts will have to be confirmed in future studies repeating this measurement on multiple samples with larger amounts of DNA. Another limitation of this work is the lack of ALDH-positive stem cell DNA from untreated animals. Because of the low DNA yield obtained from these cells, this would have required inclusion of a large number of untreated animals. Because NNK-induced DNA adducts have been extensively studied in the past and there is no report of detection of POB adducts and O6-methyl-G in untreated animals, we focused on investigating the presence of these adducts in ALDH-positive stem cells of treated mice. A negative control, however, should be included in future larger studies. DNA adducts play a key role in chemically induced carcinogenesis. The miscoding properties of O6-methyl-G are well-established. This adduct pairs with thymidine during replication, causing an O6-methyl-G-T mismatch and G → A transition mutations commonly observed in the K-ras gene isolated from tumors of mice treated with NNK.19 Recent studies have shown that O2-POB-dT is mutagenic and is associated with TA to AT mutations in Chinese hamster ovary cells and with TA to GC and TA to AT transversion mutations in bacteria, suggesting that O2-POB-dT could be involved in mutations of AT base pairs that lead to oncogene activation.10 The mutagenicity of 7-POB-G needs to be clarified. This adduct can spontaneously depurinate, leaving abasic sites that ultimately can result into GC to AT transitions and GC to TA transversion mutations. However, the role of all these mutations in ALDH-positive stem cells has not yet been explored. To the best of our knowledge, this is the first report of the identification of NNK-derived DNA adducts in ALDH-positive lung stem cells. DNA adducts formed in these cells may be significant because these may be the cells from which lung tumors originate. Further studies of the kinetics of formation of the adducts, on cytochrome P450 and phase 2 enzyme

expression, and DNA repair efficiency of ALDH-positive lung stem cells are warranted. Studies of the mutagenic consequences of the DNA adducts formed in these cells may also provide more insight into lung carcinogenesis mechanisms.

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ASSOCIATED CONTENT

S Supporting Information *

Materials and detailed experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: (612) 624-4240. Fax: (612) 626-5135. E-mail: [email protected]. Funding

This study was funded by Faculty Start-up funds to F.K. from the Masonic Cancer Center and the College of Veterinary Medicine, University of Minnesota. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Robert Carlson for assistance with graphic design. REFERENCES

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dx.doi.org/10.1021/tx400054s | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX