Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets Nagarajan Kannana, Long V. Nguyena, Maisam Makarema, Yifei Donga, Kingsley Shiha, Peter Eirewa, Afshin Raoufa,1, Joanne T. Emermanb, and Connie J. Eavesa,c,2 a Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 1L3; and Departments of bCellular and Physiological Sciences and cMedical Genetics, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research at Princess Margaret Cancer Centre, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada, and approved April 9, 2014 (received for review March 5, 2014)
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human epithelial stem and progenitor cells mammary differentiation 3D clonogenic assay superoxide dismutase peroxiredoxin
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ellular synthesis of different reactive oxygen species (ROS) results primarily from the incomplete reduction of molecular oxygen in mitochondria to generate free radical superoxide anions. ROS also are produced when cells are exposed to different environmental sources of oxidative stressors, including ionizing radiation. Together, these regulate many normal cellular processes and also contribute to DNA damage, tumorigenesis, and cell death (1–3). In mice, the inner layer of “luminal” epithelial cells of the normal adult mammary gland have been found to contain higher levels of ROS than the outer “basal” layer of epithelial cells (4). The basis for these differences in ROS levels in the two cell types has been attributed to differences in their content of mitochondria; however, their stress response mechanisms have not been defined. Even less is known about ROS levels and their control in the normal adult human mammary gland, which consists of a similar continuous bilayered epithelial network of ducts and terminal alveolae. MCF10A cells are an immortal but nontumorigenic human mammary cell line that, when grown in 3D Matrigel cultures, generates multilayered spheres in which a lumen forms owing to the acquisition of lethal levels of ROS by the inner cells (5). These studies have suggested that ROS regulation plays an important role in the structural morphogenesis and homeostasis of normal adult human mammary tissue. Here we report the results of experiments designed to investigate the levels of ROS and their control in highly purified populations of viable luminal progenitors (LPs) and basal cells (BCs) isolated from normal human breast tissue (6–8). Our results confirm the different levels of ROS identified in the corresponding subsets of mouse mammary cells and further characterize the distinct antioxidant
www.pnas.org/cgi/doi/10.1073/pnas.1403813111
mechanisms operative in the human cells and their associated differential sensitivity to treatments that elevate ROS levels. Results Differing ROS Levels in Different Subsets of Primary Human Mammary + + −/low MUC1− BCs Epithelial Cells. CD49f CD90(THY1) EpCAM
and CD49f+CD90(THY1)−EpCAM+MUC1+ LPs were purified by fluorescence-activated cell sorting (FACS) at ≥98% purity from normal human reduction mammoplasty samples depleted of hematopoietic cells, endothelial cells, and stromal cells (SCs) (Fig. 1 A and B). These LP and BC subsets were highly enriched in cells with lineage-restricted and bilineage clonogenic activity in vitro, respectively (∼20%; Fig. S1A). Some BCs can also regenerate complete bilayered gland structures containing clonogenic progeny in xenotransplanted immunodeficient mice (8, 9). The inner luminal epithelial layer of the normal human mammary gland also contains many cells devoid of clonogenic activity. These cells, known as luminal cells (LCs), can be isolated as a phenotypically distinct population based on their lack of CD49f and CD90(THY1) expression and high EpCAM and MUC1 expression. LPs are thus assumed to represent an intermediate stage of differentiation between BCs and LCs. FACS analysis of dihydroethidium (DHE)-stained cells showed a higher free-radical superoxide anion (O2°) content in the purified LPs compared with the purified BCs (Fig. 1C). Parallel measurements of intracellular H2O2, generally assumed to be the major intracellular nonradical ROS produced by Significance Our study reveals lineage-specific mechanisms of ROS control and associated sensitivity to oxidative DNA damage in the basal and luminal progenitor-enriched subsets of normal human mammary cells. We show that the primitive luminal cells contain more mitochondria, show greater uptake of O2, sustain and withstand higher levels of ROS, and have mechanisms that allow them to accrue mutagenic levels of oxidative DNA damage. These findings support a growing body of data suggesting the involvement of primitive luminal cells in the generation of human breast cancers. Author contributions: N.K. and C.J.E. designed research; N.K., L.V.N., Y.D., and K.S. performed research; J.T.E. organized accrual of primary tissue; N.K., M.M., P.E., A.R., J.T.E., and C.J.E. analyzed data; and N.K. and C.J.E. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1
Present address: Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada R3E 0T5.
2
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1403813111/-/DCSupplemental.
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Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated “steady-state” normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.
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Fig. 1. Isolation of normal human mammary cells at different stages of differentiation and their changing levels of ROS. (A and B) FACS profiles and gating strategies used to isolate the three different viable (DAPI−) human mammary (CD31−CD45−) subpopulations studied. BCs: CD49f+EpCAM-/low (28) or CD49fhiTHY1+MUC1− cells (clonogenic cell frequency, 18 ± 3%; n = 10). LPs: CD49f+EpCAM+ (28) or CD49fhiMUC1+THY1− cells (clonogenic cell frequency, 22 ± 5%; n = 10). LCs: CD49f-/lowEpCAM+ cells. Mammary SCs: CD49f−EpCAM− cells. (C) Representative FACS histograms of DHE-stained BCs and LPs showing their different O2° levels and comparing the fold increase in median fluorescent intensity (MFI) values of LPs vs. matching BCs. n = 7; P = 0.001. (D) Representative FACS histograms of 2′,7′ –dichlorofluorescein diacetate (DCFDA)-stained BCs and LPs and the fold increase in MFI values of LPs vs. matching BCs. n = 9; P < 0.0001. (E) Western blot analysis of mitochondria-specific β-F1-ATPase protein in each of the three mammary epithelial subpopulations studied (n = 3), with histone H3 as the internal loading control. (F) Comparison of the mitochondrial DNA content relative to genomic DNA as measured by qRT-PCR in extracts of paired isolates of BCs and LPs. Results for LPs are shown relative to the results for BCs in the same sample. n = 6; P = 0.01. (G) Representative FACS histograms of MitoTracker dye-stained BCs and LPs and a comparison of the fold increase in MFI of the LPs vs. matching BCs. n = 9; P < 0.0005. (H) Representative FACS histograms of mitochondrial membrane potential (mΔψ) measured by TMRE staining and a comparison of the fold increase in MFI of the LPs vs. matching BCs. n = 3; P < 0.05. (I) Representative plots of O2 consumption of 105 BCs and matched LPs measured using a Seahorse respirometer in one of four experiments that yielded similar results. Unequal slopes; P < 0.0001.
spontaneous or catalyzed dismutation of mitochondrial O2°, revealed higher levels in the LPs (Fig. 1D). The higher ROS in LPs is consistent with the higher number of mitochondria that they contain, as indicated by their higher levels of mitochondrial-specific β-F1ATPase seen on Western blot analysis (Fig. 1E), higher levels of mitochondrial-specific DNA on quantitative RT-PCR (qRTPCR) analysis (Fig. 1F), and more intense fluorescence when stained with MitoTracker dye (Fig. 1G). The LPs also showed greater sequestration of the tetramethylrhodamine ethyl ester (TMRE) dye (Fig. 1H) and more rapid O2 consumption (Fig. 1I), indicative of increased mitochondrial activity in the LPs. Different Antioxidant Mechanisms Are Active in Different Subsets of Normal Human Mammary Cells. We next asked whether the dif-
ferent subsets of normal human mammary cells would show differences in the mechanisms used to produce and control ROS. We first compared the levels of SOD1, SOD2, and SOD3 present in FACS-purified BCs, LPs, and LCs, because these three enzymes act spatially to maintain steady-state concentrations of O2° through conversion to H2O2 (10). Western blot analysis showed consistent elevations of all three SODs in both LPs and LCs compared with BCs, in agreement with the finding that the total SOD activity in LPs was higher than in the BCs (Fig. 2 A and B). We also investigated the levels of both glutathione-dependent and glutathione-independent enzymes that reduce intracellular H2O2. The former included the glutathione peroxidases (GPX) 1–8 and 1-cysteine peroxiredoxinperoxidase (PRDX) 6, and the latter included catalase and 2cysteine peroxiredoxin-peroxidases, PRDX1–5 (11, 12). Western blot analyses revealed higher levels of many of the antioxidantperoxidases in the LPs and LCs compared with the BCs (Fig. 2A), consistent with the increased total GPX activity detected in LPs compared with BCs (Fig. 2C). Biochemical determination of intracellular levels of NAD(P)H, the ultimate electron donor for antioxidant mechanisms (13) 7790 | www.pnas.org/cgi/doi/10.1073/pnas.1403813111
showed higher levels in LPs (Fig. 2D). Transcript levels for the catalytic subunit of γ-glutamyl-cysteinyl ligase (GCLC), a ratelimiting enzyme in glutathione biosynthesis (14), also were higher in LPs (Fig. 2E). Elevated glutathione levels in LPs were confirmed by FACS-based measurement of monochlorobimanestained cells (Fig. 2F). Taken together, these findings indicate that the LPs in the normal human mammary gland have a stronger antioxidant defense profile than the BCs. In contrast, both qRT-PCR and Western blot analyses showed that the mitochondrial enzyme glutathione peroxidase 2 (GPX2) is present almost exclusively in BCs (Fig. 2 A and G), as suggested by previous serial analyses of gene expression and microarray analyses of their transcript levels (Fig. S2A) (7). On Western blot analysis, GPX2 was largely undetectable in either luminal subset or in the SCs (Fig. S2B). Interestingly, BCs consistently showed higher transcript levels of the antioxidant transcription factor NRF2 and its response element, γ-glutamylcysteinyl ligase modulatory subunit (GCLM) (Fig. 2E). These observations suggest that the BCs of the normal human mammary gland may rely primarily on their lower, albeit tightly regulated, glutathione levels and GPX2 to deal with the lower levels of ROS that they produce compared with the LPs and LCs. In contrast, the latter make use of different, more effective mechanisms to survive the higher levels of ROS that they sustain. Differing Effects of Perturbing Endogenous Control of Oxidative Stress in Different Subsets of Normal Human Mammary Cells. To test
these predictions, we first compared the effect of lowering the level of intracellular glutathione on the proliferative activity of BCs and LPs by adding buthionine sulfoximine (BSO), a potent and selective inhibitor of glutathione synthesis (15), or vehicle directly into the media in which the cells were assayed (Fig. 3A and Fig. S1B). BSO concentrations that had little or no effect on LPs markedly, selectively, and specifically reduced the yield from BCs of total viable cells in 4-d bulk cultures (Fig. 3B) and of Kannan et al.
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colonies produced in either 2D (at 20% O2) or 3D (at 5% O2) 10-d cultures (Fig. 3 C–F). However, the addition of either N-acetyl cysteine (NAC) or Trolox completely reversed the toxic effect of BSO on the BCs and had no effect on the LPs (Fig. 3C). We also tested the effect of pretreating purified BCs and LPs with BSO for 2 d in vitro (under 2D conditions) on their subsequent clonogenic activity assessed either immediately, in the absence of BSO, or, in the case of the BCs only, at 4 wk after being transplanted under the kidney capsules of immunodeficient mice (Fig. 3G). The results showed that BCs with both in vivo and in vitro proliferative activity are highly sensitive to even short-term exposure to BSO (Fig. 3H). As an alternative approach, we depleted GPX2 by transducing bulk normal human mammary cells with a lentivirus encoding a reporter fluorescent protein (VENUS) with or without a shRNA directed against GPX2 transcripts (shRNA GPX2) (Fig. 3I) and then compared the numbers of colonies obtained from the transduced (VENUS+) cells (Fig. 3J). The results of these experiments also showed a much greater shRNA GPX2-mediated reduction of colony yields from the BCs compared with the LPs. These findings indicate that the more primitive human mammary BCs are much more reliant on glutathione-dependent mechanisms to survive even the relatively low levels of ROS that they possess. In contrast, the LPs’ multiple glutathioneindependent antioxidant mechanisms appear sufficient to ensure their survival and clonogenic activity despite their much higher ROS levels. Kannan et al.
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Fig. 2. Differences in the antioxidant mechanisms active at different stages of normal human mammary cell differentiation. (A) Representative Western blots showing the relative levels of the enzymes indicated in all three matching epithelial subpopulations from three samples out of a total of six samples analyzed that all yielded similar results. Histone H3 served as the loading control. (B) Fold increase in total SOD activity in LPs vs. BCs. n = 3; P < 0.001. (C) Fold increase in total glutathione peroxidase activity in LPs vs. BCs. n = 3; P < 0.05. (D) NAD(P) H levels in extracts of equal numbers of BCs and LPs, determined from paired samples by spectrophotometry using the Biovision kit. n = 3; P = 0.01. (E) Fold increase in qRT-PCR–determined transcript levels of the catalytic subunit of GCL (GCLC; n = 10; P < 0.05), its modulatory subunit (GCLM; n = 10; P < 0.0001), and transcription factor Nuclear Factor (Erythroid-Derived 2)-Like 2 (NRF2; n = 8; P < 0.001) in LPs compared with matched BCs. (F) Representative FACS histograms of monochlorobimane-stained BCs and LPs showing their different intracellular reduced-glutathione levels and the fold increase in MFI of LPs vs. matching BCs. n = 7; P < 0.0005. (G) Comparison of qRT-PCR–determined transcript levels of GPX2 (n = 9; P < 0.005) and TP63 (n = 8; P < 0.01) in BCs and LPs.
Differing Effects of Exogenously Derived Oxidative Stress on Different Subsets of Normal Human Mammary Cells. To test the possibility
that LPs might also be more resistant to perturbations that increase their intracellular ROS levels, we next compared the effect of increasing concentrations of H2O2 on the viable cell output of purified BCs and LPs in 4-d bulk cultures and on their clonogenic activities in both 2D and 3D assays (Fig. 4 A–D). Compared with the BCs, the LPs were more resistant to H2O2 exposure in all three assays. Despite this differential toxicity, the differentiated features of the 10-d progeny of the H2O2-treated cells were unchanged compared with untreated controls (Fig. S3). In addition, the selective H2O2 toxicity in BCs was partially neutralized by pretreatment of BCs with NAC or Trolox (Fig. 4C). Ionizing radiation also mediates its lethal effects primarily by producing intracellular ROS, which then cause double-strand breaks in the DNA (16). Responses of mammary cells to ionizing radiation are of particular interest, because this treatment remains a current mainstay in the management of breast cancer. Comparing the effect of increasing X-ray doses on the subsequent clonogenic activity of BCs and LPs (in 2D assays) revealed that the LPs were again more resistant (Fig. 4E). Interestingly, LPs also expressed higher levels of OGG1, MTH1 (also known as NUDT1), and MUTYH(MYH) (Fig. 4F and Fig. S4), enzymes that promote cell survival by reducing the toxic, oxidized nucleotides generated by ROS from free intracellular pools or that have been incorporated into cellular DNA (17–19). PNAS | May 27, 2014 | vol. 111 | no. 21 | 7791
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Given the elevated levels of intracellular ROS characteristic of LPs, along with the long-established association between chronic oxidative stress and oxidative damage to DNA, we then asked whether the LPs would exhibit evidence of oxidative DNA damage even without treatments designed to alter their ROS levels. H2O2 in the presence of transition metal ions, such as iron or copper, is known to cause oxidative damage to DNA via Fenton’s reaction, which generates short-lived hydroxyl radicals (OH°) continuously at low levels in living cells (20). One indicator of such oxidative damage is the formation of genomic 8-oxo-deoxyguanosine (8-oxo-dG) nucleotides (21–23). FACS measurement of genomic 8-oxo-dG levels in the DNA from purified LPs and BCs revealed higher levels in the LPs (Fig. 4G). Thus, LPs that survive oxidative insults presumably continue to accumulate additional oxidative DNA damage (Fig. 4H). Discussion Delineating homeostatic ROS control mechanisms in human mammary BCs and luminal epithelial cells may provide new insights relevant to both normal and malignant breast tissue. Our 7792 | www.pnas.org/cgi/doi/10.1073/pnas.1403813111
LP
Fig. 3. Effects of perturbing endogenous ROS control mechanisms on normal human mammary cells at different stages of differentiation. (A) Schematic showing experimental strategies used to inhibit reduction of intracellular ROS. (B) Effect of BSO (Sigma-Aldrich) on purified BC- and LP-derived cells in 4-d bulk cultures. (C) Purified BCs and LPs were plated in 2D-clonogenic assays with 1.5 mM (two experiments) or 6 mM (three experiments) NAC (Sigma-Aldrich) or 50 μM Trolox (Calbiochem) and 50 μM BSO added 3 h later. Colonies were counted after 8–10 d (total of five experiments). *P < 0.01; **P < 0.001. (D) (Upper) Confocal section of a 3D colony generated from unmanipulated purified BCs or LPs. Cells were stained with a high-affinity F-actin probe (phalloidin) conjugated to tetramethylrhodamine (Molecular Probes). (Lower) H&E-stained section of 3D cultures. (E) Effect of BSO on colony formation by purified BCs and LPs assayed in the 3D system. Values are percent of controls containing no BSO. n = 5; unequal slopes; P < 0.005. (F) Representative photomicrographs of day 12–14 cultures from E. (G) Experimental design for testing the effects of an in vitro 48-h exposure to 100 μM BSO on the immediate clonogenic activity of BCs and LPs (n = 2) and on cells that produce clonogenic cells in transplanted immunodeficient mice (n = 3). (H) Results of experiments performed as shown in G. Black bars represent in vivo regenerated clonogenic cells. (I) Western blot showing the reduction of GPX2 protein expression in transduced HepG2 cells exposed to either the shRNA or control lentivirus and analyzed 3 d later. GAPDH served as the loading control. (J) Effect of GPX2 suppression on the clonogenic activity of mammary cells transduced with a fluorescent reporter (VENUS)encoded lentivirus with or without shRNA targeting GPX2. Transduced cells were selected by FACS at 3 d after virus exposure and plated in 2D-clonogenic assays. Colony yields from shRNA GPX2 transduced cells are shown as percent of values obtained in assays of the same number of control transduced cells. n = 3; P < 0.01.
findings establish the normal human luminal epithelium as a distinct site of oxidative stress resistance in the presence of high intracellular ROS levels mediated by a unique enzymatic repertoire. These features distinguish LPs from BCs, which include very primitive bipotent cells with both luminal and myoepithelial differentiation abilities (24). The unique properties of the LPs include up-regulated levels and activity of SODs and their superoxide substrates, with an accompanying switch in mechanisms used to control ROS levels and promote cell survival in the presence of increased ROS levels. Specifically, this “switch” involves the replacement of a highly glutathione-dependent mechanism primarily mediated by GPX2 in the BCs with a largely glutathione-independent mechanism in the LPs. It is important to note that the observations made here are for cells obtained from “resting” normal adult human mammary glands. Exmaining whether different results will be found for mammary cells subject to the strong hormonal influences of pregnancy and initiation or cessation of lactation will be of interest. The present findings also set the stage for future investigations of the transcriptional regulation of the switch in ROS Kannan et al.
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Fig. 4. Effects of exogenous perturbations of ROS levels on normal human mammary cells at various stages of differentiation. (A) Effect of H2O2 on purified BC- and LP-derived cells in 4-d bulk cultures. (B) Effect of H2O2 on colony formation by BCs and LPs in 2D colony assays. Values are percent of controls containing no H2O2. n = 3; unequal slopes; P < 0.0001. (C) Purified BCs and LPs were plated in 2D clonogenic assays with 50 μM Trolox or 1.5 mM (two experiments) or 6 mM (five experiments) NAC, with 50 μM H2O2 added 3 h later. Colonies were counted after 8–10 d. n = 7; *P < 0.001; **P < 0.0001. (D) Same design as in B, but with assays performed in 3D cultures. n = 4; unequal slopes; P < 0.005. (E) Effect of 300 kVp (1 Gy/min) X-rays on subsequent colony formation by unseparated mammary cells in 2D assays. BC- and LP-derived colonies were distinguished morphologically. Values are percent of controls given a sham treatment. n = 3; unequal slopes; P < 0.0001. (F) Representative Western blots showing the relative levels of OGG1, MTH1, and MUTYH in three matching subpopulations from three samples out of a total of six samples analyzed that all yielded similar results. Histone H3 served as the loading control. (G) Representative FACS histogram of BC and LP cells stained for the 8-oxo-dG DNA adduct using a specific FITC-labeled probe (Left) and fold change in MFIs in LPs relative to matching BCs (Right). n = 4; P < 0.005. Arrows indicate LPs and BCs treated similarly but unstained for the 8-oxo-dG DNA adduct. (H) Model illustrating the mechanisms underlying the changes in production, control, and responses to altered ROS levels in BCs and LPs.
control that demarcates the generation of LPs from BCs. Various transcription factors show dramatic differences in expression in these two subsets, of which TP63, NOTCH3, and GATA3 are notable examples (7, 9, 25, 26). Of these, TP63 has been reported to increase the tolerance of breast cancer cells to oxidative stress by transactivating GPX2 (27). It is also possible that the glutathione-independent and oxidative DNA damage control mechanisms that confer resistance to oxidative stress described here as characteristic of normal LPs and LCs may operate in their malignant derivatives, making them more resistant to oxidative treatments. The glutathione-independence of the LPs and their more differentiated LC progeny is associated with up-regulated expression of numerous enzymes that, in combination, appear able to counteract at least some of the potentially lethally mutagenic effects of the ROS levels that these cells produce. These observations add to a growing body of data revealing other mechanisms that predispose normal human luminal cells to DNA damage (28). Thus, the up-regulated expression of multiple antioxidant enzymes at the point of mammary progenitor cell restriction to the luminal lineage could contribute to multiple mechanisms that promote the death of LCs and allow lumen formation (5). Consistent with this hypothesis is our present finding of increased expression in LPs and LCs of unique PRDXperoxidases that facilitate a type of “floodgate” control of ROS levels owing to these enzymes’ innate capability to be inactivated selectively in response to various stimuli (29, 30). Materials and Methods Cells. Histologically confirmed normal anonymized tissue from women undergoing cosmetic reduction mammoplasty was obtained following procedures approved by the University of British Columbia’s Ethics Review Board.
Kannan et al.
Tissue was processed and viable single cells were isolated as described previously (31). In brief, reduction tissues were first minced with a scalpel and dissociated in Ham’s F-12 medium and DMEM (1:1 vol/vol; Stem Cell Technologies) supplemented with 2% wt/vol BSA (Fraction V; Life Technologies), 300 U/mL collagenase (Sigma-Aldrich), and 100 U/mL hyaluronidase (SigmaAldrich), from which an epithelial organoid-rich pellet was obtained after centrifugation at 80 × g for 4 min. Single-cell suspensions were then obtained after further incubation in 1 mM EDTA supplemented with 2.5 mg/mL trypsin (Stem Cell Technologies), 5 mg/mL dispase (Stem Cell Technologies), and 100 μg/mL DNase1 (Sigma-Aldrich), followed by filtration of the suspension through a 40-μm strainer. Mammary cells were then stained with antibodies listed in Table S1, and subsets were isolated as described previously (7, 8), using a protocol in which hematopoietic and endothelial cells were removed using fluorochrome-conjugated antibodies to CD45 and CD31, respectively. Cells were also stained with DAPI to eliminate dead (DAPI+) cells. SCs were eliminated based on absent or reduced expression of EpCAM and/ or CD49f. Viable subsets of epithelial cells were then isolated at ≥98% purity using a FACSAria or Influx II cell sorter (BD Biosciences), as described in Fig. 1 A and B. In Vitro and in Vivo Growth Assays. Bulk cultures for toxicity experiments were initiated by seeding 5,000 purified BCs and LPs on collagen-coated wells in 96-well plates in SF7 media supplemented with 5% FBS, followed by colorimetric assessment for viable cells after a 4-h incubation with 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), as described previously (32). The 2D and 3D clonogenic assays were performed as described in Fig. S1. Intracellular Flow Cytometry. For live-cell analyses, cells were either costained with antibodies to allow the isolation of subsets or were first purified and then incubated for 30 min at 37 °C with 100 nM MitoTracker, 10 μM DCFDA, 5 μM dihydroethidium, 400 nM TMRE (all from Invitrogen), or 50 μM monochlorobimane (Sigma-Aldrich). For the detection of 8-oxo-dG DNA adducts, FACS-purified cells were secondarily fixed, permeabilized, and then stained with a specific Calbiochem 8-oxo-dG–binding FITC probe (EMD Millipore).
PNAS | May 27, 2014 | vol. 111 | no. 21 | 7793
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Stained cells were analyzed using a FACSAria, FACSInfluxII, or Fortessa machine (BD Biosciences). O2 Consumption. For measurement of O2 consumption, 105 purified cells were seeded in Seahorse XF96 cell culture plates (with and without a Matrigel coating) and cultured for 24 h in SF7 media supplemented with 5% FBS. On the next day, the medium was changed to 150 μL of bicarbonate-free assay medium (adjusted to 17 mM glucose). O2 levels in the medium were measured at 15-s intervals for 3 min with a Seahorse Biosciences XFe 96 Analyzer. Antioxidant Activity Assays. A total SOD activity kit (Enzo Life Sciences) and total glutathione peroxidase activity kit (Cedarlane) were used to measure respective antioxidant activities in purified cells.
Lentiviral Transduction. A human GPX2 targeting shRNA (Open Biosystems) was cloned into the pLKO.1-VENUS lentiviral vector, and virus-containing supernatants were generated and used to infect test cells as described previously (7). Transduced (VENUS+) cells were isolated 2–3 d later and assayed for colony formation. The shRNA activity of the lenticonstruct was confirmed by Western blot analyses of GPX2 levels in extracts of transduced human hepatocarcinoma HepG2 cells cultured for 2–3 d before isolation of the transduced (VENUS+) cells. Graphs and Statistics. GraphPad Prism 6 software was used for statistical comparisons and graph generation. Values are reported as mean ± SEM, and P values were generated using the Student t test. The number of independent experiments with cells from different donors is shown in brackets.
qRT-PCR and Western Blot Analyses. Cells were rapidly lysed for RNA and protein isolation and analyzed by standard methods (33). The antibodies and primers used are listed in Tables S1 and S2, respectively.
ACKNOWLEDGMENTS. We acknowledge the excellent help of D. Wilkinson in collecting and processing samples of normal human mammary tissue. We thank Drs. J. Sproul and N. Van Laeken for providing the reduction mammoplasty material, and G. Edin, M. Hale, D. Ko, and W. Xu for technical and FACS assistance. N.K. held a Canadian Breast Cancer Foundation (CBCF) British Columbia/Yukon Postdoctoral Fellowship. This work was also supported by operating grants from the Canadian Breast Cancer Research Alliance, funded by the Canadian Cancer Society, and from the British Columbia/Yukon division of the CBCF.
1. Nathan C, Ding A (2010) SnapShot: Reactive oxygen intermediates (ROI). Cell 140(6): 951, e2. 2. Cooke MS, Evans MD, Dizdaroglu M, Lunec J (2003) Oxidative DNA damage: Mechanisms, mutation, and disease. FASEB J 17(10):1195–1214. 3. Kryston TB, Georgiev AB, Pissis P, Georgakilas AG (2011) Role of oxidative stress and DNA damage in human carcinogenesis. Mutat Res 711(1-2):193–201. 4. Diehn M, et al. (2009) Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature 458(7239):780–783. 5. Schafer ZT, et al. (2009) Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461(7260):109–113. 6. Stingl J, Raouf A, Eirew P, Eaves CJ (2006) Deciphering the mammary epithelial cell hierarchy. Cell Cycle 5(14):1519–1522. 7. Raouf A, et al. (2008) Transcriptome analysis of the normal human mammary cell commitment and differentiation process. Cell Stem Cell 3(1):109–118. 8. Eirew P, et al. (2008) A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability. Nat Med 14(12):1384–1389. 9. Lim E, et al. (2009) Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat Med 15(8):907–913. 10. Oberley LW, Buettner GR (1979) Role of superoxide dismutase in cancer: A review. Cancer Res 39(4):1141–1149. 11. McQueen CA, ed (2010) Comprehensive Toxicology (Elsevier, Kidlington, UK), 2nd Ed, pp 325–328. 12. Rhee SG, Woo HA, Kil IS, Bae SH (2012) Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides. J Biol Chem 287(7):4403–4410. 13. Kirsch M, De Groot H (2001) NAD(P)H, a directly operating antioxidant? FASEB J 15(9): 1569–1574. 14. Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52(1):711–760. 15. Griffith OW, Meister A (1979) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem 254(16): 7558–7560. 16. Riley PA (1994) Free radicals in biology: Oxidative stress and the effects of ionizing radiation. Int J Radiat Biol 65(1):27–33. 17. Gros L, Saparbaev MK, Laval J (2002) Enzymology of the repair of free radicals-induced DNA damage. Oncogene 21(58):8905–8925.
18. Rai P, et al. (2009) Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence. Proc Natl Acad Sci USA 106(1):169–174. 19. David SS, O’Shea VL, Kundu S (2007) Base-excision repair of oxidative DNA damage. Nature 447(7147):941–950. 20. Marnett LJ (2000) Oxyradicals and DNA damage. Carcinogenesis 21(3):361–370. 21. Maki H, Sekiguchi M (1992) MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis. Nature 355(6357):273–275. 22. Cheng KC, Cahill DS, Kasai H, Nishimura S, Loeb LA (1992) 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G→T and A→C substitutions. J Biol Chem 267(1):166–172. 23. Grollman AP, Moriya M (1993) Mutagenesis by 8-oxoguanine: An enemy within. Trends Genet 9(7):246–249. 24. Nguyen LV, et al. (2014) Clonal analysis via barcoding reveals diverse growth and differentiation of transplanted mouse and human mammary stem cells. Cell Stem Cell 14(2):253–263. 25. Asselin-Labat M-L, et al. (2007) Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol 9(2):201–209. 26. Kouros-Mehr H, Slorach EM, Sternlicht MD, Werb Z (2006) GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell 127(5):1041–1055. 27. Yan W, Chen X (2006) GPX2, a direct target of p63, inhibits oxidative stress-induced apoptosis in a p53-dependent manner. J Biol Chem 281(12):7856–7862. 28. Kannan N, et al. (2013) The luminal progenitor compartment of the normal human mammary gland constitutes a unique site of telomere dysfunction. Stem Cell Rep 1(1): 28–37. 29. Wood ZA, Poole LB, Karplus PA (2003) Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science 300(5619):650–653. 30. Jarvis RM, Hughes SM, Ledgerwood EC (2012) Peroxiredoxin 1 functions as a signal peroxidase to receive, transduce, and transmit peroxide signals in mammalian cells. Free Radic Biol Med 53(7):1522–1530. 31. Stingl J, Emerman JT, Eaves CJ (2005) Enzymatic dissociation and culture of normal human mammary tissue to detect progenitor activity. Methods Mol Biol 290:249–263. 32. Kawamura E, et al. (2013) Identification of novel small molecule inhibitors of centrosome clustering in cancer cells. Oncotarget 4(10):1763–1776. 33. Eirew P, et al. (2012) Aldehyde dehydrogenase activity is a biomarker of primitive normal human mammary luminal cells. Stem Cells 30(2):344–348.
5
NAD(P)H Levels. NAD(P)H was measured on lysed aliquots of 10 FACS-purified cells using a commercially available kit (BioVision Research Products).
7794 | www.pnas.org/cgi/doi/10.1073/pnas.1403813111
Kannan et al.
Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research at Princess Margaret Cancer Centre, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada, and approved April 9, 2014 (received for review March 5, 2014)
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human epithelial stem and progenitor cells mammary differentiation 3D clonogenic assay superoxide dismutase peroxiredoxin
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ellular synthesis of different reactive oxygen species (ROS) results primarily from the incomplete reduction of molecular oxygen in mitochondria to generate free radical superoxide anions. ROS also are produced when cells are exposed to different environmental sources of oxidative stressors, including ionizing radiation. Together, these regulate many normal cellular processes and also contribute to DNA damage, tumorigenesis, and cell death (1–3). In mice, the inner layer of “luminal” epithelial cells of the normal adult mammary gland have been found to contain higher levels of ROS than the outer “basal” layer of epithelial cells (4). The basis for these differences in ROS levels in the two cell types has been attributed to differences in their content of mitochondria; however, their stress response mechanisms have not been defined. Even less is known about ROS levels and their control in the normal adult human mammary gland, which consists of a similar continuous bilayered epithelial network of ducts and terminal alveolae. MCF10A cells are an immortal but nontumorigenic human mammary cell line that, when grown in 3D Matrigel cultures, generates multilayered spheres in which a lumen forms owing to the acquisition of lethal levels of ROS by the inner cells (5). These studies have suggested that ROS regulation plays an important role in the structural morphogenesis and homeostasis of normal adult human mammary tissue. Here we report the results of experiments designed to investigate the levels of ROS and their control in highly purified populations of viable luminal progenitors (LPs) and basal cells (BCs) isolated from normal human breast tissue (6–8). Our results confirm the different levels of ROS identified in the corresponding subsets of mouse mammary cells and further characterize the distinct antioxidant
www.pnas.org/cgi/doi/10.1073/pnas.1403813111
mechanisms operative in the human cells and their associated differential sensitivity to treatments that elevate ROS levels. Results Differing ROS Levels in Different Subsets of Primary Human Mammary + + −/low MUC1− BCs Epithelial Cells. CD49f CD90(THY1) EpCAM
and CD49f+CD90(THY1)−EpCAM+MUC1+ LPs were purified by fluorescence-activated cell sorting (FACS) at ≥98% purity from normal human reduction mammoplasty samples depleted of hematopoietic cells, endothelial cells, and stromal cells (SCs) (Fig. 1 A and B). These LP and BC subsets were highly enriched in cells with lineage-restricted and bilineage clonogenic activity in vitro, respectively (∼20%; Fig. S1A). Some BCs can also regenerate complete bilayered gland structures containing clonogenic progeny in xenotransplanted immunodeficient mice (8, 9). The inner luminal epithelial layer of the normal human mammary gland also contains many cells devoid of clonogenic activity. These cells, known as luminal cells (LCs), can be isolated as a phenotypically distinct population based on their lack of CD49f and CD90(THY1) expression and high EpCAM and MUC1 expression. LPs are thus assumed to represent an intermediate stage of differentiation between BCs and LCs. FACS analysis of dihydroethidium (DHE)-stained cells showed a higher free-radical superoxide anion (O2°) content in the purified LPs compared with the purified BCs (Fig. 1C). Parallel measurements of intracellular H2O2, generally assumed to be the major intracellular nonradical ROS produced by Significance Our study reveals lineage-specific mechanisms of ROS control and associated sensitivity to oxidative DNA damage in the basal and luminal progenitor-enriched subsets of normal human mammary cells. We show that the primitive luminal cells contain more mitochondria, show greater uptake of O2, sustain and withstand higher levels of ROS, and have mechanisms that allow them to accrue mutagenic levels of oxidative DNA damage. These findings support a growing body of data suggesting the involvement of primitive luminal cells in the generation of human breast cancers. Author contributions: N.K. and C.J.E. designed research; N.K., L.V.N., Y.D., and K.S. performed research; J.T.E. organized accrual of primary tissue; N.K., M.M., P.E., A.R., J.T.E., and C.J.E. analyzed data; and N.K. and C.J.E. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1
Present address: Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada R3E 0T5.
2
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1403813111/-/DCSupplemental.
PNAS | May 27, 2014 | vol. 111 | no. 21 | 7789–7794
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Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated “steady-state” normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.
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Fig. 1. Isolation of normal human mammary cells at different stages of differentiation and their changing levels of ROS. (A and B) FACS profiles and gating strategies used to isolate the three different viable (DAPI−) human mammary (CD31−CD45−) subpopulations studied. BCs: CD49f+EpCAM-/low (28) or CD49fhiTHY1+MUC1− cells (clonogenic cell frequency, 18 ± 3%; n = 10). LPs: CD49f+EpCAM+ (28) or CD49fhiMUC1+THY1− cells (clonogenic cell frequency, 22 ± 5%; n = 10). LCs: CD49f-/lowEpCAM+ cells. Mammary SCs: CD49f−EpCAM− cells. (C) Representative FACS histograms of DHE-stained BCs and LPs showing their different O2° levels and comparing the fold increase in median fluorescent intensity (MFI) values of LPs vs. matching BCs. n = 7; P = 0.001. (D) Representative FACS histograms of 2′,7′ –dichlorofluorescein diacetate (DCFDA)-stained BCs and LPs and the fold increase in MFI values of LPs vs. matching BCs. n = 9; P < 0.0001. (E) Western blot analysis of mitochondria-specific β-F1-ATPase protein in each of the three mammary epithelial subpopulations studied (n = 3), with histone H3 as the internal loading control. (F) Comparison of the mitochondrial DNA content relative to genomic DNA as measured by qRT-PCR in extracts of paired isolates of BCs and LPs. Results for LPs are shown relative to the results for BCs in the same sample. n = 6; P = 0.01. (G) Representative FACS histograms of MitoTracker dye-stained BCs and LPs and a comparison of the fold increase in MFI of the LPs vs. matching BCs. n = 9; P < 0.0005. (H) Representative FACS histograms of mitochondrial membrane potential (mΔψ) measured by TMRE staining and a comparison of the fold increase in MFI of the LPs vs. matching BCs. n = 3; P < 0.05. (I) Representative plots of O2 consumption of 105 BCs and matched LPs measured using a Seahorse respirometer in one of four experiments that yielded similar results. Unequal slopes; P < 0.0001.
spontaneous or catalyzed dismutation of mitochondrial O2°, revealed higher levels in the LPs (Fig. 1D). The higher ROS in LPs is consistent with the higher number of mitochondria that they contain, as indicated by their higher levels of mitochondrial-specific β-F1ATPase seen on Western blot analysis (Fig. 1E), higher levels of mitochondrial-specific DNA on quantitative RT-PCR (qRTPCR) analysis (Fig. 1F), and more intense fluorescence when stained with MitoTracker dye (Fig. 1G). The LPs also showed greater sequestration of the tetramethylrhodamine ethyl ester (TMRE) dye (Fig. 1H) and more rapid O2 consumption (Fig. 1I), indicative of increased mitochondrial activity in the LPs. Different Antioxidant Mechanisms Are Active in Different Subsets of Normal Human Mammary Cells. We next asked whether the dif-
ferent subsets of normal human mammary cells would show differences in the mechanisms used to produce and control ROS. We first compared the levels of SOD1, SOD2, and SOD3 present in FACS-purified BCs, LPs, and LCs, because these three enzymes act spatially to maintain steady-state concentrations of O2° through conversion to H2O2 (10). Western blot analysis showed consistent elevations of all three SODs in both LPs and LCs compared with BCs, in agreement with the finding that the total SOD activity in LPs was higher than in the BCs (Fig. 2 A and B). We also investigated the levels of both glutathione-dependent and glutathione-independent enzymes that reduce intracellular H2O2. The former included the glutathione peroxidases (GPX) 1–8 and 1-cysteine peroxiredoxinperoxidase (PRDX) 6, and the latter included catalase and 2cysteine peroxiredoxin-peroxidases, PRDX1–5 (11, 12). Western blot analyses revealed higher levels of many of the antioxidantperoxidases in the LPs and LCs compared with the BCs (Fig. 2A), consistent with the increased total GPX activity detected in LPs compared with BCs (Fig. 2C). Biochemical determination of intracellular levels of NAD(P)H, the ultimate electron donor for antioxidant mechanisms (13) 7790 | www.pnas.org/cgi/doi/10.1073/pnas.1403813111
showed higher levels in LPs (Fig. 2D). Transcript levels for the catalytic subunit of γ-glutamyl-cysteinyl ligase (GCLC), a ratelimiting enzyme in glutathione biosynthesis (14), also were higher in LPs (Fig. 2E). Elevated glutathione levels in LPs were confirmed by FACS-based measurement of monochlorobimanestained cells (Fig. 2F). Taken together, these findings indicate that the LPs in the normal human mammary gland have a stronger antioxidant defense profile than the BCs. In contrast, both qRT-PCR and Western blot analyses showed that the mitochondrial enzyme glutathione peroxidase 2 (GPX2) is present almost exclusively in BCs (Fig. 2 A and G), as suggested by previous serial analyses of gene expression and microarray analyses of their transcript levels (Fig. S2A) (7). On Western blot analysis, GPX2 was largely undetectable in either luminal subset or in the SCs (Fig. S2B). Interestingly, BCs consistently showed higher transcript levels of the antioxidant transcription factor NRF2 and its response element, γ-glutamylcysteinyl ligase modulatory subunit (GCLM) (Fig. 2E). These observations suggest that the BCs of the normal human mammary gland may rely primarily on their lower, albeit tightly regulated, glutathione levels and GPX2 to deal with the lower levels of ROS that they produce compared with the LPs and LCs. In contrast, the latter make use of different, more effective mechanisms to survive the higher levels of ROS that they sustain. Differing Effects of Perturbing Endogenous Control of Oxidative Stress in Different Subsets of Normal Human Mammary Cells. To test
these predictions, we first compared the effect of lowering the level of intracellular glutathione on the proliferative activity of BCs and LPs by adding buthionine sulfoximine (BSO), a potent and selective inhibitor of glutathione synthesis (15), or vehicle directly into the media in which the cells were assayed (Fig. 3A and Fig. S1B). BSO concentrations that had little or no effect on LPs markedly, selectively, and specifically reduced the yield from BCs of total viable cells in 4-d bulk cultures (Fig. 3B) and of Kannan et al.
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colonies produced in either 2D (at 20% O2) or 3D (at 5% O2) 10-d cultures (Fig. 3 C–F). However, the addition of either N-acetyl cysteine (NAC) or Trolox completely reversed the toxic effect of BSO on the BCs and had no effect on the LPs (Fig. 3C). We also tested the effect of pretreating purified BCs and LPs with BSO for 2 d in vitro (under 2D conditions) on their subsequent clonogenic activity assessed either immediately, in the absence of BSO, or, in the case of the BCs only, at 4 wk after being transplanted under the kidney capsules of immunodeficient mice (Fig. 3G). The results showed that BCs with both in vivo and in vitro proliferative activity are highly sensitive to even short-term exposure to BSO (Fig. 3H). As an alternative approach, we depleted GPX2 by transducing bulk normal human mammary cells with a lentivirus encoding a reporter fluorescent protein (VENUS) with or without a shRNA directed against GPX2 transcripts (shRNA GPX2) (Fig. 3I) and then compared the numbers of colonies obtained from the transduced (VENUS+) cells (Fig. 3J). The results of these experiments also showed a much greater shRNA GPX2-mediated reduction of colony yields from the BCs compared with the LPs. These findings indicate that the more primitive human mammary BCs are much more reliant on glutathione-dependent mechanisms to survive even the relatively low levels of ROS that they possess. In contrast, the LPs’ multiple glutathioneindependent antioxidant mechanisms appear sufficient to ensure their survival and clonogenic activity despite their much higher ROS levels. Kannan et al.
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Fig. 2. Differences in the antioxidant mechanisms active at different stages of normal human mammary cell differentiation. (A) Representative Western blots showing the relative levels of the enzymes indicated in all three matching epithelial subpopulations from three samples out of a total of six samples analyzed that all yielded similar results. Histone H3 served as the loading control. (B) Fold increase in total SOD activity in LPs vs. BCs. n = 3; P < 0.001. (C) Fold increase in total glutathione peroxidase activity in LPs vs. BCs. n = 3; P < 0.05. (D) NAD(P) H levels in extracts of equal numbers of BCs and LPs, determined from paired samples by spectrophotometry using the Biovision kit. n = 3; P = 0.01. (E) Fold increase in qRT-PCR–determined transcript levels of the catalytic subunit of GCL (GCLC; n = 10; P < 0.05), its modulatory subunit (GCLM; n = 10; P < 0.0001), and transcription factor Nuclear Factor (Erythroid-Derived 2)-Like 2 (NRF2; n = 8; P < 0.001) in LPs compared with matched BCs. (F) Representative FACS histograms of monochlorobimane-stained BCs and LPs showing their different intracellular reduced-glutathione levels and the fold increase in MFI of LPs vs. matching BCs. n = 7; P < 0.0005. (G) Comparison of qRT-PCR–determined transcript levels of GPX2 (n = 9; P < 0.005) and TP63 (n = 8; P < 0.01) in BCs and LPs.
Differing Effects of Exogenously Derived Oxidative Stress on Different Subsets of Normal Human Mammary Cells. To test the possibility
that LPs might also be more resistant to perturbations that increase their intracellular ROS levels, we next compared the effect of increasing concentrations of H2O2 on the viable cell output of purified BCs and LPs in 4-d bulk cultures and on their clonogenic activities in both 2D and 3D assays (Fig. 4 A–D). Compared with the BCs, the LPs were more resistant to H2O2 exposure in all three assays. Despite this differential toxicity, the differentiated features of the 10-d progeny of the H2O2-treated cells were unchanged compared with untreated controls (Fig. S3). In addition, the selective H2O2 toxicity in BCs was partially neutralized by pretreatment of BCs with NAC or Trolox (Fig. 4C). Ionizing radiation also mediates its lethal effects primarily by producing intracellular ROS, which then cause double-strand breaks in the DNA (16). Responses of mammary cells to ionizing radiation are of particular interest, because this treatment remains a current mainstay in the management of breast cancer. Comparing the effect of increasing X-ray doses on the subsequent clonogenic activity of BCs and LPs (in 2D assays) revealed that the LPs were again more resistant (Fig. 4E). Interestingly, LPs also expressed higher levels of OGG1, MTH1 (also known as NUDT1), and MUTYH(MYH) (Fig. 4F and Fig. S4), enzymes that promote cell survival by reducing the toxic, oxidized nucleotides generated by ROS from free intracellular pools or that have been incorporated into cellular DNA (17–19). PNAS | May 27, 2014 | vol. 111 | no. 21 | 7791
MEDICAL SCIENCES
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Given the elevated levels of intracellular ROS characteristic of LPs, along with the long-established association between chronic oxidative stress and oxidative damage to DNA, we then asked whether the LPs would exhibit evidence of oxidative DNA damage even without treatments designed to alter their ROS levels. H2O2 in the presence of transition metal ions, such as iron or copper, is known to cause oxidative damage to DNA via Fenton’s reaction, which generates short-lived hydroxyl radicals (OH°) continuously at low levels in living cells (20). One indicator of such oxidative damage is the formation of genomic 8-oxo-deoxyguanosine (8-oxo-dG) nucleotides (21–23). FACS measurement of genomic 8-oxo-dG levels in the DNA from purified LPs and BCs revealed higher levels in the LPs (Fig. 4G). Thus, LPs that survive oxidative insults presumably continue to accumulate additional oxidative DNA damage (Fig. 4H). Discussion Delineating homeostatic ROS control mechanisms in human mammary BCs and luminal epithelial cells may provide new insights relevant to both normal and malignant breast tissue. Our 7792 | www.pnas.org/cgi/doi/10.1073/pnas.1403813111
LP
Fig. 3. Effects of perturbing endogenous ROS control mechanisms on normal human mammary cells at different stages of differentiation. (A) Schematic showing experimental strategies used to inhibit reduction of intracellular ROS. (B) Effect of BSO (Sigma-Aldrich) on purified BC- and LP-derived cells in 4-d bulk cultures. (C) Purified BCs and LPs were plated in 2D-clonogenic assays with 1.5 mM (two experiments) or 6 mM (three experiments) NAC (Sigma-Aldrich) or 50 μM Trolox (Calbiochem) and 50 μM BSO added 3 h later. Colonies were counted after 8–10 d (total of five experiments). *P < 0.01; **P < 0.001. (D) (Upper) Confocal section of a 3D colony generated from unmanipulated purified BCs or LPs. Cells were stained with a high-affinity F-actin probe (phalloidin) conjugated to tetramethylrhodamine (Molecular Probes). (Lower) H&E-stained section of 3D cultures. (E) Effect of BSO on colony formation by purified BCs and LPs assayed in the 3D system. Values are percent of controls containing no BSO. n = 5; unequal slopes; P < 0.005. (F) Representative photomicrographs of day 12–14 cultures from E. (G) Experimental design for testing the effects of an in vitro 48-h exposure to 100 μM BSO on the immediate clonogenic activity of BCs and LPs (n = 2) and on cells that produce clonogenic cells in transplanted immunodeficient mice (n = 3). (H) Results of experiments performed as shown in G. Black bars represent in vivo regenerated clonogenic cells. (I) Western blot showing the reduction of GPX2 protein expression in transduced HepG2 cells exposed to either the shRNA or control lentivirus and analyzed 3 d later. GAPDH served as the loading control. (J) Effect of GPX2 suppression on the clonogenic activity of mammary cells transduced with a fluorescent reporter (VENUS)encoded lentivirus with or without shRNA targeting GPX2. Transduced cells were selected by FACS at 3 d after virus exposure and plated in 2D-clonogenic assays. Colony yields from shRNA GPX2 transduced cells are shown as percent of values obtained in assays of the same number of control transduced cells. n = 3; P < 0.01.
findings establish the normal human luminal epithelium as a distinct site of oxidative stress resistance in the presence of high intracellular ROS levels mediated by a unique enzymatic repertoire. These features distinguish LPs from BCs, which include very primitive bipotent cells with both luminal and myoepithelial differentiation abilities (24). The unique properties of the LPs include up-regulated levels and activity of SODs and their superoxide substrates, with an accompanying switch in mechanisms used to control ROS levels and promote cell survival in the presence of increased ROS levels. Specifically, this “switch” involves the replacement of a highly glutathione-dependent mechanism primarily mediated by GPX2 in the BCs with a largely glutathione-independent mechanism in the LPs. It is important to note that the observations made here are for cells obtained from “resting” normal adult human mammary glands. Exmaining whether different results will be found for mammary cells subject to the strong hormonal influences of pregnancy and initiation or cessation of lactation will be of interest. The present findings also set the stage for future investigations of the transcriptional regulation of the switch in ROS Kannan et al.
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Fig. 4. Effects of exogenous perturbations of ROS levels on normal human mammary cells at various stages of differentiation. (A) Effect of H2O2 on purified BC- and LP-derived cells in 4-d bulk cultures. (B) Effect of H2O2 on colony formation by BCs and LPs in 2D colony assays. Values are percent of controls containing no H2O2. n = 3; unequal slopes; P < 0.0001. (C) Purified BCs and LPs were plated in 2D clonogenic assays with 50 μM Trolox or 1.5 mM (two experiments) or 6 mM (five experiments) NAC, with 50 μM H2O2 added 3 h later. Colonies were counted after 8–10 d. n = 7; *P < 0.001; **P < 0.0001. (D) Same design as in B, but with assays performed in 3D cultures. n = 4; unequal slopes; P < 0.005. (E) Effect of 300 kVp (1 Gy/min) X-rays on subsequent colony formation by unseparated mammary cells in 2D assays. BC- and LP-derived colonies were distinguished morphologically. Values are percent of controls given a sham treatment. n = 3; unequal slopes; P < 0.0001. (F) Representative Western blots showing the relative levels of OGG1, MTH1, and MUTYH in three matching subpopulations from three samples out of a total of six samples analyzed that all yielded similar results. Histone H3 served as the loading control. (G) Representative FACS histogram of BC and LP cells stained for the 8-oxo-dG DNA adduct using a specific FITC-labeled probe (Left) and fold change in MFIs in LPs relative to matching BCs (Right). n = 4; P < 0.005. Arrows indicate LPs and BCs treated similarly but unstained for the 8-oxo-dG DNA adduct. (H) Model illustrating the mechanisms underlying the changes in production, control, and responses to altered ROS levels in BCs and LPs.
control that demarcates the generation of LPs from BCs. Various transcription factors show dramatic differences in expression in these two subsets, of which TP63, NOTCH3, and GATA3 are notable examples (7, 9, 25, 26). Of these, TP63 has been reported to increase the tolerance of breast cancer cells to oxidative stress by transactivating GPX2 (27). It is also possible that the glutathione-independent and oxidative DNA damage control mechanisms that confer resistance to oxidative stress described here as characteristic of normal LPs and LCs may operate in their malignant derivatives, making them more resistant to oxidative treatments. The glutathione-independence of the LPs and their more differentiated LC progeny is associated with up-regulated expression of numerous enzymes that, in combination, appear able to counteract at least some of the potentially lethally mutagenic effects of the ROS levels that these cells produce. These observations add to a growing body of data revealing other mechanisms that predispose normal human luminal cells to DNA damage (28). Thus, the up-regulated expression of multiple antioxidant enzymes at the point of mammary progenitor cell restriction to the luminal lineage could contribute to multiple mechanisms that promote the death of LCs and allow lumen formation (5). Consistent with this hypothesis is our present finding of increased expression in LPs and LCs of unique PRDXperoxidases that facilitate a type of “floodgate” control of ROS levels owing to these enzymes’ innate capability to be inactivated selectively in response to various stimuli (29, 30). Materials and Methods Cells. Histologically confirmed normal anonymized tissue from women undergoing cosmetic reduction mammoplasty was obtained following procedures approved by the University of British Columbia’s Ethics Review Board.
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Tissue was processed and viable single cells were isolated as described previously (31). In brief, reduction tissues were first minced with a scalpel and dissociated in Ham’s F-12 medium and DMEM (1:1 vol/vol; Stem Cell Technologies) supplemented with 2% wt/vol BSA (Fraction V; Life Technologies), 300 U/mL collagenase (Sigma-Aldrich), and 100 U/mL hyaluronidase (SigmaAldrich), from which an epithelial organoid-rich pellet was obtained after centrifugation at 80 × g for 4 min. Single-cell suspensions were then obtained after further incubation in 1 mM EDTA supplemented with 2.5 mg/mL trypsin (Stem Cell Technologies), 5 mg/mL dispase (Stem Cell Technologies), and 100 μg/mL DNase1 (Sigma-Aldrich), followed by filtration of the suspension through a 40-μm strainer. Mammary cells were then stained with antibodies listed in Table S1, and subsets were isolated as described previously (7, 8), using a protocol in which hematopoietic and endothelial cells were removed using fluorochrome-conjugated antibodies to CD45 and CD31, respectively. Cells were also stained with DAPI to eliminate dead (DAPI+) cells. SCs were eliminated based on absent or reduced expression of EpCAM and/ or CD49f. Viable subsets of epithelial cells were then isolated at ≥98% purity using a FACSAria or Influx II cell sorter (BD Biosciences), as described in Fig. 1 A and B. In Vitro and in Vivo Growth Assays. Bulk cultures for toxicity experiments were initiated by seeding 5,000 purified BCs and LPs on collagen-coated wells in 96-well plates in SF7 media supplemented with 5% FBS, followed by colorimetric assessment for viable cells after a 4-h incubation with 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), as described previously (32). The 2D and 3D clonogenic assays were performed as described in Fig. S1. Intracellular Flow Cytometry. For live-cell analyses, cells were either costained with antibodies to allow the isolation of subsets or were first purified and then incubated for 30 min at 37 °C with 100 nM MitoTracker, 10 μM DCFDA, 5 μM dihydroethidium, 400 nM TMRE (all from Invitrogen), or 50 μM monochlorobimane (Sigma-Aldrich). For the detection of 8-oxo-dG DNA adducts, FACS-purified cells were secondarily fixed, permeabilized, and then stained with a specific Calbiochem 8-oxo-dG–binding FITC probe (EMD Millipore).
PNAS | May 27, 2014 | vol. 111 | no. 21 | 7793
MEDICAL SCIENCES
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Stained cells were analyzed using a FACSAria, FACSInfluxII, or Fortessa machine (BD Biosciences). O2 Consumption. For measurement of O2 consumption, 105 purified cells were seeded in Seahorse XF96 cell culture plates (with and without a Matrigel coating) and cultured for 24 h in SF7 media supplemented with 5% FBS. On the next day, the medium was changed to 150 μL of bicarbonate-free assay medium (adjusted to 17 mM glucose). O2 levels in the medium were measured at 15-s intervals for 3 min with a Seahorse Biosciences XFe 96 Analyzer. Antioxidant Activity Assays. A total SOD activity kit (Enzo Life Sciences) and total glutathione peroxidase activity kit (Cedarlane) were used to measure respective antioxidant activities in purified cells.
Lentiviral Transduction. A human GPX2 targeting shRNA (Open Biosystems) was cloned into the pLKO.1-VENUS lentiviral vector, and virus-containing supernatants were generated and used to infect test cells as described previously (7). Transduced (VENUS+) cells were isolated 2–3 d later and assayed for colony formation. The shRNA activity of the lenticonstruct was confirmed by Western blot analyses of GPX2 levels in extracts of transduced human hepatocarcinoma HepG2 cells cultured for 2–3 d before isolation of the transduced (VENUS+) cells. Graphs and Statistics. GraphPad Prism 6 software was used for statistical comparisons and graph generation. Values are reported as mean ± SEM, and P values were generated using the Student t test. The number of independent experiments with cells from different donors is shown in brackets.
qRT-PCR and Western Blot Analyses. Cells were rapidly lysed for RNA and protein isolation and analyzed by standard methods (33). The antibodies and primers used are listed in Tables S1 and S2, respectively.
ACKNOWLEDGMENTS. We acknowledge the excellent help of D. Wilkinson in collecting and processing samples of normal human mammary tissue. We thank Drs. J. Sproul and N. Van Laeken for providing the reduction mammoplasty material, and G. Edin, M. Hale, D. Ko, and W. Xu for technical and FACS assistance. N.K. held a Canadian Breast Cancer Foundation (CBCF) British Columbia/Yukon Postdoctoral Fellowship. This work was also supported by operating grants from the Canadian Breast Cancer Research Alliance, funded by the Canadian Cancer Society, and from the British Columbia/Yukon division of the CBCF.
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NAD(P)H Levels. NAD(P)H was measured on lysed aliquots of 10 FACS-purified cells using a commercially available kit (BioVision Research Products).
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