Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets

Abstract

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.

Keywords: 3D clonogenic assay; human epithelial stem and progenitor cells; mammary differentiation; peroxiredoxin; superoxide dismutase.

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 CD49f^hiTHY1⁺MUC1⁻ cells (clonogenic cell frequency, 18 ± 3%; n = 10). LPs: CD49f⁺EpCAM⁺ (28) or CD49f^hiMUC1⁺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.

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.

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.

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.