Induction of erythroid differentiation in human erythroleukemia cells by depletion of malic enzyme 2 - PubMed (original) (raw)

Induction of erythroid differentiation in human erythroleukemia cells by depletion of malic enzyme 2

Jian-Guo Ren et al. PLoS One. 2010.

Abstract

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for leukemia therapy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Effects on K562 cell proliferation of three independent shRNA hairpins targeting ME2.

A: Western blot analysis using an ME2 antibody of lysate from respective pools of cells transduced with three independent ME2 shRNA lentiviruses, and following selection of puromycin for 10 days as described under “Materials and Methods”. Data are representative of two independent experiments. All three pools showed marked ME2 silencing. B: Cell proliferation in K562 cells transduced with the indicated shRNA lentiviral constructs as described in “A”. Data are representative of three independent experiments. C: Western blot analysis of cellular extracts in single clone K562 cells as described under “Materials and Methods” demonstrated effective knockdown of ME2 levels. Data are representative of two independent experiments. D: Cell proliferation of K562 single cell clones with ME2 knockdown derived from the corresponding pools as described in “C”. Data are representative of three independent experiments.

Figure 2. Stable knockdown of endogenous ME2 levels in K562 cells induces erythroid differentiation.

A: Expression levels of the erythroid marker glycophorin A on the surface of control (pLKO) and ME2 knockdown cells (shME2-1, shME2-2 and shME2-3) were compared using a mouse FITC-conjugated anti-human glycophorin A antibody. As a negative control (Neg Ctrl), cells were incubated with FITC-conjugated control IgG. As a positive control, K562 cells were transduced with ATP citrate lyase (ACL) shRNA lentiviral particles (ACL inhibition is known to cause erythroid differentiation in K562 cells – referenced in the text), and incubated with mouse FITC-conjugated anti-human glycophorin A antibody. The control clone was generated by stable transduction of control pLKO vector, while clones shME2-1, shME2-2 and shME2-3 were generated using the pLKO-ME2 shRNA lentivirus. Data are representative of three independent experiments. B: The percentage of hemoglobin-expressing cells in control (pLKO) and ME2 knockdown (shME2-1, shME2-2 and shME2-3) cell populations was determined by benzedrine staining. Plotted is the mean ± SD from triplicate samples from a representative experiment. Insert: cell pellets from ME2 knockdown cells. 1: pLKO; 2: shME2-1; 3: shME2-2; 4: shME2-3. Increased brown color is clearly visible in lanes 2, 3 and 4. C: Expression levels of the megakaryocytic marker CD10 on the surface of control (pLKO) and ME2 knockdown cells (shME2-1, shME2-2 and shME2-3) were compared using a mouse FITC-conjugated anti-human CD10 antibody. As a negative control (Neg Ctrl), cells were incubated with FITC-conjugated control IgG. The control clone was generated by stable transduction of control pLKO vector, while clones shME2-1, shME2-2 and shME2-3 were generated using the pLKO-ME2 shRNA let virus. Data are representative of three independent experiments.

Figure 3. Stable knockdown of endogenous ME2 levels in K562 cells results in apoptosis in vitro and suppresses tumor formation from K562 cells in vivo.

A: Knockdown of ME2 induces apoptosis in K562 as detected using the annexin V reagent. Data are expressed as mean ± SD, n = 3. B: Stable knockdown of ME2 in K562 cells failed to generate tumors in nude mice. Approximately 107 ME2 deficient or control K562 cells resuspended in 200 µl of a serum-free culture medium/Matrigel mixture (1∶1) were subcutaneously implanted into female athymic nude mice as described under “Materials and Methods”. Tumor-bearing mice were sacrificed after 4 weeks and the mice photographed before excision and weighing. a, Left (L): pLKO; Right (R): shME2-2: b, L and R: shME2-3. Tumors formed only in the pLKO transduced cells.

Figure 4. Depletion of endogenous ME2 enhances ROS generation, increases NAD+/NADH and NADP/NADPH ratios and decreases ATP levels.

A: Accumulation of mitochondrially generated superoxide in K562 ME2 knockdown cells as detected by MitoSOX. Data are representative of two independent experiments. B: Increased ROS in K562 ME2 knockdown cells detected by flow cytometry using CM-H2DCF-DA. Each histogram is representative of three experiments. C. Comparison of oxidative damage to cardiolipin in ME2 knockdown versus control K562 cells. M1 indicates subpopulation of cells that lost NAO signal due to cardiolipin oxidation. D. Depletion of ME2 inhibits ATP production in K562 cells. Data are expressed as mean ± SD, n = 3. E: Depletion of ME2 increases NAD+/NADH ratio. a, NAD+ and NADH were measured by NAD/NADH Assay Kit (Abcam, San Francisco, CA) as described in “Materials and Methods”. Data are expressed as mean ± SD, n = 3. b, NAD+ and NADH were measured by LC-MS methods as described in “Materials and Methods”. F. Depletion of ME2 increases NADP/NADPH ratio in ME2 knockdown cells. NADP and NADPH were measured by LC-MS methods as described in “Materials and Methods”.

Figure 5. The antioxidant NAC cannot rescue ME2 knockdown induced erythroid differentiation in K562 cells.

A: 5 mM NAC completely rescues ROS generation in K562 cells as detected by flow cytometry using CM-H2DCF-DA. Each histogram is representative of three experiments. B: ROS inhibition by 5.0 mM NAC did not rescue ME2 knockdown induced erythroid differentiation in K562 cells. Each histogram is representative of three experiments.

Figure 6. Supplementation by exogenous amino-oxyacetate in medium cannot induce erythroid differentiation but does induce cell death.

A. Cells were treated with different concentrations of amino-oxyacetate and expression levels of the erythroid marker glycophorin A on the surface of K562 cells were assessed using a mouse FITC-conjugated anti-human glycophorin A antibody. Each histogram is representative of three experiments. B. K562 cells with or without ME2 knockdown were incubated with different concentrations of AOA for 72 h. Cell death was assessed by flow cytometry. Top: pLKO; Bottom: shME2-3. Data are representative of two independent experiments.

Figure 7. Effects of ME2 knockdown on signaling pathways, and GATA-1 and vimentin expression.

K562 cells with or without ME2 knockdown were lysed with RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA) containing 1 mM PMSF and a protease inhibitor cocktail and subjected to centrifugation at 15,000× g for 10 min at 4°C to remove debris. After lysis, equal aliquots of protein lysate were resolved by Western blotting. Western blots were probed with anti-phospho-ERK1/2, anti-ERK1, anti-p-AKT308, anti-AKT472, anti-AKT1/2, anti-GATA-1, anti-vimentin and anti-β-tubulin. A, phospho-ERK1/2 activity in ME2 knockdown K562 cells. B, Phospho-AKT detection in ME2 knockdown K562 cells. C, 10 µM PI3K inhibitor, LY294002, inhibits p-AKT activity. D, LY294002 rescue of differentiation in ME2 knockdown K562 cells. E, The effect of LY294002 on the proliferation of K562 cells with or without ME2 knockdown. F, The expression difference of GATA-1 and vimentin in ME2 knockdown cells. Data are representative of three independent experiments.

Figure 8. Knockdown of ME2 alters pyrimidine metabolism in K562 cells.

The metabolites were measured by LC-MS method as described in “Materials and Methods”.

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Induction of erythroid differentiation in human erythroleukemia cells by depletion of malic enzyme 2 - PubMed (original) (raw)