MUC1-C oncoprotein suppresses reactive oxygen species-induced terminal differentiation of acute myelogenous leukemia cells

Abstract

Acute myeloid leukemia (AML) cells are characterized by unlimited self-renewal and an impaired capacity to undergo terminal differentiation. The MUC1 oncoprotein is aberrantly expressed in AML cells; however, there has been no evidence for involvement of MUC1 in myeloid leukemogenesis. Cell-penetrating peptide inhibitors of the MUC1-C subunit block its oligomerization and thereby oncogenic function. The present results demonstrate that treatment of human MOLM-14 and MV4-11 AML cells with these inhibitors is associated with arrest of growth, induction of late apoptosis/necrosis, and loss of self-renewal capacity. Similar results were obtained with primary blasts from patients with AML. Inhibition of MUC1-C was associated with increases in reactive oxygen species (ROS) and depletion of glutathione. Increases in ROS have been linked to induction of hematopoietic cell differentiation along the myeloid lineage. In this regard, inhibition of MUC1-C was associated with induction of a terminally differentiated myeloid phenotype in AML cell lines and primary blasts by an ROS-dependent mechanism. These findings indicate that MUC1-C function is of importance to AML cell self-renewal and that inhibition of MUC1-C represents a potential therapeutic approach to induce terminal differentiation of AML cells.

Figure 1

Growth of MOLM-14 and MV4–11 AML cells is inhibited by GO-201. (A) MOLM-14 (top panels) and MV4-11 (bottom panels) were incubated with a control IgG or anti–MUC1-N (MAb DF3) and analyzed by flow cytometry. The percentage of MUC1-positive cells is indicated in the panels. (B) Lysates from MOLM-14 and MV4-11 cells were subjected to immunoblotting with the indicated antibodies. (C) Amino acid sequences of GO-201 and CP-1. MOLM-14 cells were left untreated (♦), and treated with 5μM GO-201 (■) or 5μM CP-1 (▵) each day for the indicated days. Viable cell number as determined by trypan blue exclusion is expressed as the mean ± SD of 3 determinations. (D) MV4-11 cells were left untreated (♦), and treated with 5μM GO-201 (■) or 5μM CP-1 (▵) each day for the indicated days. Viable cell number was determined by trypan blue exclusion.

Figure 2

GO-203 is effective in inhibiting growth of AML cells. (A) Amino acid sequences of GO-202, GO-203, and CP-3. MOLM-14 cells were left untreated (♦), and treated with 5μM GO-203 (■) or 5μM CP-2 (▵) each day for the indicated days. Viable cell number was determined by trypan blue exclusion. (B) MV4–11 cells were left untreated (♦), and treated with 5μM GO-203 (■) or 5μM CP-2 (▵) each day for the indicated days. Viable cell number was determined by trypan blue exclusion. (C) AML patient 1 cells were incubated with a control IgG or anti–MUC1-N (MAb DF3) and analyzed by flow cytometry. The percentage of MUC1-positive cells is indicated in the panels. (D) AML patient 1 cells were left untreated (♦), and treated with 5μM GO-203 (■) or 5μM CP-2 (▵) each day for the indicated days. Viable cell number was determined by trypan blue exclusion.

Figure 3

Redox balance is disrupted in AML cells by GO-203 treatment. (A) MOLM-14 cells were left untreated, and treated with 5μM GO-203 or 5μM CP-2 each day for 3 days. The cells were then incubated with c-H₂DCFDA for 30 minutes. Fluorescence of oxidized DCF was measured by flow cytometry (left). The results are expressed as the relative H₂O₂ level (mean ± SD for 3 determinations) compared with that obtained with control cells (right). (B) MV4-11 cells were left untreated, and treated with 5μM GO-203 or 5μM CP-2 each day for 3 days. Oxidation of DCF was measured by flow cytometry. The results are expressed as the relative H₂O₂ level (mean ± SD for 3 determinations) compared with that obtained with control cells. (C-D) MOLM-14 (C) and MV4-11 (D) cells were left untreated, and treated each day with 5μM GO-203 or 5μM CP-2. The cells were harvested at the indicated times and analyzed for GSH levels. The results are expressed as GSH levels/10⁶ cells.

Figure 4

GO-203 induces late apoptosis/necrosis of AML cells. (A) MOLM-14 cells were left untreated, and treated with 5μM GO-203 or 5μM CP-2 each day for 3 days. The cells were stained with PI and annexin V, and analyzed by flow cytometry. The percentage of MOLM-14 cells staining with both PI and annexin V is indicated in the upper right panels. (B-D) MOLM-14 (B), MV4-11 (C) and AML patient 1 (D) cells were left untreated, and treated each day with 5μM GO-203 or 5μM CP-2. The MOLM-14, MV4-11 and AML patient 1 cells were harvested on days 3, 3, and 4, respectively. The results obtained from staining with PI/annexin V are expressed as the percentage of cells (mean ± SD of 3 determinations) with late apoptosis/necrosis (left). The cells were also seeded in agar in the absence of drug and colonies were counted at 14 days. The results are expressed as the number of colonies (mean ± SD of 3 determinations) (right).

Figure 5

GO-203–induced disruption of redox balance and death is reversed by NAC. (A) MOLM-14 cells were left untreated, and treated with 5μM GO-203 or 5μM CP-2 each day for 3 days. The GO-203–treated cells were also incubated concurrently with 5mM NAC. The cells were analyzed for GSH and hydrogen peroxide levels (A). The results are expressed as GSH levels/10⁶ cells (mean ± SD of 3 determinations) (A, left) and relative hydrogen peroxide levels (mean ± SD of 3 determinations) compared with that obtained with control cells (A, right). The cells were also stained with PI/annexin V (B). The results are expressed as the percentage of cells (mean ± SD of 3 determinations) with late apoptosis/necrosis (B). (C-D) MV4-11 (C) and AML patient 1 (D) cells were left untreated, and treated with 5μM GO-203 or 5μM CP-2 each day for 3 (C) and 4 (D) days. The GO-203–treated cells were also incubated in the presence of 5mM NAC. The cells were stained with PI/annexin V. The results are expressed as the percentage of cells (mean ± SD of 3 determinations) with late apoptosis/necrosis.

Figure 6

AML cells respond to GO-203 with induction of ROS-mediated myeloid differentiation. (A) MOLM-14 cells were left untreated (□), and treated with 2.5μM GO-203 (■) or 2.5μM CP-2 (▩) each day for the indicated days. Cells were analyzed for the percentage with expression of CD11b (left) and CD11c (right). (B) MOLM-14 cells were left untreated, and treated with 2.5μM GO-203 or 2.5μM CP-2 each day for 6 days. Lysates were immunoblotted with the indicated antibodies (left). Cells were seeded in agar in the absence of drug and colonies were counted at 14 days. The results are expressed as the number of colonies (mean ± SD of 3 determinations) (right). (C) AML patient 1 cells were left untreated (□), and treated with 2.5μM GO-203 (■) or 2.5μM CP-2 (▩) each day for the indicated days. Cells were analyzed for the percentage with expression of CD11c (left). On day 6, the cells were seeded in agar in the absence of drug and colonies were counted at 14 days. The results are expressed as the number of colonies (mean ± SD of 3 determinations) (right). (D) MOLM-14 (left) and MV4-11 (right) cells were treated with 2.5μM GO-203 in the absence (□) and presence (■) of 5mM NAC for 6 days. The cells were then analyzed for the percentage with expression of CD11b and CD11c.