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. 2009 Nov 2;187(3):429-42.
doi: 10.1083/jcb.200904049. Epub 2009 Oct 26.

MCL-1-dependent leukemia cells are more sensitive to chemotherapy than BCL-2-dependent counterparts

Joslyn K Brunelle  1 , Jeremy Ryan, Derek Yecies, Joseph T Opferman, Anthony Letai
Affiliations

MCL-1-dependent leukemia cells are more sensitive to chemotherapy than BCL-2-dependent counterparts

Joslyn K Brunelle et al. J Cell Biol. .

Abstract

Myeloid cell leukemia sequence 1 (MCL-1) and B cell leukemia/lymphoma 2 (BCL-2) are anti-apoptotic proteins in the BCL-2 protein family often expressed in cancer. To compare the function of MCL-1 and BCL-2 in maintaining cancer survival, we constructed complementary mouse leukemia models based on Emu-Myc expression in which either BCL-2 or MCL-1 are required for leukemia maintenance. We show that the principal anti-apoptotic mechanism of both BCL-2 and MCL-1 in these leukemias is to sequester pro-death BH3-only proteins rather than BAX and BAK. We find that the MCL-1-dependent leukemias are more sensitive to a wide range of chemotherapeutic agents acting by disparate mechanisms. In common across these varied treatments is that MCL-1 protein levels rapidly decrease in a proteosome-dependent fashion, whereas those of BCL-2 are stable. We demonstrate for the first time that two anti-apoptotic proteins can enable tumorigenesis equally well, but nonetheless differ in their influence on chemosensitivity.

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Figures

Figure 1.
Figure 1.
MCL-1 overexpression targeted to hematopoietic cells by the H2K promoter in conjunction with c-myc results in the development of leukemia in mice. (A) Levels of MCL-1 protein expression in wild-type and H2K-Mcl-1 mouse spleenocytes. (B) Kaplan-Meier plot showing the survival in number of days for Mcl-1/Eμ-Myc, H2K-Mcl-1 only, Eμ-Myc only, and wild-type mice. This is from a cohort of mice containing seven of each genotype. (C) Number of white blood cells per μl of blood from Mcl-1/Eμ-Myc, H2K-Mcl-1 only, Eμ-Myc only, and wild-type mice. This is from a cohort of mice containing seven of each genotype.
Figure 2.
Figure 2.
Pathology reveals that the normal architecture of spleen and bone marrow is replaced by a monotonous population of lymphoblasts in Mcl-1/Eμ-Myc mice, similar to BCL-2/Eμ-Myc mice. (A) Wildtype. (B) H2K-Mcl-1 only. (C) Eμ-Myc only. (D) Mcl-1/Eμ-Myc. (E) BCL-2/Eμ-Myc. Bar for the spleen samples is 100 µm, and bar for the bone marrow samples is 50 µm.
Figure 3.
Figure 3.
BH3 profiling distinguishes BCL-2– and MCL-1–dependent leukemias. Each profile is an average and standard deviation of three independent experiments, except for the JC1 whole-cell assay. The JC1 whole-cell assay is one experiment done in triplicate, with the average of the three repeats presented here. (A) BH3 profile of Mcl-1/Eμ-Myc primary leukemia cells. (B) BH3 profile of Mcl-1/Eμ-Myc cell line. (C) BH3 profile of Mcl-1/Eμ-Myc cell line using the JC1 whole-cell assay. (D) BH3 profile of BCL-2/ Eμ-Myc primary leukemia cells. (E) BH3 profile of BCL-2/Eμ-Myc cell line. (F) BH3 profile of BCL-2/Eμ-Myc cell line using the JC1 whole-cell assay. (G) Cell lines were treated with doxycycline for 7 d and survival assessed as the Annexin V negative population, measured by FACS. Data presented are from a single experiment; the experiment was repeated twice.
Figure 4.
Figure 4.
MCL-1 and BCL-2 are primed with BIM and PUMA in leukemia cells. (A) Coimmunoprecipiation of MCL-1 from CHAPS lysate of white blood cell primary sample or cell line from Mcl-1/Eμ-Myc mouse. Blot was probed for BIM, PUMA, BID, BAX, BAK, and MCL-1. (B) Coimmunoprecipiation of BCL-2 from CHAPS lysate of white blood cell primary sample or cell line from BCL-2/Eμ-Myc mouse. Blot was probed for BIM, PUMA, BID, BAX, BAK, and BCL-2.
Figure 5.
Figure 5.
BH3 profiling of Eμ-Myc tumors reveals variable dependence on anti-apoptotic proteins. Data presented are from one single experiment. (A) Eμ-Myc tumor #859 shows a MCL-1 profile. (B) Eμ-Myc tumor #1107 shows a profile where MCL-1 > BCL-2/BCL-XL. (C) Eμ-Myc tumor #1433 shows a profile where MCL-1 > BCL-2/BCL-XL. (D) Eμ-Myc tumor #1656 shows a profile where BCL-2/BCL-XL > MCL-1. (E) Western blot containing CHAPS lysates of the Eμ-Myc tumors in order to compare protein levels of anti-apoptotic proteins BCL-2, BCL-XL, and MCL-1. Note low BCL-2 protein level in Eμ-Myc tumor #859.
Figure 6.
Figure 6.
Higher BIM and PUMA expression observed in MCL-1– and BCL-2–overexpressing leukemias. Western blot comparing protein levels among three Eμ-Myc tumors, two BCL-2/Eμ-Myc leukemias, and three Mcl-1/Eμ-Myc leukemias. (A) The proteins blotted were MCL-1, BCL-2, BIM, PUMA, BID, BAD, BAX, and BAK. Please note that the BCL-2 transgene in the BCL-2/EμMyc lymphomas is human. (B) The proteins blotted were BAD, Phospho-Bad (Ser112), and Actin.
Figure 7.
Figure 7.
MCL-1–dependent leukemia lines are more sensitive to chemotherapy drugs than BCL-2–dependent leukemia lines. Dose response curves use the following drug treatments: MNNG, Vincristine, Staurosporin, Etoposide, MG132, Fludarabine, Flavopiridol, and Bortezomib. After 24 h of drug treatment, the cells were stained with Annexin/PI and the percentage of viable cells was graphed. Each time point is an average and standard deviation of three independent experiments. For the statistics, the three Mcl-1 cell lines and the three Bcl-2 cell lines were averaged and area under the curve was determined. P value was analyzed by using a paired t test of area under the curve.
Figure 8.
Figure 8.
Varied chemotherapeutics induce MCL-1 before cell death. All cell lines pretreated with 20 μM of caspase inhibitor Q-VD-OPH for 1 h. CHAPS lysates were prepared and MCL-1, BCL-2, BIM, and Actin protein levels were determined using Western blotting. (A) Mcl-1/Eμ-Myc cell lines treated with 1 μM Bortezomib or 1 μM Flavopiridol for 0, 8, and 16 h. (B) Mcl-1/Eμ-Myc cell lines treated with 1 μM Etoposide for 0, 8, 16, and 24 h. (C) Mcl-1/Eμ-Myc cell lines treated with 1 μM Vincristine for 0, 8, 16, and 24 h. (D and E) BCL-2/Eμ-Myc cell lines treated with 1 μM Etoposide or 1 μM Vincristine for 0, 8, 16, and 24 h. (F) BCL-2/Eμ-Myc cell lines treated with 1 μM Bortezomib for 0, 8, and 16 h. (G) BCL-2/Eμ-Myc cell lines treated with 1 μM Flavopiridol for 0, 8, and 16 h.
Figure 9.
Figure 9.
The Mcl-1/Eμ-Myc cell line dies faster than the BCL-2/Eμ-Myc cell line. (A) Annexin/PI measurement used to determine cell viability of Mcl-1/Eμ-Myc cell line using Etoposide, Flavopiridol, Vincristine, and DMSO drug treatments with and without 20 μM Q-VD-OPH. Data presented are an average and standard deviation of three independent experiments. (B) Dose-response curve of Mcl-1/Eμ-Myc cell line treated with 1 μM of Etoposide, Flavopiridol, or Vincristine over 8, 16, 24, and 48 h. Data presented are an average and standard deviation of three independent experiments. (C) Dose-response curve of BCL-2/Eμ-Myc cell line treated with 1 μM Etoposide, Flavopiridol, or Vincristine over 8, 16, 24, and 48 h. Data presented are an average and standard deviation of three independent experiments.
Figure 10.
Figure 10.
MCL-1 half-life is unchanged by flavopiridol treatment; MCL-1– and BCL-2–dependent mitochondria demonstrate equivalent pretreatment “priming.” (A) A Mcl-1/Eμ-Myc cell line pretreated with 20 μM of caspase inhibitor Q-VD-OPH for 1 h, followed by treatment with 20 μM cycloheximide (CHX). CHAPS lysates were made after each hour of treatment. (B) A Mcl-1/Eμ-Myc cell line pretreated with 20 μM of caspase inhibitor Q-VD-OPH for 1 h, followed by a combined treatment with 20 μM cycloheximide (CHX) and 1 μM Flavopiridol. CHAPS lysates were made after each hour of treatment. (C) BH3 profiling of a Mcl-1/Eμ-Myc and BCL-2/Eμ-Myc cell line using decreasing concentrations of the PUMA peptide. BAD and NOXA peptides were included as controls. Three independent experiments were averaged. (D) BH3 profiling by the whole-cell JC1 method at the 60-min time point. The results of the three Bcl-2 cell lines and three Mcl-1 cell lines were averaged for the following peptides: Puma, Bad, Noxa25, and FCCP.
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