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Myeloid leukemia factor 1 interfered with Bcl-XL to promote apoptosis and its function was regulated by 14-3-3

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Abstract

Myeloid leukemia factor 1 (MLF1) was involved in t(3;5) chromosomal rearrangement and aberrantly expressed in myelodysplastic syndromes/acute myeloid leukemia patients. Ex vivo experiments showed that the lymphocytes from the Mlf1-deficient mice were more resistant to apoptotic stimulations than the wild-type cells. Furthermore, the ectopically expressed MLF1 induced apoptosis in the cell models. These findings revealed that MLF1 was required for the cells to respond to the apoptotic stimulations. Ex vivo experiments also demonstrated that cytokine withdrawal significantly up-regulated Mlf1’s expression and promoted its association with B cell lymphoma-extra large (Bcl-XL) in the lymphocytes, at the same time reduced the association of Bax with Bcl-XL The same effects were also observed in the cells that over-expressed MLF1. However, these effects were observed in Mlf1 null lymphocytes as well as the cells over-expressing Bcl-XL. In addition, MLF1’s proapoptosis could be completely prevented by co-expression of Bcl-XL and significantly attenuated in Bax/Bak double null cells. These data, taken together, strongly suggested that in response to the stresses, up-regulated Mlf1 promoted its association with Bcl-XL and reduced the available Bcl-XL for associating with Bax, which resulted in releasing Bax from the Bcl-XL and apoptosis in turn. Lastly, we showed that MLF1 was negatively regulated by 14-3-3 and revealed that 14-3-3 bound to MLF1 and physically blocked MLF1’s Bcl-2 homology domain 3 (BH3) as well as Bcl-XL from associating with MLF1. Our findings suggested that ectopically expressed MLF1 could be responsible for the pathological apoptosis in early myelodysplastic syndrome (MDS) patients.

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Abbreviations

AML:

acute myeloid leukemia BAX

BAX:

Bcl-2-associated X protein

Bcl-2:

B cell lymphoma gene 2

Bcl-XL:

B cell lymphoma-extra large

BH3:

Bcl-2 homology domain 3

BIM:

Bcl-2-interacting mediator

CD34:

Cluster differentiation 34

CSN3:

COP9 signalosome complex subunit 3

EV:

Empty vector

Fas:

Apoptosis stimulating fragment

GFP:

Green fluorescent protein

HFF:

Human fetal foreskin fibroblast

MDS:

Myelodysplastic syndromes

MEF:

Mouse embryonic fibroblast

MLF1:

Myeloid leukemia factor 1

NPM-MLF1:

Nucleophosmin-Myeloid leukemia factor 1

NPM:

Nucleophosmin

Q-VD-OPH:

A carboxy terminal phenoxy group conjugated to the amino acids valine and aspartate

PMA:

Phorbol 12-myristate 13-acetate

UV:

Ultra violet

WT:

Wild type

MADM:

Mlf1 adaptor molecule

MLFIP:

MLF1-interacting protein

MANP:

Mlf1-associated nuclear protein

WB:

Western blot

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Acknowledgments

This work was supported by the American Lebanese Syrian Associated Charities, St. Jude Children’s Research Hospital, and ShiJiaZhuang First Hospital. We thank James N. Ihle and Gerard P. Zambetti (Department of Biochemistry, St Jude Children’s Research Hospital) for providing Bax/Bak, p53 null MEF cells and Bim knockout mice. The authors thank the equipment support from Enhancement Program of Louisiana Board of Reagents (LEQSF(2014–15)-ENH-TR-28) (awarded to WX). Yi Sun and Stephan W Morris initiated the research project. Yi Sun, Amina Fu, Xu Wu, and Jyh-Rong Chao carried out the research project. Simon Moshiach did microinjection experiments. Yi Sun and Xu Wu wrote the paper.

Author information

Authors and Affiliations

  1. Department of Hematology, Shijiazhuang City First Hospital, 36 FanXiLu, Shijiazhuang, 050011, People’s Republic of China

    Yi Sun & Amina Fu

  2. Department of Chemistry, University of Louisiana at Lafayette, Lafayette, LA, 70504, USA

    Wu Xu

  3. Department of Biochemistry, St Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA

    Jyh-Rong Chao

  4. Department of Genetics, St Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA

    Simon Moshiach

  5. Department of Pathology, St Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA

    Yi Sun & Stephan W Morris

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  1. Yi Sun
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Correspondence to Yi Sun.

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Supplementary Fig. 1

The BH3 domain mutation disrupted MLF1-induced apoptosis. WT MLF1 and L41AMLF1-IRES-GFP were microinjected into HHF cells. Viable GFP positive cell numbers were present at each time point. The data shown represent the results from three independent experiments. All experiments were repeated three times (PPT 158 kb)

Supplementary Fig. 2

MLF1 was independent on p53 for its proapoptosis. a The MLF1-induced apoptosis was examined in p53 null MEFs. p53−/− MEFs were microinjected with MLF1-IRES-FGP. Viable GFP positive cell numbers were presented at indicated time points. The data shown in g represented the results from a minimum of three independent microinjection experiments; b As a control expression of MLF1 in the wild-type Bax/Bak null and p53 null MEFs were examined. The cells were transfected with MLF1-IRES-GFP and analyzed by WB with anti-Mlf1 antibodies after 24 h transfection. Expressed MLF1 was indicated. All experiments were repeated three times (PPT 155 kb)

Supplementary Fig. 3

Examination of the associations between MLF1 and Bcl-2 family proteins, Bcl-2, Mcl-1, and A1. NIH3T3 cells were transfected with Flag-tagged Bcl-2 family proteins and Myc-tagged MLF1. Total cell lysates were either directly loaded on the gels or immunoprecipitated (IP) with anti-Myc antibodies then loaded on to the gel, and immunoblotted with either anti-Flag or anti-Mfl1. Detected proteins were indicated; all experiments were repeated three times (PPT 197 kb)

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Sun, Y., Fu, A., Xu, W. et al. Myeloid leukemia factor 1 interfered with Bcl-XL to promote apoptosis and its function was regulated by 14-3-3. J Physiol Biochem 71, 807–821 (2015). https://doi.org/10.1007/s13105-015-0445-5

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  • DOI: https://doi.org/10.1007/s13105-015-0445-5

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