Mechanistic role of heat shock protein 70 in Bcr-Abl–mediated resistance to apoptosis in human acute leukemia cells

AbstractBcr-Abl–expressing primary or cultured leukemia cells display high levels of the antiapoptotic heat shock protein (hsp) 70 and are resistant to cytarabine (Ara-C), etoposide, or Apo-2L/TRAIL (TNF-related apoptosis-inducing ligand)–induced apoptosis. Conversely, a stable expression of the cDNA of hsp70 in the reverse orientation attenuated not only hsp70 but also signal transducers and activators of transcription 5 (STAT5) and Bcl-xL levels. This increased apoptosis induced by cytarabine, etoposide, or Apo-2L/TRAIL. Ectopic expression of hsp70 in HL-60 cells (HL-60/hsp70) inhibited Ara-C and etoposide-induced Bax conformation change and translocation to the mitochondria; attenuated the accumulation of cytochrome c, Smac, and Omi/HtrA2 in the cytosol; and inhibited the processing and activity of caspase-9 and caspase-3. Hsp70 was bound to death receptors 4 and 5 (DR4 and DR5) and inhibited Apo-2L/TRAIL-induced assembly and activity of the death-inducing signaling complex (DISC). HL-60/hsp70 cells exhibited increased levels and DNA binding activity of STAT5, which was associated with high levels of Pim-2 and Bcl-xL and resistance to apoptosis. Expression of the dominant negative (DN) STAT5 resensitized HL-60/hsp70 cells to cytarabine, etoposide, and Apo-2L/TRAIL–induced apoptosis. Collectively, these findings suggest that hsp70 inhibits apoptosis upstream and downstream of the mitochondria and is a promising therapeutic target for reversing drug-resistance in chronic myeloid leukemia-blast crisis and acute myeloid leukemia cells. (Blood. 2005;105:1246-1255)

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The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein.

The EMBO Journal ◽

10.1002/j.1460-2075.1990.tb08081.x ◽

1990 ◽

Vol 9 (1) ◽

pp. 69-76 ◽

Cited By ~ 146

Author(s):

A. Wilhelmsson ◽

S. Cuthill ◽

M. Denis ◽

A.C. Wikström ◽

J.A. Gustafsson ◽

...

Keyword(s):

Heat Shock ◽

Dna Binding ◽

Heat Shock Protein ◽

Binding Activity ◽

Dna Binding Activity ◽

Dioxin Receptor

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Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-xL

Blood ◽

10.1182/blood.v95.3.1014.003k04_1014_1022 ◽

2000 ◽

Vol 95 (3) ◽

pp. 1014-1022 ◽

Cited By ~ 162

Author(s):

Charles Perkins ◽

Caryn N. Kim ◽

Guofu Fang ◽

Kapil N. Bhalla

Keyword(s):

Myeloid Leukemia ◽

Blast Crisis ◽

K562 Cells ◽

Cell Types ◽

Multidrug Resistant ◽

Leukemia Cells ◽

Protein Levels ◽

Growth Inhibitory ◽

Induced Apoptosis

We investigated the in vitro growth inhibitory and apoptotic effects of clinically achievable concentrations of As2O3 (0.5 to 2.0 μmol/L) against human myeloid leukemia cells known to be resistant to a number of apoptotic stimuli. These included chronic myelocytic leukemia (CML) blast crisis K562 and HL-60/Bcr-Abl cells, which contain p210 and p185 Bcr-Abl, respectively, and HL-60 cell types that overexpress Bcl-2 (HL-60/Bcl-2), Bcl-xL(HL-60/Bcl-xL), MDR (HL-60/VCR), or MRP (HL-60/AR) protein. The growth-inhibitory IC50 values for As2O3 treatment for 7 days against all these cell types ranged from 0.8 to 1.5 μmol/L. Exposure to 2 μmol/L As2O3 for 7 days induced apoptosis of all cell types, including HL-60/Bcr-Abl and K562 cells. This was associated with the cytosolic accumulation of cyt c and preapoptotic mitochondrial events, such as the loss of inner membrane potential (▵Ψm) and the increase in reactive oxygen species (ROS). Treatment with As2O3 (2 μmol/L) generated the activities of caspases, which produced the cleavage of the BH3 domain containing proapoptotic Bid protein and poly (ADP-ribose) polymerase. Significantly, As2O3-induced apoptosis of HL-60/Bcr-Abl and K562 cells was associated with a decline in Bcr-Abl protein levels, without any significant alterations in the levels of Bcl-xL, Bax, Apaf-1, Fas, and FasL. Although As2O3 treatment caused a marked increase in the expression of the myeloid differentiation marker CD11b, it did not affect Hb levels in HL-60/Bcr-Abl, K562, or HL-60/neo cells. However, in these cells, As2O3 potently induced hyper-acetylation of the histones H3 and H4. These findings characterize As2O3 as a growth inhibiting and apoptosis-inducing agent against a variety of myeloid leukemia cells resistant to multiple apoptotic stimuli.

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Inhibition of Bcl-2 antiapoptotic members by obatoclax potently enhances sorafenib-induced apoptosis in human myeloid leukemia cells through a Bim-dependent process

Blood ◽

10.1182/blood-2011-09-378141 ◽

2012 ◽

Vol 119 (25) ◽

pp. 6089-6098 ◽

Cited By ~ 66

Author(s):

Mohamed Rahmani ◽

Mandy Mayo Aust ◽

Elisa Attkisson ◽

David C. Williams ◽

Andrea Ferreira-Gonzalez ◽

...

Keyword(s):

Myeloid Leukemia ◽

Fluorescent Protein ◽

Ectopic Expression ◽

Leukemia Cells ◽

Down Regulation ◽

Multikinase Inhibitor ◽

Cd34 Cells ◽

Human Acute Myeloid Leukemia ◽

Induced Apoptosis ◽

Green Fluorescent

Abstract Interactions between the multikinase inhibitor sorafenib and the BH3-mimetic obatoclax (GX15-070) were examined in human acute myeloid leukemia (AML) cells. Treatment with sorafenib/obatoclax induced pronounced apoptosis in and reduced the clonogenic growth of multiple AML lines and primary AML cells but not normal CD34+ cells. Sorafenib triggered rapid and pronounced Mcl-1 down-regulation accompanied by enhanced binding of Bim to Bcl-2 and Bcl-xL, effects that were abolished by obatoclax coadministration. Notably, shRNA knockdown of Bim, Bak, or Bax, but not Noxa, significantly attenuated obatoclax/sorafenib lethality, whereas ectopic expression of Mcl-1 exerted a protective effect. Furthermore, exposure of leukemia cells to sorafenib and obatoclax markedly induced autophagy, reflected by rapid and pronounced LC3 processing and LC3–green fluorescent protein (GFP) punctate formation. Multiple autophagy inhibitors or VPS34 knockdown, significantly potentiated sorafenib/obatoclax lethality, indicating a cytoprotective role for autophagy in this setting. Finally, studies in a xenograft mouse model revealed that combined sorafenib/obatoclax treatment markedly reduced tumor growth and significantly prolonged survival in association with Mcl-1 down-regulation and apoptosis induction, whereas agents administered individually had only modest effects. These findings suggest that combining sorafenib with agents that inhibit Mcl-1 and Bcl-2/Bcl-xL such as obatoclax may represent a novel and potentially effective strategy in AML.

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Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines

Blood ◽

10.1182/blood.v88.3.809.809 ◽

1996 ◽

Vol 88 (3) ◽

pp. 809-816 ◽

Cited By ~ 236

Author(s):

RM Weber-Nordt ◽

C Egen ◽

J Wehinger ◽

W Ludwig ◽

V Gouilleux-Gruart ◽

...

Keyword(s):

Dna Binding ◽

Nuclear Localization ◽

Myeloid Leukemia ◽

Epstein Barr Virus ◽

Binding Activity ◽

Leukemia Cells ◽

Stat Proteins ◽

Barr Virus ◽

Dna Binding Activity ◽

Epstein Barr

Abstract Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta- casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1–6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus- positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.

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Transcription Factor SCL Is Required for c-kit Expression and c-Kit Function in Hemopoietic Cells

Journal of Experimental Medicine ◽

10.1084/jem.188.3.439 ◽

1998 ◽

Vol 188 (3) ◽

pp. 439-450 ◽

Cited By ~ 55

Author(s):

Gorazd Krosl ◽

Gang He ◽

Martin Lefrancois ◽

Frédéric Charron ◽

Paul-Henri Roméo ◽

...

Keyword(s):

Transcription Factor ◽

Cell Survival ◽

Cell Line ◽

Ectopic Expression ◽

Binding Activity ◽

Dominant Negative ◽

Hemopoietic Cells ◽

Downstream Target ◽

Helix Loop Helix ◽

Dna Binding Activity

In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34+ hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony–stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit–dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL– or dn-SCL– expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro–B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.

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Significance of heat-shock protein (HSP) 90 expression in acute myeloid leukemia cells

Cell Stress and Chaperones ◽

10.1007/s12192-008-0035-3 ◽

2008 ◽

Vol 13 (3) ◽

pp. 357-364 ◽

Cited By ~ 57

Author(s):

Pascale Flandrin ◽

Denis Guyotat ◽

Amélie Duval ◽

Jérôme Cornillon ◽

Emmanuelle Tavernier ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Heat Shock ◽

Heat Shock Protein ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Acute Myeloid Leukemia Cells ◽

Hsp 90 ◽

Acute Myeloid

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Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines

Blood ◽

10.1182/blood.v88.3.809.bloodjournal883809 ◽

1996 ◽

Vol 88 (3) ◽

pp. 809-816 ◽

Cited By ~ 12

Author(s):

RM Weber-Nordt ◽

C Egen ◽

J Wehinger ◽

W Ludwig ◽

V Gouilleux-Gruart ◽

...

Keyword(s):

Dna Binding ◽

Nuclear Localization ◽

Myeloid Leukemia ◽

Epstein Barr Virus ◽

Binding Activity ◽

Leukemia Cells ◽

Stat Proteins ◽

Barr Virus ◽

Dna Binding Activity ◽

Epstein Barr

Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta- casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1–6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus- positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.

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