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Identification of heat shock protein 32 (Hsp32) as a novel target in acute lymphoblastic leukemia.

Cerny-Reiterer S, Meyer RA, Herrmann H, Peter B, Gleixner KV, Stefanzl G, Hadzijusufovic E, Pickl WF, Sperr WR, Melo JV, Maeda H, Jäger U, Valent P - Oncotarget (2014)

Bottom Line: Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells.A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells.In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria.

ABSTRACT
Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in oncology. We have shown that Hsp32, also known as heme oxygenase-1 (HO-1), serves as survival factor and potential target in Ph+ chronic myeloid leukemia. We here report that primary cells and cell lines derived from patients with acute lymphoblastic leukemia (ALL) express Hsp32 mRNA and the Hsp32 protein in a constitutive manner. Highly enriched CD34+/CD38- ALL stem cells also expressed Hsp32. Two Hsp32-targeting drugs, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), induced apoptosis and growth arrest in the BCR/ABL1+ cell lines, in Ph- lymphoblastic cell lines and in primary Ph+ and Ph- ALL cells. The effects of PEG-ZnPP and SMA-ZnPP on growth of leukemic cells were dose-dependent. In Ph+ ALL, major growth-inhibitory effects of the Hsp32-targeting drugs were observed in imatinib-sensitive and imatinib-resistant cells. Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells. A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells. In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

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Related in: MedlinePlus

Expression of Hsp32 in ALL cellsA: Primary ALL cells (left panel) and cell lines (right panel) were subjected to RNA isolation and RT-PCR using primers specific for Hsp32 and human ABL (control) as described in the text. Before RNA was isolated, cells were cultured in control medium (Co) or in medium containing 10 μM hemin (He) at 37°C for 8 hours. Expression of Hsp32 mRNA and ABL mRNA was determined by qPCR. The left panel shows data obtained with primary ALL cells (7 Ph+ donors and 10 Ph─ donors) and the right panel shows data obtained with Ph+ and Ph─ cell lines (Ph+: BV-173, NALM-1, TOM-1, Z-119, Ph−: Raji, Ramos, REH, BL-41). Hsp32 mRNA levels are expressed as percentage of ABL mRNA levels and represent the mean±S.D. from all donors or cell lines. Asterisk: p<0.05. B: Immunocytochemical detection of the Hsp32 protein in primary ALL cells (left panel, Ph+ patients #10 and #17; and Ph− patient #14 from Table 1) and cell lines (right panel) after incubation in control medium or hemin (10 μM) at 37°C for 8 hours. After incubation, cells were spun on cytospin slides and stained with an antibody against Hsp32 as described in the text. Images were taken using an Olympus DP21 camera connected to an Olympus BX50F4 microscope equipped with 100x/1.35 UPlan-Apo objective lense (Olympus, Hamburg, Germany). Figures were prepared using Adobe Photoshop CS2 software version 9.0 (Adobe Systems, San Jose, CA) and processed with PowerPoint software (Microsoft, Redmond, WA). C: Western blot analysis of expression of Hsp32 in Ph+ cell line Z-119 (left) and Ph─ cell line BL-41 (right). Cells were incubated with control medium (Co) or hemin (10 μM) (He) at 37°C for 8 hours. Then, cells were lysed and lysates subjected to Western blot analysis using an antibody against Hsp32 and an antibody against ß-actin as described in the text. D: Expression of HO-1 mRNA in highly enriched (sorted) CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells obtained from 3 patients with Ph− ALL (left side) and 3 patients with Ph+ ALL (p210 right side) as determined by qPCR. Cells were subjected to RNA isolation, cDNA synthesis and qPCR using primers specific for HO-1 and ABL. Results show HO-1 mRNA levels as percent of ABL mRNA levels, and represent the mean±S.D. of 3 independent experiments (3 patients).
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Figure 1: Expression of Hsp32 in ALL cellsA: Primary ALL cells (left panel) and cell lines (right panel) were subjected to RNA isolation and RT-PCR using primers specific for Hsp32 and human ABL (control) as described in the text. Before RNA was isolated, cells were cultured in control medium (Co) or in medium containing 10 μM hemin (He) at 37°C for 8 hours. Expression of Hsp32 mRNA and ABL mRNA was determined by qPCR. The left panel shows data obtained with primary ALL cells (7 Ph+ donors and 10 Ph─ donors) and the right panel shows data obtained with Ph+ and Ph─ cell lines (Ph+: BV-173, NALM-1, TOM-1, Z-119, Ph−: Raji, Ramos, REH, BL-41). Hsp32 mRNA levels are expressed as percentage of ABL mRNA levels and represent the mean±S.D. from all donors or cell lines. Asterisk: p<0.05. B: Immunocytochemical detection of the Hsp32 protein in primary ALL cells (left panel, Ph+ patients #10 and #17; and Ph− patient #14 from Table 1) and cell lines (right panel) after incubation in control medium or hemin (10 μM) at 37°C for 8 hours. After incubation, cells were spun on cytospin slides and stained with an antibody against Hsp32 as described in the text. Images were taken using an Olympus DP21 camera connected to an Olympus BX50F4 microscope equipped with 100x/1.35 UPlan-Apo objective lense (Olympus, Hamburg, Germany). Figures were prepared using Adobe Photoshop CS2 software version 9.0 (Adobe Systems, San Jose, CA) and processed with PowerPoint software (Microsoft, Redmond, WA). C: Western blot analysis of expression of Hsp32 in Ph+ cell line Z-119 (left) and Ph─ cell line BL-41 (right). Cells were incubated with control medium (Co) or hemin (10 μM) (He) at 37°C for 8 hours. Then, cells were lysed and lysates subjected to Western blot analysis using an antibody against Hsp32 and an antibody against ß-actin as described in the text. D: Expression of HO-1 mRNA in highly enriched (sorted) CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells obtained from 3 patients with Ph− ALL (left side) and 3 patients with Ph+ ALL (p210 right side) as determined by qPCR. Cells were subjected to RNA isolation, cDNA synthesis and qPCR using primers specific for HO-1 and ABL. Results show HO-1 mRNA levels as percent of ABL mRNA levels, and represent the mean±S.D. of 3 independent experiments (3 patients).

Mentions: As assessed by qPCR, primary ALL cells as well as the ALL cell lines tested were found to express Hsp32 mRNA (Figure 1A, Tables 1 and 2). Hsp32 transcripts were present in Ph+ ALL cells as well as in Ph− ALL cells (Figure 1A). Hemin was found to promote expression of Hsp23 mRNA in all ALL samples tested (Figure 1A). We were also able to show that ALL cells display the Hsp32 protein. Expression of the Hsp32 protein was demonstrable by immunocytochemistry (Figure 1B) as well as by Western blotting (Figure 1C), and hemin was found to upregulate expression of the Hsp32 protein in ALL cells (Figure 1B and 1C). Since leukemic stem cells are considered a major target of therapy, we were also interested to know whether CD34+/CD38− stem cells in ALL express Hsp32. In these experiments, we were able to show that highly enriched (sorted) CD34+/CD38− ALL stem cells as well as CD34+/CD38+ progenitor cells express Hsp32 mRNA in patients with Ph+ ALL and patients with Ph− ALL (Figure 1D).


Identification of heat shock protein 32 (Hsp32) as a novel target in acute lymphoblastic leukemia.

Cerny-Reiterer S, Meyer RA, Herrmann H, Peter B, Gleixner KV, Stefanzl G, Hadzijusufovic E, Pickl WF, Sperr WR, Melo JV, Maeda H, Jäger U, Valent P - Oncotarget (2014)

Expression of Hsp32 in ALL cellsA: Primary ALL cells (left panel) and cell lines (right panel) were subjected to RNA isolation and RT-PCR using primers specific for Hsp32 and human ABL (control) as described in the text. Before RNA was isolated, cells were cultured in control medium (Co) or in medium containing 10 μM hemin (He) at 37°C for 8 hours. Expression of Hsp32 mRNA and ABL mRNA was determined by qPCR. The left panel shows data obtained with primary ALL cells (7 Ph+ donors and 10 Ph─ donors) and the right panel shows data obtained with Ph+ and Ph─ cell lines (Ph+: BV-173, NALM-1, TOM-1, Z-119, Ph−: Raji, Ramos, REH, BL-41). Hsp32 mRNA levels are expressed as percentage of ABL mRNA levels and represent the mean±S.D. from all donors or cell lines. Asterisk: p<0.05. B: Immunocytochemical detection of the Hsp32 protein in primary ALL cells (left panel, Ph+ patients #10 and #17; and Ph− patient #14 from Table 1) and cell lines (right panel) after incubation in control medium or hemin (10 μM) at 37°C for 8 hours. After incubation, cells were spun on cytospin slides and stained with an antibody against Hsp32 as described in the text. Images were taken using an Olympus DP21 camera connected to an Olympus BX50F4 microscope equipped with 100x/1.35 UPlan-Apo objective lense (Olympus, Hamburg, Germany). Figures were prepared using Adobe Photoshop CS2 software version 9.0 (Adobe Systems, San Jose, CA) and processed with PowerPoint software (Microsoft, Redmond, WA). C: Western blot analysis of expression of Hsp32 in Ph+ cell line Z-119 (left) and Ph─ cell line BL-41 (right). Cells were incubated with control medium (Co) or hemin (10 μM) (He) at 37°C for 8 hours. Then, cells were lysed and lysates subjected to Western blot analysis using an antibody against Hsp32 and an antibody against ß-actin as described in the text. D: Expression of HO-1 mRNA in highly enriched (sorted) CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells obtained from 3 patients with Ph− ALL (left side) and 3 patients with Ph+ ALL (p210 right side) as determined by qPCR. Cells were subjected to RNA isolation, cDNA synthesis and qPCR using primers specific for HO-1 and ABL. Results show HO-1 mRNA levels as percent of ABL mRNA levels, and represent the mean±S.D. of 3 independent experiments (3 patients).
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: Expression of Hsp32 in ALL cellsA: Primary ALL cells (left panel) and cell lines (right panel) were subjected to RNA isolation and RT-PCR using primers specific for Hsp32 and human ABL (control) as described in the text. Before RNA was isolated, cells were cultured in control medium (Co) or in medium containing 10 μM hemin (He) at 37°C for 8 hours. Expression of Hsp32 mRNA and ABL mRNA was determined by qPCR. The left panel shows data obtained with primary ALL cells (7 Ph+ donors and 10 Ph─ donors) and the right panel shows data obtained with Ph+ and Ph─ cell lines (Ph+: BV-173, NALM-1, TOM-1, Z-119, Ph−: Raji, Ramos, REH, BL-41). Hsp32 mRNA levels are expressed as percentage of ABL mRNA levels and represent the mean±S.D. from all donors or cell lines. Asterisk: p<0.05. B: Immunocytochemical detection of the Hsp32 protein in primary ALL cells (left panel, Ph+ patients #10 and #17; and Ph− patient #14 from Table 1) and cell lines (right panel) after incubation in control medium or hemin (10 μM) at 37°C for 8 hours. After incubation, cells were spun on cytospin slides and stained with an antibody against Hsp32 as described in the text. Images were taken using an Olympus DP21 camera connected to an Olympus BX50F4 microscope equipped with 100x/1.35 UPlan-Apo objective lense (Olympus, Hamburg, Germany). Figures were prepared using Adobe Photoshop CS2 software version 9.0 (Adobe Systems, San Jose, CA) and processed with PowerPoint software (Microsoft, Redmond, WA). C: Western blot analysis of expression of Hsp32 in Ph+ cell line Z-119 (left) and Ph─ cell line BL-41 (right). Cells were incubated with control medium (Co) or hemin (10 μM) (He) at 37°C for 8 hours. Then, cells were lysed and lysates subjected to Western blot analysis using an antibody against Hsp32 and an antibody against ß-actin as described in the text. D: Expression of HO-1 mRNA in highly enriched (sorted) CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells obtained from 3 patients with Ph− ALL (left side) and 3 patients with Ph+ ALL (p210 right side) as determined by qPCR. Cells were subjected to RNA isolation, cDNA synthesis and qPCR using primers specific for HO-1 and ABL. Results show HO-1 mRNA levels as percent of ABL mRNA levels, and represent the mean±S.D. of 3 independent experiments (3 patients).
Mentions: As assessed by qPCR, primary ALL cells as well as the ALL cell lines tested were found to express Hsp32 mRNA (Figure 1A, Tables 1 and 2). Hsp32 transcripts were present in Ph+ ALL cells as well as in Ph− ALL cells (Figure 1A). Hemin was found to promote expression of Hsp23 mRNA in all ALL samples tested (Figure 1A). We were also able to show that ALL cells display the Hsp32 protein. Expression of the Hsp32 protein was demonstrable by immunocytochemistry (Figure 1B) as well as by Western blotting (Figure 1C), and hemin was found to upregulate expression of the Hsp32 protein in ALL cells (Figure 1B and 1C). Since leukemic stem cells are considered a major target of therapy, we were also interested to know whether CD34+/CD38− stem cells in ALL express Hsp32. In these experiments, we were able to show that highly enriched (sorted) CD34+/CD38− ALL stem cells as well as CD34+/CD38+ progenitor cells express Hsp32 mRNA in patients with Ph+ ALL and patients with Ph− ALL (Figure 1D).

Bottom Line: Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells.A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells.In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria.

ABSTRACT
Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in oncology. We have shown that Hsp32, also known as heme oxygenase-1 (HO-1), serves as survival factor and potential target in Ph+ chronic myeloid leukemia. We here report that primary cells and cell lines derived from patients with acute lymphoblastic leukemia (ALL) express Hsp32 mRNA and the Hsp32 protein in a constitutive manner. Highly enriched CD34+/CD38- ALL stem cells also expressed Hsp32. Two Hsp32-targeting drugs, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), induced apoptosis and growth arrest in the BCR/ABL1+ cell lines, in Ph- lymphoblastic cell lines and in primary Ph+ and Ph- ALL cells. The effects of PEG-ZnPP and SMA-ZnPP on growth of leukemic cells were dose-dependent. In Ph+ ALL, major growth-inhibitory effects of the Hsp32-targeting drugs were observed in imatinib-sensitive and imatinib-resistant cells. Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells. A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells. In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

Show MeSH
Related in: MedlinePlus