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HSP90 inhibitor AUY922 induces cell death by disruption of the Bcr-Abl, Jak2 and HSP90 signaling network complex in leukemia cells.

Tao W, Chakraborty SN, Leng X, Ma H, Arlinghaus RB - Genes Cancer (2015)

Bottom Line: Co-IP results showed that HSP90 directly bound to Bcr-Abl, Jak2, Stat 3 and Akt.Tyrosine phosphorylation of Bcr-Abl showed a dose-dependent decrease in 32Dp210T315I following AUY922 treatment for 16h.Our results showed that Bcr-Abl and Jak2 form HMWNC with HSP90 in CML cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
The Bcr-Abl protein is an important client protein of heat shock protein 90 (HSP90). We evaluated the inhibitory effects of the HSP90 ATPase inhibitor AUY922 on 32D mouse hematopoietic cells expressing wild-type Bcr-Abl (b3a2, 32Dp210) and mutant Bcr-Abl imatinib (IM)-resistant cell lines. Western blotting results of fractions from gel filtration column chromatography of 32Dp210 cells showed that HSP90 together with Bcr-Abl, Jak2 Stat3 and several other proteins co-eluted in peak column fractions of a high molecular weight network complex (HMWNC). Co-IP results showed that HSP90 directly bound to Bcr-Abl, Jak2, Stat 3 and Akt. The associations between HSP90 and Bcr-Abl or Bcr-Abl kinase domain mutants (T315I and E255K) were interrupted by AUY922 treatment. Tyrosine phosphorylation of Bcr-Abl showed a dose-dependent decrease in 32Dp210T315I following AUY922 treatment for 16h. AUY922 also markedly inhibited cell proliferation of both IM-sensitive 32Dp210 (IC50 =6 nM) and IM-resistant 32Dp210T315I cells (IC50 ≈6 nM) and human KBM-5R/KBM-7R cell lines (IC50 =50 nM). AUY922 caused significant G1 arrest in 32Dp210 cells but not in T315I or E255K cells. AUY922 efficiently induced apoptosis in 32Dp210 (IC50 =10 nM) and T315I or E255K lines with IC50 around 20 to 50 nM. Our results showed that Bcr-Abl and Jak2 form HMWNC with HSP90 in CML cells. Inhibition of HSP90 by AUY922 disrupted the structure of HMWNC, leading to Bcr-Abl degradation, nhibiting cell proliferation and inducing apoptosis. Thus, inhibition of HSP90 is a powerful way to inhibit not only IM-sensitive CML cells but also IM-resistant CML cells.

No MeSH data available.


Related in: MedlinePlus

HMWNC is disrupted by HSP90 inhibitor AUY922A1 and A2, HMWNC that was detected in Bcr-Abl+ cells was disrupted by AUY922 treatment. Proteins extraction of 32Dp210 cells treated with (A2 left panel) or without (A1 left panel) 0.5 μM AUY922 for 16 h were eluted from the gel filtration column. 25μl aliquot of each fraction was analyzed by Western blotting with indicated antibodies. No Fr, cell lysate without gel filtration. Right panel, image quantification of the Western blotting bands shown in left panel was graphically presented. B. Association of HSP90 and its client proteins in HMWNC. IP HSP90 column, the HMW region fractions from 32Dp210 cells were immunoprecipitated with HSP90 antibody following Western blotting with indicated antibodies. DWB, the same HMW region column fractions were directly Western blotted with the corresponding antibodies. C, The association between HSP90 and Bcr-Abl (wt, T315I or E255K) was impaired by AUY922. Left panel, cells were treated with 10 nM AUY922 for 16 h. Cell lysates were immunoprecipitated with HSP90 antibody followed by Western blotting with indicated antibodies. Right panel, the ratio of Bcr-Abl associated with HSP90. Protein levels were evaluated by image quantification. The levels of Bcr-Abl were normalized to HSP90 levels followed by normalization against non-treated controls to determine the association ratio, D. HSP90 client proteins level decreased under AUY922 treatment. Left panel, 32Dp210 cells were incubated with 0.5 μM AUY922 for 1, 3 and 6h. Cell lysates were analyzed by Western blotting with indicated antibodies. Right panel, the image quantification of protein bands normalized by HSP90.
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Figure 1: HMWNC is disrupted by HSP90 inhibitor AUY922A1 and A2, HMWNC that was detected in Bcr-Abl+ cells was disrupted by AUY922 treatment. Proteins extraction of 32Dp210 cells treated with (A2 left panel) or without (A1 left panel) 0.5 μM AUY922 for 16 h were eluted from the gel filtration column. 25μl aliquot of each fraction was analyzed by Western blotting with indicated antibodies. No Fr, cell lysate without gel filtration. Right panel, image quantification of the Western blotting bands shown in left panel was graphically presented. B. Association of HSP90 and its client proteins in HMWNC. IP HSP90 column, the HMW region fractions from 32Dp210 cells were immunoprecipitated with HSP90 antibody following Western blotting with indicated antibodies. DWB, the same HMW region column fractions were directly Western blotted with the corresponding antibodies. C, The association between HSP90 and Bcr-Abl (wt, T315I or E255K) was impaired by AUY922. Left panel, cells were treated with 10 nM AUY922 for 16 h. Cell lysates were immunoprecipitated with HSP90 antibody followed by Western blotting with indicated antibodies. Right panel, the ratio of Bcr-Abl associated with HSP90. Protein levels were evaluated by image quantification. The levels of Bcr-Abl were normalized to HSP90 levels followed by normalization against non-treated controls to determine the association ratio, D. HSP90 client proteins level decreased under AUY922 treatment. Left panel, 32Dp210 cells were incubated with 0.5 μM AUY922 for 1, 3 and 6h. Cell lysates were analyzed by Western blotting with indicated antibodies. Right panel, the image quantification of protein bands normalized by HSP90.

Mentions: We have reported the presence of the HMWNC of signaling molecules in Bcr-Abl+ cells [37], To further explore the components of the network complex in Bcr-Abl+ cells, lysates of 32Dp210 cell were fractioned by gel filtration column chromatography as previously described [37]. Fractions from the high molecular weight region (HMW) (fractions No. 8-17) and as well as the lower molecular weight region (LMW) (fractions No. 18-28) were analyzed by Western blotting. We found HSP90 and its client proteins including Bcr-Abl, Jak2, Stat3, and Akt were present in the same gel fractions of HMW (fraction No. 12-15), which were defined as the HMWNC with an estimated molecular weight of 2-6 million Dalton (Fig. 1A1, left panel). Image quantification of Western blotting bands showed that the gel column fractions containing highest levels of HSP90 and its client proteins (Bcr-Abl, Jak2, Stat3 and Akt) eluted in the same fractions of the column fractionation (Fig. 1A1, right panel). This suggests HSP90 and its client proteins are associated with each other in the HMWNC.


HSP90 inhibitor AUY922 induces cell death by disruption of the Bcr-Abl, Jak2 and HSP90 signaling network complex in leukemia cells.

Tao W, Chakraborty SN, Leng X, Ma H, Arlinghaus RB - Genes Cancer (2015)

HMWNC is disrupted by HSP90 inhibitor AUY922A1 and A2, HMWNC that was detected in Bcr-Abl+ cells was disrupted by AUY922 treatment. Proteins extraction of 32Dp210 cells treated with (A2 left panel) or without (A1 left panel) 0.5 μM AUY922 for 16 h were eluted from the gel filtration column. 25μl aliquot of each fraction was analyzed by Western blotting with indicated antibodies. No Fr, cell lysate without gel filtration. Right panel, image quantification of the Western blotting bands shown in left panel was graphically presented. B. Association of HSP90 and its client proteins in HMWNC. IP HSP90 column, the HMW region fractions from 32Dp210 cells were immunoprecipitated with HSP90 antibody following Western blotting with indicated antibodies. DWB, the same HMW region column fractions were directly Western blotted with the corresponding antibodies. C, The association between HSP90 and Bcr-Abl (wt, T315I or E255K) was impaired by AUY922. Left panel, cells were treated with 10 nM AUY922 for 16 h. Cell lysates were immunoprecipitated with HSP90 antibody followed by Western blotting with indicated antibodies. Right panel, the ratio of Bcr-Abl associated with HSP90. Protein levels were evaluated by image quantification. The levels of Bcr-Abl were normalized to HSP90 levels followed by normalization against non-treated controls to determine the association ratio, D. HSP90 client proteins level decreased under AUY922 treatment. Left panel, 32Dp210 cells were incubated with 0.5 μM AUY922 for 1, 3 and 6h. Cell lysates were analyzed by Western blotting with indicated antibodies. Right panel, the image quantification of protein bands normalized by HSP90.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: HMWNC is disrupted by HSP90 inhibitor AUY922A1 and A2, HMWNC that was detected in Bcr-Abl+ cells was disrupted by AUY922 treatment. Proteins extraction of 32Dp210 cells treated with (A2 left panel) or without (A1 left panel) 0.5 μM AUY922 for 16 h were eluted from the gel filtration column. 25μl aliquot of each fraction was analyzed by Western blotting with indicated antibodies. No Fr, cell lysate without gel filtration. Right panel, image quantification of the Western blotting bands shown in left panel was graphically presented. B. Association of HSP90 and its client proteins in HMWNC. IP HSP90 column, the HMW region fractions from 32Dp210 cells were immunoprecipitated with HSP90 antibody following Western blotting with indicated antibodies. DWB, the same HMW region column fractions were directly Western blotted with the corresponding antibodies. C, The association between HSP90 and Bcr-Abl (wt, T315I or E255K) was impaired by AUY922. Left panel, cells were treated with 10 nM AUY922 for 16 h. Cell lysates were immunoprecipitated with HSP90 antibody followed by Western blotting with indicated antibodies. Right panel, the ratio of Bcr-Abl associated with HSP90. Protein levels were evaluated by image quantification. The levels of Bcr-Abl were normalized to HSP90 levels followed by normalization against non-treated controls to determine the association ratio, D. HSP90 client proteins level decreased under AUY922 treatment. Left panel, 32Dp210 cells were incubated with 0.5 μM AUY922 for 1, 3 and 6h. Cell lysates were analyzed by Western blotting with indicated antibodies. Right panel, the image quantification of protein bands normalized by HSP90.
Mentions: We have reported the presence of the HMWNC of signaling molecules in Bcr-Abl+ cells [37], To further explore the components of the network complex in Bcr-Abl+ cells, lysates of 32Dp210 cell were fractioned by gel filtration column chromatography as previously described [37]. Fractions from the high molecular weight region (HMW) (fractions No. 8-17) and as well as the lower molecular weight region (LMW) (fractions No. 18-28) were analyzed by Western blotting. We found HSP90 and its client proteins including Bcr-Abl, Jak2, Stat3, and Akt were present in the same gel fractions of HMW (fraction No. 12-15), which were defined as the HMWNC with an estimated molecular weight of 2-6 million Dalton (Fig. 1A1, left panel). Image quantification of Western blotting bands showed that the gel column fractions containing highest levels of HSP90 and its client proteins (Bcr-Abl, Jak2, Stat3 and Akt) eluted in the same fractions of the column fractionation (Fig. 1A1, right panel). This suggests HSP90 and its client proteins are associated with each other in the HMWNC.

Bottom Line: Co-IP results showed that HSP90 directly bound to Bcr-Abl, Jak2, Stat 3 and Akt.Tyrosine phosphorylation of Bcr-Abl showed a dose-dependent decrease in 32Dp210T315I following AUY922 treatment for 16h.Our results showed that Bcr-Abl and Jak2 form HMWNC with HSP90 in CML cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
The Bcr-Abl protein is an important client protein of heat shock protein 90 (HSP90). We evaluated the inhibitory effects of the HSP90 ATPase inhibitor AUY922 on 32D mouse hematopoietic cells expressing wild-type Bcr-Abl (b3a2, 32Dp210) and mutant Bcr-Abl imatinib (IM)-resistant cell lines. Western blotting results of fractions from gel filtration column chromatography of 32Dp210 cells showed that HSP90 together with Bcr-Abl, Jak2 Stat3 and several other proteins co-eluted in peak column fractions of a high molecular weight network complex (HMWNC). Co-IP results showed that HSP90 directly bound to Bcr-Abl, Jak2, Stat 3 and Akt. The associations between HSP90 and Bcr-Abl or Bcr-Abl kinase domain mutants (T315I and E255K) were interrupted by AUY922 treatment. Tyrosine phosphorylation of Bcr-Abl showed a dose-dependent decrease in 32Dp210T315I following AUY922 treatment for 16h. AUY922 also markedly inhibited cell proliferation of both IM-sensitive 32Dp210 (IC50 =6 nM) and IM-resistant 32Dp210T315I cells (IC50 ≈6 nM) and human KBM-5R/KBM-7R cell lines (IC50 =50 nM). AUY922 caused significant G1 arrest in 32Dp210 cells but not in T315I or E255K cells. AUY922 efficiently induced apoptosis in 32Dp210 (IC50 =10 nM) and T315I or E255K lines with IC50 around 20 to 50 nM. Our results showed that Bcr-Abl and Jak2 form HMWNC with HSP90 in CML cells. Inhibition of HSP90 by AUY922 disrupted the structure of HMWNC, leading to Bcr-Abl degradation, nhibiting cell proliferation and inducing apoptosis. Thus, inhibition of HSP90 is a powerful way to inhibit not only IM-sensitive CML cells but also IM-resistant CML cells.

No MeSH data available.


Related in: MedlinePlus