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Functional characterization of the ectopically expressed olfactory receptor 2AT4 in human myelogenous leukemia

View Article: PubMed Central - PubMed

ABSTRACT

The olfactory receptor (OR) family was found to be expressed mainly in the nasal epithelium. In the last two decades members of the OR family were detected to be functional expressed in different parts of the human body such as in liver, prostate or intestine cancer cells. Here, we detected the expression of several ORs in the human chronic myelogenous leukemia (CML) cell line K562 and in white blood cells of clinically diagnosed acute myeloid leukemia (AML) patients by RT-PCR and next-generation sequencing. With calcium-imaging, we characterized in greater detail the cell biological role of one OR (OR2AT4) in leukemia. In both cell systems, the OR2AT4 agonist Sandalore-evoked strong Ca2+ influx via the adenylate cyclase-cAMP-mediated pathway. The OR2AT4 antagonist Phenirat prevented the Sandalore-induced intracellular Ca2+ increase. Western blot and flow cytometric experiments revealed that stimulation of OR2AT4 reduced the proliferation by decreasing p38-MAPK phosphorylation and induced apoptosis via phosphorylation of p44/42-MAPK. Furthermore, Sandalore increased the number of hemoglobin-containing cells in culture. We described for the first time an OR-mediated pathway in CML and AML that can regulate proliferation, apoptosis and differentiation after activation. This mechanism offers novel therapeutic options for the treatment of AML.

No MeSH data available.


ORs are expressed in the K562 and in the white blood cells of AML patients. (a) K562 RNA-Seq data revealed 7 ORs expressed (>1 FPKM). (b) With qPCR experiements we compared the expression strength of ORs in K562 to ORs in AML-patient blood. Some ORs, such as the OR2AT4, were higher expressed in AML than in K562 (n=3). (c) The OR2AT4 protein (35 kDa) is detected in the K562 membrane but not in the cytosolic fraction. (d) We confirmed the expression of the ORs in K562 and AML-patient blood by RT-PCR. Same ORs were found in cell types. (e) Immunofluorescence staining of OR2AT4 in K562 cells was analyzed with a confocal microscope. Anti-OR2AT4, red; DAPI, blue; scale bars, 20 μm.
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fig1: ORs are expressed in the K562 and in the white blood cells of AML patients. (a) K562 RNA-Seq data revealed 7 ORs expressed (>1 FPKM). (b) With qPCR experiements we compared the expression strength of ORs in K562 to ORs in AML-patient blood. Some ORs, such as the OR2AT4, were higher expressed in AML than in K562 (n=3). (c) The OR2AT4 protein (35 kDa) is detected in the K562 membrane but not in the cytosolic fraction. (d) We confirmed the expression of the ORs in K562 and AML-patient blood by RT-PCR. Same ORs were found in cell types. (e) Immunofluorescence staining of OR2AT4 in K562 cells was analyzed with a confocal microscope. Anti-OR2AT4, red; DAPI, blue; scale bars, 20 μm.

Mentions: For the initial detection of ectopically expressed ORs in myelogenous leukemia, we reanalyzed a free online available NGS data set from the CML cell line K562 (SRR1207231). In total, the expression of 7 ORs >1 FPKM could be shown (Figure 1a). In Figure 1b we compared the expression strength of ORs in K562 cells between ORs in AML-patient blood cells. Our results showed that most of the ORs detected >1 FPKM were higher expressed in all tested AML-patient blood samples. Our western blot analysis additionally verified the expression of the OR2AT4 protein in membrane components of K562 cells (Figure 1c). We validated by RT-PCR in K562 cDNA and in white blood cells of AML patients’ cDNA the expression of the ORs (Figure 1d). One of the highest expressed ORs in K562 and AML patients’ cDNA was the OR2AT4. Immunocytochemical staining of OR2AT4 showed its expression in K562 cells (Figure 1e). In addition, we investigated the expression of members of the common G-protein-coupled signaling pathways (Supplementary Figure 1). Here, we detected the expression of different adenylate cyclases, G-proteins, protein lipase C (PLC) and PKA.


Functional characterization of the ectopically expressed olfactory receptor 2AT4 in human myelogenous leukemia
ORs are expressed in the K562 and in the white blood cells of AML patients. (a) K562 RNA-Seq data revealed 7 ORs expressed (>1 FPKM). (b) With qPCR experiements we compared the expression strength of ORs in K562 to ORs in AML-patient blood. Some ORs, such as the OR2AT4, were higher expressed in AML than in K562 (n=3). (c) The OR2AT4 protein (35 kDa) is detected in the K562 membrane but not in the cytosolic fraction. (d) We confirmed the expression of the ORs in K562 and AML-patient blood by RT-PCR. Same ORs were found in cell types. (e) Immunofluorescence staining of OR2AT4 in K562 cells was analyzed with a confocal microscope. Anti-OR2AT4, red; DAPI, blue; scale bars, 20 μm.
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fig1: ORs are expressed in the K562 and in the white blood cells of AML patients. (a) K562 RNA-Seq data revealed 7 ORs expressed (>1 FPKM). (b) With qPCR experiements we compared the expression strength of ORs in K562 to ORs in AML-patient blood. Some ORs, such as the OR2AT4, were higher expressed in AML than in K562 (n=3). (c) The OR2AT4 protein (35 kDa) is detected in the K562 membrane but not in the cytosolic fraction. (d) We confirmed the expression of the ORs in K562 and AML-patient blood by RT-PCR. Same ORs were found in cell types. (e) Immunofluorescence staining of OR2AT4 in K562 cells was analyzed with a confocal microscope. Anti-OR2AT4, red; DAPI, blue; scale bars, 20 μm.
Mentions: For the initial detection of ectopically expressed ORs in myelogenous leukemia, we reanalyzed a free online available NGS data set from the CML cell line K562 (SRR1207231). In total, the expression of 7 ORs >1 FPKM could be shown (Figure 1a). In Figure 1b we compared the expression strength of ORs in K562 cells between ORs in AML-patient blood cells. Our results showed that most of the ORs detected >1 FPKM were higher expressed in all tested AML-patient blood samples. Our western blot analysis additionally verified the expression of the OR2AT4 protein in membrane components of K562 cells (Figure 1c). We validated by RT-PCR in K562 cDNA and in white blood cells of AML patients’ cDNA the expression of the ORs (Figure 1d). One of the highest expressed ORs in K562 and AML patients’ cDNA was the OR2AT4. Immunocytochemical staining of OR2AT4 showed its expression in K562 cells (Figure 1e). In addition, we investigated the expression of members of the common G-protein-coupled signaling pathways (Supplementary Figure 1). Here, we detected the expression of different adenylate cyclases, G-proteins, protein lipase C (PLC) and PKA.

View Article: PubMed Central - PubMed

ABSTRACT

The olfactory receptor (OR) family was found to be expressed mainly in the nasal epithelium. In the last two decades members of the OR family were detected to be functional expressed in different parts of the human body such as in liver, prostate or intestine cancer cells. Here, we detected the expression of several ORs in the human chronic myelogenous leukemia (CML) cell line K562 and in white blood cells of clinically diagnosed acute myeloid leukemia (AML) patients by RT-PCR and next-generation sequencing. With calcium-imaging, we characterized in greater detail the cell biological role of one OR (OR2AT4) in leukemia. In both cell systems, the OR2AT4 agonist Sandalore-evoked strong Ca2+ influx via the adenylate cyclase-cAMP-mediated pathway. The OR2AT4 antagonist Phenirat prevented the Sandalore-induced intracellular Ca2+ increase. Western blot and flow cytometric experiments revealed that stimulation of OR2AT4 reduced the proliferation by decreasing p38-MAPK phosphorylation and induced apoptosis via phosphorylation of p44/42-MAPK. Furthermore, Sandalore increased the number of hemoglobin-containing cells in culture. We described for the first time an OR-mediated pathway in CML and AML that can regulate proliferation, apoptosis and differentiation after activation. This mechanism offers novel therapeutic options for the treatment of AML.

No MeSH data available.