Contributions
Abstract: PB1909
Type: Publication Only
Background
The effect of hypoxia on protein translation is mediated in part through inhibition of mammalian target of rapamycin complex 1 (mTORC1), but the principle mechanism for hypoxia-induced mTORC1 inhibition however was not elucidated until recently.
Aims
In order to elucidate leukemic stem cell behaviour including self-renewal, apoptosis, proliferation and molecular pathway involved in these mechanisms we analysed K562 under hypoxic condition. We focused our studies on mTOR pathaway, expecially on mTORC2, which ability to activate directly AKT by Ser473 phosphorylation makes it an interestly candidate in cell self-renewal scenario.
Methods
K562 erythroleukemia cells were maintained in RPMI 1640, supplemented with 10%FCS. Hypoxia exposure was performed by incubation in 5% CO2, 1% Oxygen, 94% N2 condition for different time point (20 and 40 hours). Cell viability and death were performed by MTT assay and Annexin V identification by FACS analysis. Total cell lyses, nuclear and cytoplasm fractions were used to carry out western blot to analyse mTORC1 and mTORC2 pathway and to investigate cellular delocalization of TOR component in hypoxia condition. Immunofluorescence were performed on K562 slices after permeabilization and subsequent primary and FITC-secondary antibody incubation to detect mTOR component localization. RNA were extract by TRIzol method and used to analyse genes known to be involved in hypoxia pathway.
Results
In agreement with previous works, the hypoxic culture of k562 led to decreased in proliferation and increased in CD34 expression, suggesting the ability of cell line to take a “stem phenotype” under hypoxic condition. Moreover we have observed a reduction of the S phase of the cellluar cycle, and subsequent accumulation on G2/M phase. We observed that after hypoxia exposition mTORC1 pathway is strongly abrogated, as documented by reduction in 4EBP, ribosomal protein S6 and mTOR phosphorylation. Conversely mTORC2 resulted significantly activated, with increased in AKT (ser473), NDRG and SGK phosphorylation, reduction in GSK phosphorilation and increased in total Rac protein. This phenomenon is accompanied by a strong and significant relocation of mTOR, Rictor and Akt in the nucleus. Interesting these peculiar localization has been observed by immunofluorescence on CD34+ CML cells suggesting therefore that it could be a typical behaviour of cells localizing in hypoxic environment. The quantitative analysis on different hypoxic treatments showed a significant increase in VEGF (known to be regulated by HIF1) and SOX genes (SOX2 and SOX17) which would appear to be negatively regulated by mTORC1. Furthermore the computational analysis on HIF promoter showed the presence of many HMG-box motifs, typical of SOX genes, suggesting a possible link between mTORC1 inactivation, mTORC2 and AKT nuclear activation and HIF expression in hypoxic K562 cells.
Conclusion
We observed that hypoxic treatment leads to a strong mTORC1 decreased activity, inhibits proliferation and promotes survival and stem cell phenotype in K562, with an increased in mTORC2 activity and its relocalization in nucleus. How Rictor and mTORC2 complex act in nucleus is still unknown. A recent work performed on S. Pombae showed that Rictor is able to rilocalize in nucleus by direct link to AKT and E2F transcription factor, favouring its transcription activiy. Our future propose will be to investigate the role of E2F, which have emerged as essential regulators of stem cell fate control in a number of lineages, in maintaining leukaemia stem cells.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): Chronic myeloid leukemia, Hypoxia-sensing, mTOR
Abstract: PB1909
Type: Publication Only
Background
The effect of hypoxia on protein translation is mediated in part through inhibition of mammalian target of rapamycin complex 1 (mTORC1), but the principle mechanism for hypoxia-induced mTORC1 inhibition however was not elucidated until recently.
Aims
In order to elucidate leukemic stem cell behaviour including self-renewal, apoptosis, proliferation and molecular pathway involved in these mechanisms we analysed K562 under hypoxic condition. We focused our studies on mTOR pathaway, expecially on mTORC2, which ability to activate directly AKT by Ser473 phosphorylation makes it an interestly candidate in cell self-renewal scenario.
Methods
K562 erythroleukemia cells were maintained in RPMI 1640, supplemented with 10%FCS. Hypoxia exposure was performed by incubation in 5% CO2, 1% Oxygen, 94% N2 condition for different time point (20 and 40 hours). Cell viability and death were performed by MTT assay and Annexin V identification by FACS analysis. Total cell lyses, nuclear and cytoplasm fractions were used to carry out western blot to analyse mTORC1 and mTORC2 pathway and to investigate cellular delocalization of TOR component in hypoxia condition. Immunofluorescence were performed on K562 slices after permeabilization and subsequent primary and FITC-secondary antibody incubation to detect mTOR component localization. RNA were extract by TRIzol method and used to analyse genes known to be involved in hypoxia pathway.
Results
In agreement with previous works, the hypoxic culture of k562 led to decreased in proliferation and increased in CD34 expression, suggesting the ability of cell line to take a “stem phenotype” under hypoxic condition. Moreover we have observed a reduction of the S phase of the cellluar cycle, and subsequent accumulation on G2/M phase. We observed that after hypoxia exposition mTORC1 pathway is strongly abrogated, as documented by reduction in 4EBP, ribosomal protein S6 and mTOR phosphorylation. Conversely mTORC2 resulted significantly activated, with increased in AKT (ser473), NDRG and SGK phosphorylation, reduction in GSK phosphorilation and increased in total Rac protein. This phenomenon is accompanied by a strong and significant relocation of mTOR, Rictor and Akt in the nucleus. Interesting these peculiar localization has been observed by immunofluorescence on CD34+ CML cells suggesting therefore that it could be a typical behaviour of cells localizing in hypoxic environment. The quantitative analysis on different hypoxic treatments showed a significant increase in VEGF (known to be regulated by HIF1) and SOX genes (SOX2 and SOX17) which would appear to be negatively regulated by mTORC1. Furthermore the computational analysis on HIF promoter showed the presence of many HMG-box motifs, typical of SOX genes, suggesting a possible link between mTORC1 inactivation, mTORC2 and AKT nuclear activation and HIF expression in hypoxic K562 cells.
Conclusion
We observed that hypoxic treatment leads to a strong mTORC1 decreased activity, inhibits proliferation and promotes survival and stem cell phenotype in K562, with an increased in mTORC2 activity and its relocalization in nucleus. How Rictor and mTORC2 complex act in nucleus is still unknown. A recent work performed on S. Pombae showed that Rictor is able to rilocalize in nucleus by direct link to AKT and E2F transcription factor, favouring its transcription activiy. Our future propose will be to investigate the role of E2F, which have emerged as essential regulators of stem cell fate control in a number of lineages, in maintaining leukaemia stem cells.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): Chronic myeloid leukemia, Hypoxia-sensing, mTOR