TELOMERASE MRNA IN T CELL LEUKEMIA CELL LINE (JURKAT) DERIVED EXOSOMES TRANSFORMS NONMALIGNANT FIBROBLASTS INTO TELOMERASE POSITIVE CELLS
(Abstract release date: 05/19/16)
EHA Library. Lahav M. 06/09/16; 132677; E1128
Dr. Meir Lahav
Contributions
Contributions
Abstract
Abstract: E1128
Type: Eposter Presentation
Background
Exosomes are small (30-100nm) vesicles secreted from all cell types serving as inter- cell communicators by affecting biological processes in 'recipient' cells upon their uptake. Telomerase is transcriptionally repressed in non malignant somatic cells thus enabling cellular senescence caused by telomere shortening. Telomerase activation is a hallmark of cancer cells providing them with endless replicative potential. Telomerase activation is associated also with other major cellular changes such as resistance to DNA damage, protection from apoptosis and others. We demonstrate that hTERT mRNA is transferred via exosomes from Jurkat cells into telomerase negative primary fibroblasts. The mRNA is translated into active telomerase causing several major changes in those cells. These findings have potential implications for tumor cells – microenvironment interactions.
Aims
To assess the presence of hTERT mRNA, the transcript of the enzyme telomerase, in exosomes secreted from Jurkat cells; to study the possible effects of this transcript on the phenotype of fibroblasts cells upon exposure to exosomal hTERT mRNA.
Methods
Exosomes were isolated by exosome isolation kit (exoquick). Real time PCR was performed to assess the hTERT mRNA levels in exosomes and in the cells. Verification of exosome isolation and telomerase levels were estimated by Western blotting. Telomerase activity was assessed by Real time PCR based TRAP assay. Cellular proliferation and senescence status were assessed by Trypan blue exclusion and b-Galactosidase activity assays, respectively. Nuclear morphology was visualized after DAPI staining. DNA damage response was evaluated by counting gH2AX foci accumulation following phleomycin insult.
Results
The current study demonstrates for the first time that hTERT mRNA, the transcript of the enzyme telomerase, is shuttled from 'donor' cancer cells via exosomes into 'recipient' telomerase negative fibroblasts, where it is translated into a fully active enzyme. All tested cells secreted exosomal hTERT mRNA in accordance with the endogenous levels of their hTERT mRNA and telomerase activity. A non-cancer fibroblasts cell line in which telomerase was ectopically expressed secreted relatively high levels of exosomal hTERT mRNA as well. A similar ex-vivo shuttle of exosomal hTERT mRNA isolated from the serum of a patient with pancreatic cancer into primary fibroblasts was demonstrated as well. Telomerase activity induced phenotypic changes in the recipient fibroblasts including extension of life span and postponing senescence. In addition, telomerase activity protected the fibroblasts from DNA damage induced by Phleomycin and also protected from apoptosis, indicating that telomerase extracurricular activities are also manifested in the recipient cells.
Conclusion
The shuttle of telomerase from cancer cells into fibroblasts and the induction of these changes may contribute to the formation of cancer associated fibroblasts representing a unique aspect of the cross talk between tumors and their microenvironment. These processes bear a therapeutic potential targets and as such serve as a promising research avenue.
Session topic: E-poster
Type: Eposter Presentation
Background
Exosomes are small (30-100nm) vesicles secreted from all cell types serving as inter- cell communicators by affecting biological processes in 'recipient' cells upon their uptake. Telomerase is transcriptionally repressed in non malignant somatic cells thus enabling cellular senescence caused by telomere shortening. Telomerase activation is a hallmark of cancer cells providing them with endless replicative potential. Telomerase activation is associated also with other major cellular changes such as resistance to DNA damage, protection from apoptosis and others. We demonstrate that hTERT mRNA is transferred via exosomes from Jurkat cells into telomerase negative primary fibroblasts. The mRNA is translated into active telomerase causing several major changes in those cells. These findings have potential implications for tumor cells – microenvironment interactions.
Aims
To assess the presence of hTERT mRNA, the transcript of the enzyme telomerase, in exosomes secreted from Jurkat cells; to study the possible effects of this transcript on the phenotype of fibroblasts cells upon exposure to exosomal hTERT mRNA.
Methods
Exosomes were isolated by exosome isolation kit (exoquick). Real time PCR was performed to assess the hTERT mRNA levels in exosomes and in the cells. Verification of exosome isolation and telomerase levels were estimated by Western blotting. Telomerase activity was assessed by Real time PCR based TRAP assay. Cellular proliferation and senescence status were assessed by Trypan blue exclusion and b-Galactosidase activity assays, respectively. Nuclear morphology was visualized after DAPI staining. DNA damage response was evaluated by counting gH2AX foci accumulation following phleomycin insult.
Results
The current study demonstrates for the first time that hTERT mRNA, the transcript of the enzyme telomerase, is shuttled from 'donor' cancer cells via exosomes into 'recipient' telomerase negative fibroblasts, where it is translated into a fully active enzyme. All tested cells secreted exosomal hTERT mRNA in accordance with the endogenous levels of their hTERT mRNA and telomerase activity. A non-cancer fibroblasts cell line in which telomerase was ectopically expressed secreted relatively high levels of exosomal hTERT mRNA as well. A similar ex-vivo shuttle of exosomal hTERT mRNA isolated from the serum of a patient with pancreatic cancer into primary fibroblasts was demonstrated as well. Telomerase activity induced phenotypic changes in the recipient fibroblasts including extension of life span and postponing senescence. In addition, telomerase activity protected the fibroblasts from DNA damage induced by Phleomycin and also protected from apoptosis, indicating that telomerase extracurricular activities are also manifested in the recipient cells.
Conclusion
The shuttle of telomerase from cancer cells into fibroblasts and the induction of these changes may contribute to the formation of cancer associated fibroblasts representing a unique aspect of the cross talk between tumors and their microenvironment. These processes bear a therapeutic potential targets and as such serve as a promising research avenue.
Session topic: E-poster
Abstract: E1128
Type: Eposter Presentation
Background
Exosomes are small (30-100nm) vesicles secreted from all cell types serving as inter- cell communicators by affecting biological processes in 'recipient' cells upon their uptake. Telomerase is transcriptionally repressed in non malignant somatic cells thus enabling cellular senescence caused by telomere shortening. Telomerase activation is a hallmark of cancer cells providing them with endless replicative potential. Telomerase activation is associated also with other major cellular changes such as resistance to DNA damage, protection from apoptosis and others. We demonstrate that hTERT mRNA is transferred via exosomes from Jurkat cells into telomerase negative primary fibroblasts. The mRNA is translated into active telomerase causing several major changes in those cells. These findings have potential implications for tumor cells – microenvironment interactions.
Aims
To assess the presence of hTERT mRNA, the transcript of the enzyme telomerase, in exosomes secreted from Jurkat cells; to study the possible effects of this transcript on the phenotype of fibroblasts cells upon exposure to exosomal hTERT mRNA.
Methods
Exosomes were isolated by exosome isolation kit (exoquick). Real time PCR was performed to assess the hTERT mRNA levels in exosomes and in the cells. Verification of exosome isolation and telomerase levels were estimated by Western blotting. Telomerase activity was assessed by Real time PCR based TRAP assay. Cellular proliferation and senescence status were assessed by Trypan blue exclusion and b-Galactosidase activity assays, respectively. Nuclear morphology was visualized after DAPI staining. DNA damage response was evaluated by counting gH2AX foci accumulation following phleomycin insult.
Results
The current study demonstrates for the first time that hTERT mRNA, the transcript of the enzyme telomerase, is shuttled from 'donor' cancer cells via exosomes into 'recipient' telomerase negative fibroblasts, where it is translated into a fully active enzyme. All tested cells secreted exosomal hTERT mRNA in accordance with the endogenous levels of their hTERT mRNA and telomerase activity. A non-cancer fibroblasts cell line in which telomerase was ectopically expressed secreted relatively high levels of exosomal hTERT mRNA as well. A similar ex-vivo shuttle of exosomal hTERT mRNA isolated from the serum of a patient with pancreatic cancer into primary fibroblasts was demonstrated as well. Telomerase activity induced phenotypic changes in the recipient fibroblasts including extension of life span and postponing senescence. In addition, telomerase activity protected the fibroblasts from DNA damage induced by Phleomycin and also protected from apoptosis, indicating that telomerase extracurricular activities are also manifested in the recipient cells.
Conclusion
The shuttle of telomerase from cancer cells into fibroblasts and the induction of these changes may contribute to the formation of cancer associated fibroblasts representing a unique aspect of the cross talk between tumors and their microenvironment. These processes bear a therapeutic potential targets and as such serve as a promising research avenue.
Session topic: E-poster
Type: Eposter Presentation
Background
Exosomes are small (30-100nm) vesicles secreted from all cell types serving as inter- cell communicators by affecting biological processes in 'recipient' cells upon their uptake. Telomerase is transcriptionally repressed in non malignant somatic cells thus enabling cellular senescence caused by telomere shortening. Telomerase activation is a hallmark of cancer cells providing them with endless replicative potential. Telomerase activation is associated also with other major cellular changes such as resistance to DNA damage, protection from apoptosis and others. We demonstrate that hTERT mRNA is transferred via exosomes from Jurkat cells into telomerase negative primary fibroblasts. The mRNA is translated into active telomerase causing several major changes in those cells. These findings have potential implications for tumor cells – microenvironment interactions.
Aims
To assess the presence of hTERT mRNA, the transcript of the enzyme telomerase, in exosomes secreted from Jurkat cells; to study the possible effects of this transcript on the phenotype of fibroblasts cells upon exposure to exosomal hTERT mRNA.
Methods
Exosomes were isolated by exosome isolation kit (exoquick). Real time PCR was performed to assess the hTERT mRNA levels in exosomes and in the cells. Verification of exosome isolation and telomerase levels were estimated by Western blotting. Telomerase activity was assessed by Real time PCR based TRAP assay. Cellular proliferation and senescence status were assessed by Trypan blue exclusion and b-Galactosidase activity assays, respectively. Nuclear morphology was visualized after DAPI staining. DNA damage response was evaluated by counting gH2AX foci accumulation following phleomycin insult.
Results
The current study demonstrates for the first time that hTERT mRNA, the transcript of the enzyme telomerase, is shuttled from 'donor' cancer cells via exosomes into 'recipient' telomerase negative fibroblasts, where it is translated into a fully active enzyme. All tested cells secreted exosomal hTERT mRNA in accordance with the endogenous levels of their hTERT mRNA and telomerase activity. A non-cancer fibroblasts cell line in which telomerase was ectopically expressed secreted relatively high levels of exosomal hTERT mRNA as well. A similar ex-vivo shuttle of exosomal hTERT mRNA isolated from the serum of a patient with pancreatic cancer into primary fibroblasts was demonstrated as well. Telomerase activity induced phenotypic changes in the recipient fibroblasts including extension of life span and postponing senescence. In addition, telomerase activity protected the fibroblasts from DNA damage induced by Phleomycin and also protected from apoptosis, indicating that telomerase extracurricular activities are also manifested in the recipient cells.
Conclusion
The shuttle of telomerase from cancer cells into fibroblasts and the induction of these changes may contribute to the formation of cancer associated fibroblasts representing a unique aspect of the cross talk between tumors and their microenvironment. These processes bear a therapeutic potential targets and as such serve as a promising research avenue.
Session topic: E-poster
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