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Characterization and identification of PARM-1 as a new potential oncogene.

Charfi C, Levros LC, Edouard E, Rassart E - Mol. Cancer (2013)

Bottom Line: Moreover, deletion mutants of human PARM-1 without either extracellular or cytoplasmic portions seem to retain the ability to induce anchorage-independent growth of NIH/3T3 cells.In addition, PARM-1 increases ERK1/2, but more importantly AKT and STAT3 phosphorylation.Our results strongly suggest the oncogenic potential of PARM-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Biologie Moléculaire, Département des Sciences Biologiques, Centre BioMed, Université du Québec à Montréal, Case Postale 8888, Succursale Centre-ville, Montréal, QC H3C-3P8, Canada.

ABSTRACT

Background: The Graffi murine retrovirus is a powerful tool to find leukemia associated oncogenes. Using DNA microarrays, we recently identified several genes specifically deregulated in T- and B-leukemias induced by this virus.

Results: In the present study, probsets associated with T-CD8+ leukemias were analyzed and we validated the expression profile of the Parm-1 gene. PARM-1 is a member of the mucin family. We showed that human PARM-1 is an intact secreted protein accumulating predominantly, such as murine PARM-1, at the Golgi and in the early and late endosomes. PARM-1 colocalization with α-tubulin suggests that its trafficking within the cell involves the microtubule cytoskeleton. Also, the protein co-localizes with caveolin-1 which probably mediates its internalization. Transient transfection of both mouse and human Parm-1 cDNAs conferred anchorage- and serum-independent growth and enhanced cell proliferation. Moreover, deletion mutants of human PARM-1 without either extracellular or cytoplasmic portions seem to retain the ability to induce anchorage-independent growth of NIH/3T3 cells. In addition, PARM-1 increases ERK1/2, but more importantly AKT and STAT3 phosphorylation.

Conclusions: Our results strongly suggest the oncogenic potential of PARM-1.

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Related in: MedlinePlus

mPARM-1 and hPARM-1 proteins activate ERK1/2, PI3K/AKT, and STAT3 signaling pathways in NIH/3T3 cells. NIH/3T3 cells were either untransfected or transfected with empty vector, hParm-1-pcDNA3.1A/Myc-His, mParm-1-pcDNA3.1A, respectively. After 48 h, cell lysates were extracted with specific buffer containing proteinase and phosphatase inhibitors and 30 μg of proteins were resolved by 12% SDS-PAGE. Following protein transfer, PVDF membranes were probed with (a) anti-phospho-ERK1/2 (Thr202/Tyr204), (b) anti-phospho-AKT or (c) anti-phospho-STAT3 (All from cell Signaling; 1:1000) antibodies. Loading of equal amount of protein was verified using anti-anti-p44/42 MAPkinase (ERK), anti-AKT, anti-STAT3 (All from cell Signaling; 1:1000) and anti-β-actin antibodies. Expression of murine and human PARM-1 proteins was verified using an anti-Myc antibody (exemplified with only one membrane). Experiments were performed in triplicate.
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Figure 7: mPARM-1 and hPARM-1 proteins activate ERK1/2, PI3K/AKT, and STAT3 signaling pathways in NIH/3T3 cells. NIH/3T3 cells were either untransfected or transfected with empty vector, hParm-1-pcDNA3.1A/Myc-His, mParm-1-pcDNA3.1A, respectively. After 48 h, cell lysates were extracted with specific buffer containing proteinase and phosphatase inhibitors and 30 μg of proteins were resolved by 12% SDS-PAGE. Following protein transfer, PVDF membranes were probed with (a) anti-phospho-ERK1/2 (Thr202/Tyr204), (b) anti-phospho-AKT or (c) anti-phospho-STAT3 (All from cell Signaling; 1:1000) antibodies. Loading of equal amount of protein was verified using anti-anti-p44/42 MAPkinase (ERK), anti-AKT, anti-STAT3 (All from cell Signaling; 1:1000) and anti-β-actin antibodies. Expression of murine and human PARM-1 proteins was verified using an anti-Myc antibody (exemplified with only one membrane). Experiments were performed in triplicate.

Mentions: We showed that both PARM-1 proteins promote NIH/3T3 cells proliferation but the implication of a specific pathway by this protein remains to be determined. Activations of ERK1/2 [20], AKT [21] and STAT3 [22] dependent signaling pathway are often linked to cell proliferation. The analysis of the phosphorylation levels of ERK1/2, AKT and STAT3 in cell lysates from NIH/3T3 fibroblasts overexpressing mPARM-1 or hPARM-1 showed an up-regulation of their phosphorylation state (especially for AKT and STAT3) (Figure 7a-c) indicating that PARM-1 affect and activate the ERK1/2, AKT, and STAT3 dependent signaling pathways.


Characterization and identification of PARM-1 as a new potential oncogene.

Charfi C, Levros LC, Edouard E, Rassart E - Mol. Cancer (2013)

mPARM-1 and hPARM-1 proteins activate ERK1/2, PI3K/AKT, and STAT3 signaling pathways in NIH/3T3 cells. NIH/3T3 cells were either untransfected or transfected with empty vector, hParm-1-pcDNA3.1A/Myc-His, mParm-1-pcDNA3.1A, respectively. After 48 h, cell lysates were extracted with specific buffer containing proteinase and phosphatase inhibitors and 30 μg of proteins were resolved by 12% SDS-PAGE. Following protein transfer, PVDF membranes were probed with (a) anti-phospho-ERK1/2 (Thr202/Tyr204), (b) anti-phospho-AKT or (c) anti-phospho-STAT3 (All from cell Signaling; 1:1000) antibodies. Loading of equal amount of protein was verified using anti-anti-p44/42 MAPkinase (ERK), anti-AKT, anti-STAT3 (All from cell Signaling; 1:1000) and anti-β-actin antibodies. Expression of murine and human PARM-1 proteins was verified using an anti-Myc antibody (exemplified with only one membrane). Experiments were performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750824&req=5

Figure 7: mPARM-1 and hPARM-1 proteins activate ERK1/2, PI3K/AKT, and STAT3 signaling pathways in NIH/3T3 cells. NIH/3T3 cells were either untransfected or transfected with empty vector, hParm-1-pcDNA3.1A/Myc-His, mParm-1-pcDNA3.1A, respectively. After 48 h, cell lysates were extracted with specific buffer containing proteinase and phosphatase inhibitors and 30 μg of proteins were resolved by 12% SDS-PAGE. Following protein transfer, PVDF membranes were probed with (a) anti-phospho-ERK1/2 (Thr202/Tyr204), (b) anti-phospho-AKT or (c) anti-phospho-STAT3 (All from cell Signaling; 1:1000) antibodies. Loading of equal amount of protein was verified using anti-anti-p44/42 MAPkinase (ERK), anti-AKT, anti-STAT3 (All from cell Signaling; 1:1000) and anti-β-actin antibodies. Expression of murine and human PARM-1 proteins was verified using an anti-Myc antibody (exemplified with only one membrane). Experiments were performed in triplicate.
Mentions: We showed that both PARM-1 proteins promote NIH/3T3 cells proliferation but the implication of a specific pathway by this protein remains to be determined. Activations of ERK1/2 [20], AKT [21] and STAT3 [22] dependent signaling pathway are often linked to cell proliferation. The analysis of the phosphorylation levels of ERK1/2, AKT and STAT3 in cell lysates from NIH/3T3 fibroblasts overexpressing mPARM-1 or hPARM-1 showed an up-regulation of their phosphorylation state (especially for AKT and STAT3) (Figure 7a-c) indicating that PARM-1 affect and activate the ERK1/2, AKT, and STAT3 dependent signaling pathways.

Bottom Line: Moreover, deletion mutants of human PARM-1 without either extracellular or cytoplasmic portions seem to retain the ability to induce anchorage-independent growth of NIH/3T3 cells.In addition, PARM-1 increases ERK1/2, but more importantly AKT and STAT3 phosphorylation.Our results strongly suggest the oncogenic potential of PARM-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Biologie Moléculaire, Département des Sciences Biologiques, Centre BioMed, Université du Québec à Montréal, Case Postale 8888, Succursale Centre-ville, Montréal, QC H3C-3P8, Canada.

ABSTRACT

Background: The Graffi murine retrovirus is a powerful tool to find leukemia associated oncogenes. Using DNA microarrays, we recently identified several genes specifically deregulated in T- and B-leukemias induced by this virus.

Results: In the present study, probsets associated with T-CD8+ leukemias were analyzed and we validated the expression profile of the Parm-1 gene. PARM-1 is a member of the mucin family. We showed that human PARM-1 is an intact secreted protein accumulating predominantly, such as murine PARM-1, at the Golgi and in the early and late endosomes. PARM-1 colocalization with α-tubulin suggests that its trafficking within the cell involves the microtubule cytoskeleton. Also, the protein co-localizes with caveolin-1 which probably mediates its internalization. Transient transfection of both mouse and human Parm-1 cDNAs conferred anchorage- and serum-independent growth and enhanced cell proliferation. Moreover, deletion mutants of human PARM-1 without either extracellular or cytoplasmic portions seem to retain the ability to induce anchorage-independent growth of NIH/3T3 cells. In addition, PARM-1 increases ERK1/2, but more importantly AKT and STAT3 phosphorylation.

Conclusions: Our results strongly suggest the oncogenic potential of PARM-1.

Show MeSH
Related in: MedlinePlus