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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

Effect of mPARM-1 and hPARM-1 on cell cycle of NIH/3T3 cells. (a) Synchronized NIH/3T3 cells were either untransfected, or transfected with the empty vector (pcDNA3.1A/Myc-His), murine or human Parm-1 constructs. Cells were fixed at 72h post-transfection, stained with propidium iodide and analyzed for cell cycle phase distribution. Percentages of cells in different phases of cell cycle were determined with the ModFit software. (b) Proliferation of control pcDNA3.1A/Myc-His, mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A transfected NIH/3T3 cells was analyzed using BrdU, 48h after transfection. DAPI labeled nuclei are in purple and cells that have incorporated BrdU are in green. Representative fields were photographed. (c) The percentage of proliferating cells was determined using the following formula: % of proliferating cells = (number of BrdU incorporating cells/total number of DAPI stained cells) * 100. Values were normalized relative to control cells. For a, b and c, similar results were obtained using either PARM-1 tagged GFP or Myc-His (data not shown). All results represent the average of three independent experiments. Statistical analysis was performed using one-way analysis of variance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
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Figure 5: Effect of mPARM-1 and hPARM-1 on cell cycle of NIH/3T3 cells. (a) Synchronized NIH/3T3 cells were either untransfected, or transfected with the empty vector (pcDNA3.1A/Myc-His), murine or human Parm-1 constructs. Cells were fixed at 72h post-transfection, stained with propidium iodide and analyzed for cell cycle phase distribution. Percentages of cells in different phases of cell cycle were determined with the ModFit software. (b) Proliferation of control pcDNA3.1A/Myc-His, mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A transfected NIH/3T3 cells was analyzed using BrdU, 48h after transfection. DAPI labeled nuclei are in purple and cells that have incorporated BrdU are in green. Representative fields were photographed. (c) The percentage of proliferating cells was determined using the following formula: % of proliferating cells = (number of BrdU incorporating cells/total number of DAPI stained cells) * 100. Values were normalized relative to control cells. For a, b and c, similar results were obtained using either PARM-1 tagged GFP or Myc-His (data not shown). All results represent the average of three independent experiments. Statistical analysis was performed using one-way analysis of variance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Mentions: Transfected NIH/3T3 cells were tested for cell-cycle progression by FACS analysis. We found that the percentage of NIH/3T3 cells transfected with mParm-1 or hParm-1 in S phase is enhanced by 2 fold compared to control cells (Figure 5a). Also, BrdU incorporation in NIH/3T3 cells transfected with either mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A was 50% higher than that of controls (Figure 5b and 5c) suggesting that PARM-1 is a positive cell-cycle regulator.


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

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

Effect of mPARM-1 and hPARM-1 on cell cycle of NIH/3T3 cells. (a) Synchronized NIH/3T3 cells were either untransfected, or transfected with the empty vector (pcDNA3.1A/Myc-His), murine or human Parm-1 constructs. Cells were fixed at 72h post-transfection, stained with propidium iodide and analyzed for cell cycle phase distribution. Percentages of cells in different phases of cell cycle were determined with the ModFit software. (b) Proliferation of control pcDNA3.1A/Myc-His, mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A transfected NIH/3T3 cells was analyzed using BrdU, 48h after transfection. DAPI labeled nuclei are in purple and cells that have incorporated BrdU are in green. Representative fields were photographed. (c) The percentage of proliferating cells was determined using the following formula: % of proliferating cells = (number of BrdU incorporating cells/total number of DAPI stained cells) * 100. Values were normalized relative to control cells. For a, b and c, similar results were obtained using either PARM-1 tagged GFP or Myc-His (data not shown). All results represent the average of three independent experiments. Statistical analysis was performed using one-way analysis of variance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effect of mPARM-1 and hPARM-1 on cell cycle of NIH/3T3 cells. (a) Synchronized NIH/3T3 cells were either untransfected, or transfected with the empty vector (pcDNA3.1A/Myc-His), murine or human Parm-1 constructs. Cells were fixed at 72h post-transfection, stained with propidium iodide and analyzed for cell cycle phase distribution. Percentages of cells in different phases of cell cycle were determined with the ModFit software. (b) Proliferation of control pcDNA3.1A/Myc-His, mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A transfected NIH/3T3 cells was analyzed using BrdU, 48h after transfection. DAPI labeled nuclei are in purple and cells that have incorporated BrdU are in green. Representative fields were photographed. (c) The percentage of proliferating cells was determined using the following formula: % of proliferating cells = (number of BrdU incorporating cells/total number of DAPI stained cells) * 100. Values were normalized relative to control cells. For a, b and c, similar results were obtained using either PARM-1 tagged GFP or Myc-His (data not shown). All results represent the average of three independent experiments. Statistical analysis was performed using one-way analysis of variance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
Mentions: Transfected NIH/3T3 cells were tested for cell-cycle progression by FACS analysis. We found that the percentage of NIH/3T3 cells transfected with mParm-1 or hParm-1 in S phase is enhanced by 2 fold compared to control cells (Figure 5a). Also, BrdU incorporation in NIH/3T3 cells transfected with either mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A was 50% higher than that of controls (Figure 5b and 5c) suggesting that PARM-1 is a positive cell-cycle regulator.

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