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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 proliferation and on anchorage-independent growth of NIH/3T3 cells. (a) NIH/3T3 cells were transfected with pEGFP-N1 (GFP), mParm-1-GFP and hParm-1-GFP expression vectors. After 48h post-transfection, 1x104 cells were resuspended in medium containing 2.5%, 5.0% or 10% of bovine serum. Medium was changed every two days. Cells were fixed and stained after 5 days with 0.2% methylene blue and 50% methanol and photographed at 40X magnification. (b) NIH/3T3 cells were either untransfected or transfected with pcDNA3.1A/Myc-His empty vector, mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A expression vectors. Cells (5.103) were plated in soft agar as described in «Colony formation in soft agar». After three weeks, cells were observed with an optical microscope (Ernst Leitz, 6MBH Wetzlar) and representative fields were photographed using a numerical camera (Nikon coolpix 4500; original magnification x40). (c) For each image, the number of colonies formed in soft agar was scored using NIH ImageJ software Version 1.42l. (d) Cells were untransfected or transiently transfected with pEGFP-N1 empty vector, hParm-1-GFP, ∆CT-GFP and ∆EC-GFP expression vectors. As a positive control, activated Ras (EJ 6.6) expression vector was used. For b and c, similar results were obtained using either PARM-1 tagged GFP or Myc-His (data not shown). The same experiment was done as for full-length constructs. All results represent the average of three independent experiments. For panels (c) and (d), the number of colonies in transfected cells was compared to untransfected cells and statistical analysis was performed using one-way analysis of variance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
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Figure 6: Effect of mPARM-1 and hPARM-1 on proliferation and on anchorage-independent growth of NIH/3T3 cells. (a) NIH/3T3 cells were transfected with pEGFP-N1 (GFP), mParm-1-GFP and hParm-1-GFP expression vectors. After 48h post-transfection, 1x104 cells were resuspended in medium containing 2.5%, 5.0% or 10% of bovine serum. Medium was changed every two days. Cells were fixed and stained after 5 days with 0.2% methylene blue and 50% methanol and photographed at 40X magnification. (b) NIH/3T3 cells were either untransfected or transfected with pcDNA3.1A/Myc-His empty vector, mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A expression vectors. Cells (5.103) were plated in soft agar as described in «Colony formation in soft agar». After three weeks, cells were observed with an optical microscope (Ernst Leitz, 6MBH Wetzlar) and representative fields were photographed using a numerical camera (Nikon coolpix 4500; original magnification x40). (c) For each image, the number of colonies formed in soft agar was scored using NIH ImageJ software Version 1.42l. (d) Cells were untransfected or transiently transfected with pEGFP-N1 empty vector, hParm-1-GFP, ∆CT-GFP and ∆EC-GFP expression vectors. As a positive control, activated Ras (EJ 6.6) expression vector was used. For b and c, similar results were obtained using either PARM-1 tagged GFP or Myc-His (data not shown). The same experiment was done as for full-length constructs. All results represent the average of three independent experiments. For panels (c) and (d), the number of colonies in transfected cells was compared to untransfected cells and statistical analysis was performed using one-way analysis of variance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Mentions: Over-expression of either mPARM-1 or hPARM-1-GFP in NIH/3T3 cells grown in the presence of 2.5%, 5% or 10% serum concentrations promoted cell-proliferation compared to control (Figure 6a) indicating that PARM-1 proteins mediate induction of serum-independent cell growth of NIH/3T3.


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 proliferation and on anchorage-independent growth of NIH/3T3 cells. (a) NIH/3T3 cells were transfected with pEGFP-N1 (GFP), mParm-1-GFP and hParm-1-GFP expression vectors. After 48h post-transfection, 1x104 cells were resuspended in medium containing 2.5%, 5.0% or 10% of bovine serum. Medium was changed every two days. Cells were fixed and stained after 5 days with 0.2% methylene blue and 50% methanol and photographed at 40X magnification. (b) NIH/3T3 cells were either untransfected or transfected with pcDNA3.1A/Myc-His empty vector, mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A expression vectors. Cells (5.103) were plated in soft agar as described in «Colony formation in soft agar». After three weeks, cells were observed with an optical microscope (Ernst Leitz, 6MBH Wetzlar) and representative fields were photographed using a numerical camera (Nikon coolpix 4500; original magnification x40). (c) For each image, the number of colonies formed in soft agar was scored using NIH ImageJ software Version 1.42l. (d) Cells were untransfected or transiently transfected with pEGFP-N1 empty vector, hParm-1-GFP, ∆CT-GFP and ∆EC-GFP expression vectors. As a positive control, activated Ras (EJ 6.6) expression vector was used. For b and c, similar results were obtained using either PARM-1 tagged GFP or Myc-His (data not shown). The same experiment was done as for full-length constructs. All results represent the average of three independent experiments. For panels (c) and (d), the number of colonies in transfected cells was compared to untransfected cells and statistical analysis was performed using one-way analysis of variance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
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Figure 6: Effect of mPARM-1 and hPARM-1 on proliferation and on anchorage-independent growth of NIH/3T3 cells. (a) NIH/3T3 cells were transfected with pEGFP-N1 (GFP), mParm-1-GFP and hParm-1-GFP expression vectors. After 48h post-transfection, 1x104 cells were resuspended in medium containing 2.5%, 5.0% or 10% of bovine serum. Medium was changed every two days. Cells were fixed and stained after 5 days with 0.2% methylene blue and 50% methanol and photographed at 40X magnification. (b) NIH/3T3 cells were either untransfected or transfected with pcDNA3.1A/Myc-His empty vector, mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A expression vectors. Cells (5.103) were plated in soft agar as described in «Colony formation in soft agar». After three weeks, cells were observed with an optical microscope (Ernst Leitz, 6MBH Wetzlar) and representative fields were photographed using a numerical camera (Nikon coolpix 4500; original magnification x40). (c) For each image, the number of colonies formed in soft agar was scored using NIH ImageJ software Version 1.42l. (d) Cells were untransfected or transiently transfected with pEGFP-N1 empty vector, hParm-1-GFP, ∆CT-GFP and ∆EC-GFP expression vectors. As a positive control, activated Ras (EJ 6.6) expression vector was used. For b and c, similar results were obtained using either PARM-1 tagged GFP or Myc-His (data not shown). The same experiment was done as for full-length constructs. All results represent the average of three independent experiments. For panels (c) and (d), the number of colonies in transfected cells was compared to untransfected cells and statistical analysis was performed using one-way analysis of variance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
Mentions: Over-expression of either mPARM-1 or hPARM-1-GFP in NIH/3T3 cells grown in the presence of 2.5%, 5% or 10% serum concentrations promoted cell-proliferation compared to control (Figure 6a) indicating that PARM-1 proteins mediate induction of serum-independent cell growth of NIH/3T3.

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