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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

PARM-1 protein profile and secretion by NIH/3T3 cells. Immunoblotting of lysates (30 μg) from NIH/3T3 cells transiently transfected with expression vector of hParm-1 (full-length or mutants) or of mParm-1 using (a) an anti-hPARM-1 (Sigma; 1:1000) antibody or (b) an anti-GFP (Santa Cruz Biotechnology; 1:1000) antibody. Culture supernatants from these cells were collected, centrifuged, concentrated and subjected to SDS-PAGE (12 %) and hPARM-1 protein was detected by western blot (a) using anti-hPARM-1 and (b) anti-GFP antibodies recognizing respectively the N-terminus and C-terminus of hPARM-1 fusion protein. The region below 80-kDa from the supernatant in panels a and b contains no detectable bands. The anti-β-actin (Sigma; 1:1000) was used to detect possible media contaminations by proteins from lysed cell. (c) mPARM-1 expression was also tested in cell lysates.
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Figure 3: PARM-1 protein profile and secretion by NIH/3T3 cells. Immunoblotting of lysates (30 μg) from NIH/3T3 cells transiently transfected with expression vector of hParm-1 (full-length or mutants) or of mParm-1 using (a) an anti-hPARM-1 (Sigma; 1:1000) antibody or (b) an anti-GFP (Santa Cruz Biotechnology; 1:1000) antibody. Culture supernatants from these cells were collected, centrifuged, concentrated and subjected to SDS-PAGE (12 %) and hPARM-1 protein was detected by western blot (a) using anti-hPARM-1 and (b) anti-GFP antibodies recognizing respectively the N-terminus and C-terminus of hPARM-1 fusion protein. The region below 80-kDa from the supernatant in panels a and b contains no detectable bands. The anti-β-actin (Sigma; 1:1000) was used to detect possible media contaminations by proteins from lysed cell. (c) mPARM-1 expression was also tested in cell lysates.

Mentions: The EC domain of most transmembrane mucins is released from the cell surface and we verified if this was the case for PARM-1. Culture supernatant of NIH/3T3 cells transfected with hParm-1-GFP was collected and the presence of hPARM-1 visualized by western blot using either anti-hPARM-1 (specific for the EC portion) or anti-GFP antibodies (specific for the GFP tag in C-terminal). Lysates from NIH/3T3 expressing hPARM-1-GFP were also analyzed. Using the anti-hPARM-1 antibody, hPARM-1-GFP was detected in the supernatant as a very faint band slightly lower than 100-kDa. We then used two deletion mutant constructs, one deleted for the TM and CT domains (EC-GFP, Figure 2c) and the other missing only the CT portion (∆CT-GFP, Figure 2d) of hPARM-1. Our results showed that ∆CT-GFP mutant protein was also secreted in approximately the same proportion and size as the full-length construct. However, the EC-GFP mutant was found to be secreted as two bands: one intense band of about 90-kDa and a weaker band of about 70-kDa (Figure 3a; See Additional file 2: Figure S2 to compare the molecular weight of all the obtained bands). The abundance of EC-GFP in both the cell lysate and the supernatant probably reflects protein stability (Figure 3a and Additional file 2: Figure S2). Surprisingly, anti-GFP antibodies detected the secreted protein for the three constructs at the same molecular weight as for the anti-hPARM-1 antibodies (Figure 3b and Additional file 2: Figure S2) suggesting that the protein could be entirely secreted since the GFP tag is located at the C-terminal end. We could not detect actin in these supernatants excluding contamination from lysed cells. These results suggest that PARM-1 is a secreted intact protein.


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

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

PARM-1 protein profile and secretion by NIH/3T3 cells. Immunoblotting of lysates (30 μg) from NIH/3T3 cells transiently transfected with expression vector of hParm-1 (full-length or mutants) or of mParm-1 using (a) an anti-hPARM-1 (Sigma; 1:1000) antibody or (b) an anti-GFP (Santa Cruz Biotechnology; 1:1000) antibody. Culture supernatants from these cells were collected, centrifuged, concentrated and subjected to SDS-PAGE (12 %) and hPARM-1 protein was detected by western blot (a) using anti-hPARM-1 and (b) anti-GFP antibodies recognizing respectively the N-terminus and C-terminus of hPARM-1 fusion protein. The region below 80-kDa from the supernatant in panels a and b contains no detectable bands. The anti-β-actin (Sigma; 1:1000) was used to detect possible media contaminations by proteins from lysed cell. (c) mPARM-1 expression was also tested in cell lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: PARM-1 protein profile and secretion by NIH/3T3 cells. Immunoblotting of lysates (30 μg) from NIH/3T3 cells transiently transfected with expression vector of hParm-1 (full-length or mutants) or of mParm-1 using (a) an anti-hPARM-1 (Sigma; 1:1000) antibody or (b) an anti-GFP (Santa Cruz Biotechnology; 1:1000) antibody. Culture supernatants from these cells were collected, centrifuged, concentrated and subjected to SDS-PAGE (12 %) and hPARM-1 protein was detected by western blot (a) using anti-hPARM-1 and (b) anti-GFP antibodies recognizing respectively the N-terminus and C-terminus of hPARM-1 fusion protein. The region below 80-kDa from the supernatant in panels a and b contains no detectable bands. The anti-β-actin (Sigma; 1:1000) was used to detect possible media contaminations by proteins from lysed cell. (c) mPARM-1 expression was also tested in cell lysates.
Mentions: The EC domain of most transmembrane mucins is released from the cell surface and we verified if this was the case for PARM-1. Culture supernatant of NIH/3T3 cells transfected with hParm-1-GFP was collected and the presence of hPARM-1 visualized by western blot using either anti-hPARM-1 (specific for the EC portion) or anti-GFP antibodies (specific for the GFP tag in C-terminal). Lysates from NIH/3T3 expressing hPARM-1-GFP were also analyzed. Using the anti-hPARM-1 antibody, hPARM-1-GFP was detected in the supernatant as a very faint band slightly lower than 100-kDa. We then used two deletion mutant constructs, one deleted for the TM and CT domains (EC-GFP, Figure 2c) and the other missing only the CT portion (∆CT-GFP, Figure 2d) of hPARM-1. Our results showed that ∆CT-GFP mutant protein was also secreted in approximately the same proportion and size as the full-length construct. However, the EC-GFP mutant was found to be secreted as two bands: one intense band of about 90-kDa and a weaker band of about 70-kDa (Figure 3a; See Additional file 2: Figure S2 to compare the molecular weight of all the obtained bands). The abundance of EC-GFP in both the cell lysate and the supernatant probably reflects protein stability (Figure 3a and Additional file 2: Figure S2). Surprisingly, anti-GFP antibodies detected the secreted protein for the three constructs at the same molecular weight as for the anti-hPARM-1 antibodies (Figure 3b and Additional file 2: Figure S2) suggesting that the protein could be entirely secreted since the GFP tag is located at the C-terminal end. We could not detect actin in these supernatants excluding contamination from lysed cells. These results suggest that PARM-1 is a secreted intact protein.

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