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Identification of distinctive patterns of USP19-mediated growth regulation in normal and malignant cells.

Lu Y, Bedard N, Chevalier S, Wing SS - PLoS ONE (2011)

Bottom Line: Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27(Kip1) levels.This may occur through both KPC1 dependent and independent mechanisms.Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.

View Article: PubMed Central - PubMed

Affiliation: Polypeptide Laboratory, Department of Medicine, McGill University Health Centre Research Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT
We previously reported that the USP19 deubiquitinating enzyme positively regulates proliferation in fibroblasts by stabilizing KPC1, a ubiquitin ligase for p27(Kip1). To explore whether this role of USP19 extends to other cellular systems, we tested the effects of silencing of USP19 in several human prostate and breast models, including carcinoma cell lines. Depletion of USP19 inhibited proliferation in prostate cancer DU145, PC-3 and 22RV1 cells, which was similar to the pattern established in fibroblasts in that it was due to decreased progression from G1 to S phase and associated with a stabilization of the cyclin-dependent kinase inhibitor p27(Kip1). However, in contrast to previous findings in fibroblasts, the stabilization of p27(Kip1) upon USP19 depletion was not associated with changes in the levels of the KPC1 ligase. USP19 could also regulate the growth of immortalized MCF10A breast epithelial cells through a similar mechanism. This regulatory pattern was lost, though, in breast cancer MCF7 and MDA-MB-231 cells and in prostate carcinoma LNCaP cells. Of interest, the transformation of fibroblasts through overexpression of an oncogenic form of Ras disrupted the USP19-mediated regulation of cell growth and of levels of p27(Kip1) and KPC1. Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27(Kip1) levels. This may occur through both KPC1 dependent and independent mechanisms. Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.

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The ability of depletion of USP19 to regulate cell growth and p27Kip1 is lost in FR3T3 fibroblasts transformed by constitutively active Ras.(A) Rat FR3T3 fibroblasts expressing an activated Ras oncogene (Ras-V12) or empty vector were transfected with USP19 siRNA oligonucleotides #1, #43, or nonspecific control siRNA oligonucleotide (CTL). Forty-eight hours after transfection, cells were harvested and counted. Shown are means ± SE of triplicate samples. *, P<0.001 compared to CTL. (B) USP19 depleted FR3T3-vect or FR3T3-Ras cells from experiment described in Fig. 5A were harvested. Equal amounts of protein from lysates were analyzed by immunoblotting with the indicated antibodies.
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pone-0015936-g005: The ability of depletion of USP19 to regulate cell growth and p27Kip1 is lost in FR3T3 fibroblasts transformed by constitutively active Ras.(A) Rat FR3T3 fibroblasts expressing an activated Ras oncogene (Ras-V12) or empty vector were transfected with USP19 siRNA oligonucleotides #1, #43, or nonspecific control siRNA oligonucleotide (CTL). Forty-eight hours after transfection, cells were harvested and counted. Shown are means ± SE of triplicate samples. *, P<0.001 compared to CTL. (B) USP19 depleted FR3T3-vect or FR3T3-Ras cells from experiment described in Fig. 5A were harvested. Equal amounts of protein from lysates were analyzed by immunoblotting with the indicated antibodies.

Mentions: The striking differences observed between the USP19 growth regulatory patterns in human breast cells, normal vs carcinoma, and prostate cancer cell lines as well as rat fibroblasts raised the question of whether the cell growth response to USP19 depletion may somehow be associated to oncogenic cell transformation. To test this possibility and further dissect underlying mechanisms, FR3T3 cells were transduced stably with a retrovirus expressing an activated Ras oncogene (Ras-V12) or an empty vector. The effects upon USP19 depletion were analyzed and compared to cells transfected with control oligonucleotide. As shown before, depletion of USP19 in control FR3T3 cells expressing empty vector resulted in growth inhibition (Fig. 5A) with a corresponding upregulation of p27Kip1 and reduction in KPC1 levels (Fig. 5B). However, in Ras transformed FR3T3 cells, depletion of USP19 no longer led to any of these changes (Fig. 5A and B). Therefore, the ability of USP19 to control p27Kip1 via KPC1 and thereby regulate cell growth may be abolished when cells are transformed via the Ras oncogene. Altogether, this strongly suggests that constitutive activation of the Ras signalling pathway may figure among mechanisms explaining altered USP19 function and p27Kip1 levels in cancer cells.


Identification of distinctive patterns of USP19-mediated growth regulation in normal and malignant cells.

Lu Y, Bedard N, Chevalier S, Wing SS - PLoS ONE (2011)

The ability of depletion of USP19 to regulate cell growth and p27Kip1 is lost in FR3T3 fibroblasts transformed by constitutively active Ras.(A) Rat FR3T3 fibroblasts expressing an activated Ras oncogene (Ras-V12) or empty vector were transfected with USP19 siRNA oligonucleotides #1, #43, or nonspecific control siRNA oligonucleotide (CTL). Forty-eight hours after transfection, cells were harvested and counted. Shown are means ± SE of triplicate samples. *, P<0.001 compared to CTL. (B) USP19 depleted FR3T3-vect or FR3T3-Ras cells from experiment described in Fig. 5A were harvested. Equal amounts of protein from lysates were analyzed by immunoblotting with the indicated antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3022023&req=5

pone-0015936-g005: The ability of depletion of USP19 to regulate cell growth and p27Kip1 is lost in FR3T3 fibroblasts transformed by constitutively active Ras.(A) Rat FR3T3 fibroblasts expressing an activated Ras oncogene (Ras-V12) or empty vector were transfected with USP19 siRNA oligonucleotides #1, #43, or nonspecific control siRNA oligonucleotide (CTL). Forty-eight hours after transfection, cells were harvested and counted. Shown are means ± SE of triplicate samples. *, P<0.001 compared to CTL. (B) USP19 depleted FR3T3-vect or FR3T3-Ras cells from experiment described in Fig. 5A were harvested. Equal amounts of protein from lysates were analyzed by immunoblotting with the indicated antibodies.
Mentions: The striking differences observed between the USP19 growth regulatory patterns in human breast cells, normal vs carcinoma, and prostate cancer cell lines as well as rat fibroblasts raised the question of whether the cell growth response to USP19 depletion may somehow be associated to oncogenic cell transformation. To test this possibility and further dissect underlying mechanisms, FR3T3 cells were transduced stably with a retrovirus expressing an activated Ras oncogene (Ras-V12) or an empty vector. The effects upon USP19 depletion were analyzed and compared to cells transfected with control oligonucleotide. As shown before, depletion of USP19 in control FR3T3 cells expressing empty vector resulted in growth inhibition (Fig. 5A) with a corresponding upregulation of p27Kip1 and reduction in KPC1 levels (Fig. 5B). However, in Ras transformed FR3T3 cells, depletion of USP19 no longer led to any of these changes (Fig. 5A and B). Therefore, the ability of USP19 to control p27Kip1 via KPC1 and thereby regulate cell growth may be abolished when cells are transformed via the Ras oncogene. Altogether, this strongly suggests that constitutive activation of the Ras signalling pathway may figure among mechanisms explaining altered USP19 function and p27Kip1 levels in cancer cells.

Bottom Line: Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27(Kip1) levels.This may occur through both KPC1 dependent and independent mechanisms.Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.

View Article: PubMed Central - PubMed

Affiliation: Polypeptide Laboratory, Department of Medicine, McGill University Health Centre Research Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT
We previously reported that the USP19 deubiquitinating enzyme positively regulates proliferation in fibroblasts by stabilizing KPC1, a ubiquitin ligase for p27(Kip1). To explore whether this role of USP19 extends to other cellular systems, we tested the effects of silencing of USP19 in several human prostate and breast models, including carcinoma cell lines. Depletion of USP19 inhibited proliferation in prostate cancer DU145, PC-3 and 22RV1 cells, which was similar to the pattern established in fibroblasts in that it was due to decreased progression from G1 to S phase and associated with a stabilization of the cyclin-dependent kinase inhibitor p27(Kip1). However, in contrast to previous findings in fibroblasts, the stabilization of p27(Kip1) upon USP19 depletion was not associated with changes in the levels of the KPC1 ligase. USP19 could also regulate the growth of immortalized MCF10A breast epithelial cells through a similar mechanism. This regulatory pattern was lost, though, in breast cancer MCF7 and MDA-MB-231 cells and in prostate carcinoma LNCaP cells. Of interest, the transformation of fibroblasts through overexpression of an oncogenic form of Ras disrupted the USP19-mediated regulation of cell growth and of levels of p27(Kip1) and KPC1. Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27(Kip1) levels. This may occur through both KPC1 dependent and independent mechanisms. Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.

Show MeSH
Related in: MedlinePlus