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Variable Expression of GLIPR1 Correlates with Invasive Potential in Melanoma Cells.

Awasthi A, Woolley AG, Lecomte FJ, Hung N, Baguley BC, Wilbanks SM, Jeffs AR, Tyndall JD - Front Oncol (2013)

Bottom Line: More recently, GLIPR1 was shown to be differentially expressed in other cancers including ovarian, acute myeloid leukemia, and Wilms' tumor.Elevated GLIPR1 levels were correlated with increased invasive potential, and siRNA-mediated knockdown of GLIPR1 expression resulted in reduced cell migration and proliferation in vitro.GLIPR1 staining was observed in normal skin in merocrine sweat glands, sebaceous glands, and hair follicles within the dermis.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Otago , Dunedin , New Zealand ; Department of Pathology, Dunedin School of Medicine, University of Otago , Dunedin , New Zealand ; Department of Biochemistry, Otago School of Medical Sciences, University of Otago , Dunedin , New Zealand.

ABSTRACT
GLI pathogenesis-related 1 (GLIPR1) was previously identified as an epigenetically regulated tumor suppressor in prostate cancer and, conversely, an oncoprotein in glioma. More recently, GLIPR1 was shown to be differentially expressed in other cancers including ovarian, acute myeloid leukemia, and Wilms' tumor. Here we investigated GLIPR1 expression in metastatic melanoma cell lines and tissue. GLIPR1 was variably expressed in metastatic melanoma cells, and transcript levels correlated with degree of GLIPR1 promoter methylation in vitro. Elevated GLIPR1 levels were correlated with increased invasive potential, and siRNA-mediated knockdown of GLIPR1 expression resulted in reduced cell migration and proliferation in vitro. Immunohistochemical studies of melanoma tissue microarrays showed moderate to high staining for GLIPR1 in 50% of specimens analyzed. GLIPR1 staining was observed in normal skin in merocrine sweat glands, sebaceous glands, and hair follicles within the dermis.

No MeSH data available.


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GLIPR1 siRNA knockdown decreases cellular invasion and proliferation. (A) GLIPR1 protein levels following knockdown were determined by western blotting (40 μg of total protein per lane) from cells 72 h following transfection with siRNA. GLIPR1 knockdown with siGLI (+) and control treatment with non-targeting siRNA siNT (−) are indicated above gel. Low levels of endogenous GLIPR1 prevented assessment of the extent of knockdown in NZM15, NZM12, and NZM45 by western blotting. Relative migration (B) and invasion (C) of cells across the membrane of transwell inserts was measured 24 h after siGLI. Data shown as the average number of cells per field of view ± SEM from three (B) or two (C) independent experiments. Error bars indicate SEM; *p < 0.005, **p < 0.001. No migration was observed for NZM45 (Figure 2A) and no invasion was seen for NZM45, NZM12, or NZM15 (Figure 2B). (D) Cell proliferation was quantified using MTT-based colorimetric assay. Results are mean of two independent experiments, n = 4. Results in (D) are shown as data for cells 4 days after transfection with siGLI relative to data for cells transfected with siNT. Results for (B,C) are shown for cells 24 h following transfection.
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Figure 3: GLIPR1 siRNA knockdown decreases cellular invasion and proliferation. (A) GLIPR1 protein levels following knockdown were determined by western blotting (40 μg of total protein per lane) from cells 72 h following transfection with siRNA. GLIPR1 knockdown with siGLI (+) and control treatment with non-targeting siRNA siNT (−) are indicated above gel. Low levels of endogenous GLIPR1 prevented assessment of the extent of knockdown in NZM15, NZM12, and NZM45 by western blotting. Relative migration (B) and invasion (C) of cells across the membrane of transwell inserts was measured 24 h after siGLI. Data shown as the average number of cells per field of view ± SEM from three (B) or two (C) independent experiments. Error bars indicate SEM; *p < 0.005, **p < 0.001. No migration was observed for NZM45 (Figure 2A) and no invasion was seen for NZM45, NZM12, or NZM15 (Figure 2B). (D) Cell proliferation was quantified using MTT-based colorimetric assay. Results are mean of two independent experiments, n = 4. Results in (D) are shown as data for cells 4 days after transfection with siGLI relative to data for cells transfected with siNT. Results for (B,C) are shown for cells 24 h following transfection.

Mentions: To further investigate the relationship between GLIPR1 expression and cell migration and invasion, GLIPR1 expression was decreased using siRNA. siRNA-mediated knockdown of GLIPR1 resulted in a significant decrease in the number of melanoma and glioma cells migrating across the membrane relative to non-targeting controls (Figure 3; Figure S5 in Supplementary Material). We used glioma cell lines in which GLIPR1 had previously been shown to modulate invasive behavior (11) as positive controls to compare with melanoma cell lines in our in vitro invasion assays. SNB75 glioma cells, with the highest pre-knockdown migration rate, showed about a 50% decrease in cell migration 24 h after GLIPR1 knockdown (Figure 3B). Similarly, the high GLIPR1-expressing cell lines NZM40, NZM9 and U251 showed a 20–30% decrease in migration compared to non-targeting controls. Cells with lower GLIPR1 levels (NZM12, NZM15) showed no measurable change in the already small number of cells migrating across the membrane after knockdown which reflects the intrinsically weak invasive potential of these cells.


Variable Expression of GLIPR1 Correlates with Invasive Potential in Melanoma Cells.

Awasthi A, Woolley AG, Lecomte FJ, Hung N, Baguley BC, Wilbanks SM, Jeffs AR, Tyndall JD - Front Oncol (2013)

GLIPR1 siRNA knockdown decreases cellular invasion and proliferation. (A) GLIPR1 protein levels following knockdown were determined by western blotting (40 μg of total protein per lane) from cells 72 h following transfection with siRNA. GLIPR1 knockdown with siGLI (+) and control treatment with non-targeting siRNA siNT (−) are indicated above gel. Low levels of endogenous GLIPR1 prevented assessment of the extent of knockdown in NZM15, NZM12, and NZM45 by western blotting. Relative migration (B) and invasion (C) of cells across the membrane of transwell inserts was measured 24 h after siGLI. Data shown as the average number of cells per field of view ± SEM from three (B) or two (C) independent experiments. Error bars indicate SEM; *p < 0.005, **p < 0.001. No migration was observed for NZM45 (Figure 2A) and no invasion was seen for NZM45, NZM12, or NZM15 (Figure 2B). (D) Cell proliferation was quantified using MTT-based colorimetric assay. Results are mean of two independent experiments, n = 4. Results in (D) are shown as data for cells 4 days after transfection with siGLI relative to data for cells transfected with siNT. Results for (B,C) are shown for cells 24 h following transfection.
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Figure 3: GLIPR1 siRNA knockdown decreases cellular invasion and proliferation. (A) GLIPR1 protein levels following knockdown were determined by western blotting (40 μg of total protein per lane) from cells 72 h following transfection with siRNA. GLIPR1 knockdown with siGLI (+) and control treatment with non-targeting siRNA siNT (−) are indicated above gel. Low levels of endogenous GLIPR1 prevented assessment of the extent of knockdown in NZM15, NZM12, and NZM45 by western blotting. Relative migration (B) and invasion (C) of cells across the membrane of transwell inserts was measured 24 h after siGLI. Data shown as the average number of cells per field of view ± SEM from three (B) or two (C) independent experiments. Error bars indicate SEM; *p < 0.005, **p < 0.001. No migration was observed for NZM45 (Figure 2A) and no invasion was seen for NZM45, NZM12, or NZM15 (Figure 2B). (D) Cell proliferation was quantified using MTT-based colorimetric assay. Results are mean of two independent experiments, n = 4. Results in (D) are shown as data for cells 4 days after transfection with siGLI relative to data for cells transfected with siNT. Results for (B,C) are shown for cells 24 h following transfection.
Mentions: To further investigate the relationship between GLIPR1 expression and cell migration and invasion, GLIPR1 expression was decreased using siRNA. siRNA-mediated knockdown of GLIPR1 resulted in a significant decrease in the number of melanoma and glioma cells migrating across the membrane relative to non-targeting controls (Figure 3; Figure S5 in Supplementary Material). We used glioma cell lines in which GLIPR1 had previously been shown to modulate invasive behavior (11) as positive controls to compare with melanoma cell lines in our in vitro invasion assays. SNB75 glioma cells, with the highest pre-knockdown migration rate, showed about a 50% decrease in cell migration 24 h after GLIPR1 knockdown (Figure 3B). Similarly, the high GLIPR1-expressing cell lines NZM40, NZM9 and U251 showed a 20–30% decrease in migration compared to non-targeting controls. Cells with lower GLIPR1 levels (NZM12, NZM15) showed no measurable change in the already small number of cells migrating across the membrane after knockdown which reflects the intrinsically weak invasive potential of these cells.

Bottom Line: More recently, GLIPR1 was shown to be differentially expressed in other cancers including ovarian, acute myeloid leukemia, and Wilms' tumor.Elevated GLIPR1 levels were correlated with increased invasive potential, and siRNA-mediated knockdown of GLIPR1 expression resulted in reduced cell migration and proliferation in vitro.GLIPR1 staining was observed in normal skin in merocrine sweat glands, sebaceous glands, and hair follicles within the dermis.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Otago , Dunedin , New Zealand ; Department of Pathology, Dunedin School of Medicine, University of Otago , Dunedin , New Zealand ; Department of Biochemistry, Otago School of Medical Sciences, University of Otago , Dunedin , New Zealand.

ABSTRACT
GLI pathogenesis-related 1 (GLIPR1) was previously identified as an epigenetically regulated tumor suppressor in prostate cancer and, conversely, an oncoprotein in glioma. More recently, GLIPR1 was shown to be differentially expressed in other cancers including ovarian, acute myeloid leukemia, and Wilms' tumor. Here we investigated GLIPR1 expression in metastatic melanoma cell lines and tissue. GLIPR1 was variably expressed in metastatic melanoma cells, and transcript levels correlated with degree of GLIPR1 promoter methylation in vitro. Elevated GLIPR1 levels were correlated with increased invasive potential, and siRNA-mediated knockdown of GLIPR1 expression resulted in reduced cell migration and proliferation in vitro. Immunohistochemical studies of melanoma tissue microarrays showed moderate to high staining for GLIPR1 in 50% of specimens analyzed. GLIPR1 staining was observed in normal skin in merocrine sweat glands, sebaceous glands, and hair follicles within the dermis.

No MeSH data available.


Related in: MedlinePlus