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Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family.

Lin ML, Park JH, Nishidate T, Nakamura Y, Katagiri T - Breast Cancer Res. (2007)

Bottom Line: Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney).We also found that MELK physically interacted with Bcl-GL through its amino-terminal region.Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Introduction: Cancer therapies directed at specific molecular targets in signaling pathways of cancer cells, such as tamoxifen, aromatase inhibitors and trastuzumab, have proven useful for treatment of advanced breast cancers. However, increased risk of endometrial cancer with long-term tamoxifen administration and of bone fracture due to osteoporosis in postmenopausal women undergoing aromatase inhibitor therapy are recognized side effects. These side effects as well as drug resistance make it necessary to search for novel molecular targets for drugs on the basis of well-characterized mechanisms of action.

Methods: Using accurate genome-wide expression profiles of breast cancers, we found maternal embryonic leucine-zipper kinase (MELK) to be significantly overexpressed in the great majority of breast cancer cells. To assess whether MELK has a role in mammary carcinogenesis, we knocked down the expression of endogenous MELK in breast cancer cell lines using mammalian vector-based RNA interference. Furthermore, we identified a long isoform of Bcl-G (Bcl-GL), a pro-apoptotic member of the Bcl-2 family, as a possible substrate for MELK by pull-down assay with recombinant wild-type and kinase-dead MELK. Finally, we performed TUNEL assays and FACS analysis, measuring proportions of apoptotic cells, to investigate whether MELK is involved in the apoptosis cascade through the Bcl-GL-related pathway.

Results: Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney). Suppression of MELK expression by small interfering RNA significantly inhibited growth of human breast cancer cells. We also found that MELK physically interacted with Bcl-GL through its amino-terminal region. Immunocomplex kinase assay showed that Bcl-GL was specifically phosphorylated by MELK in vitro. TUNEL assays and FACS analysis revealed that overexpression of wild-type MELK suppressed Bcl-GL-induced apoptosis, while that of D150A-MELK did not.

Conclusion: Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL. The kinase activity of MELK could be a promising molecular target for development of therapy for patients with breast cancers.

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MELK involvement in the apoptosis cascade through Bcl-GL. HA (hemagglutinin)-tagged MELK (HA-wild-type (WT)-MELK or HA-kinase-dead (D150A)-MELK) and Flag-tagged Bcl-GL (Flag-Bcl-GL) expression vectors were co-transfected into COS7 cells for 24 hours. (a) The expression of MELK and Bcl-GL proteins in the co-transfected cells were examined by western blot analysis. (b) TUNEL assays after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Apoptotic cells were measured by counting of TUNEL staining (means ± standard deviation, n = 3; P = 0.0001; unpaired t-test). (c) Representative images of TUNEL assays. Cells were labeled with DAPI (4',6-diamidino-2-phenylindole) for counting of total cell number. Apoptotic cells with DNA strand breaks were labeled with green fluorescence. (d) FACS analysis of cells collected after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Proportions of apoptotic cells are indicated as percentages of sub-G1 populations. Each value represents the average of three experiments (means ± standard deviation, n = 3).
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Figure 5: MELK involvement in the apoptosis cascade through Bcl-GL. HA (hemagglutinin)-tagged MELK (HA-wild-type (WT)-MELK or HA-kinase-dead (D150A)-MELK) and Flag-tagged Bcl-GL (Flag-Bcl-GL) expression vectors were co-transfected into COS7 cells for 24 hours. (a) The expression of MELK and Bcl-GL proteins in the co-transfected cells were examined by western blot analysis. (b) TUNEL assays after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Apoptotic cells were measured by counting of TUNEL staining (means ± standard deviation, n = 3; P = 0.0001; unpaired t-test). (c) Representative images of TUNEL assays. Cells were labeled with DAPI (4',6-diamidino-2-phenylindole) for counting of total cell number. Apoptotic cells with DNA strand breaks were labeled with green fluorescence. (d) FACS analysis of cells collected after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Proportions of apoptotic cells are indicated as percentages of sub-G1 populations. Each value represents the average of three experiments (means ± standard deviation, n = 3).

Mentions: Because MELK can physically interact with and phosphorylate Bcl-GL (Figures 3d and 4a), we hypothesized that it might be involved in the apoptosis cascade through the Bcl-GL-related pathway. To investigate this hypothesis, we transiently co-transfected two plasmid clones designed to express HA-tagged MELK (WT or D150A) and Flag-tagged Bcl-GL into COS7 cells, and then performed a TUNEL assay and FACS analysis to measure the proportions of apoptotic cells (see Material and methods). We first confirmed the exogenous expression of MELK and Bcl-GL in COS7 cells by western blot analysis (Figure 5a). As indicated in Figure 5b,c, the overexpression of full-length Bcl-GL (HA-Mock+Flag-Bcl-GL) significantly increased the proportion of TUNEL-positive cells compared with the cells transfected with the mock plasmids (HA-Mock+Flag-Mock), indicating that Bcl-GL induces apoptosis, as described previously [14]. In contrast, the co-overexpression of WT-MELK with Bcl-GL (HA-WT-MELK+Flag-Bcl-GL) reduced the proportion of TUNEL-positive cells compared with over-expression of Bcl-GL alone (HA-Mock+Flag-Bcl-GL) (P = 0.0001, unpaired t-test). However, the co-overexpression of D150A-MELK with Bcl-GL (HA-D150A-MELK+Flag-Bcl-GL) did not affect the proportion of TUNEL-positive cells. As shown in Figure 5d, FACS analysis of the cells under the same conditions also confirmed that the overexpression of Bcl-GL increased the sub-G1 population of cells compared with the mock-transfected cells. Similarly to the TUNEL analysis, the overexpression of WT-MELK with Bcl-GL reduced the proportion of sub-G1 cells (P = 0.03, unpaired t-test), while D150A-MELK increased the sub-G1 population. Our results imply that the kinase activity of MELK may play a critical role in the regulation of the pro-apoptotic function of Bcl-GL.


Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family.

Lin ML, Park JH, Nishidate T, Nakamura Y, Katagiri T - Breast Cancer Res. (2007)

MELK involvement in the apoptosis cascade through Bcl-GL. HA (hemagglutinin)-tagged MELK (HA-wild-type (WT)-MELK or HA-kinase-dead (D150A)-MELK) and Flag-tagged Bcl-GL (Flag-Bcl-GL) expression vectors were co-transfected into COS7 cells for 24 hours. (a) The expression of MELK and Bcl-GL proteins in the co-transfected cells were examined by western blot analysis. (b) TUNEL assays after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Apoptotic cells were measured by counting of TUNEL staining (means ± standard deviation, n = 3; P = 0.0001; unpaired t-test). (c) Representative images of TUNEL assays. Cells were labeled with DAPI (4',6-diamidino-2-phenylindole) for counting of total cell number. Apoptotic cells with DNA strand breaks were labeled with green fluorescence. (d) FACS analysis of cells collected after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Proportions of apoptotic cells are indicated as percentages of sub-G1 populations. Each value represents the average of three experiments (means ± standard deviation, n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1851384&req=5

Figure 5: MELK involvement in the apoptosis cascade through Bcl-GL. HA (hemagglutinin)-tagged MELK (HA-wild-type (WT)-MELK or HA-kinase-dead (D150A)-MELK) and Flag-tagged Bcl-GL (Flag-Bcl-GL) expression vectors were co-transfected into COS7 cells for 24 hours. (a) The expression of MELK and Bcl-GL proteins in the co-transfected cells were examined by western blot analysis. (b) TUNEL assays after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Apoptotic cells were measured by counting of TUNEL staining (means ± standard deviation, n = 3; P = 0.0001; unpaired t-test). (c) Representative images of TUNEL assays. Cells were labeled with DAPI (4',6-diamidino-2-phenylindole) for counting of total cell number. Apoptotic cells with DNA strand breaks were labeled with green fluorescence. (d) FACS analysis of cells collected after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Proportions of apoptotic cells are indicated as percentages of sub-G1 populations. Each value represents the average of three experiments (means ± standard deviation, n = 3).
Mentions: Because MELK can physically interact with and phosphorylate Bcl-GL (Figures 3d and 4a), we hypothesized that it might be involved in the apoptosis cascade through the Bcl-GL-related pathway. To investigate this hypothesis, we transiently co-transfected two plasmid clones designed to express HA-tagged MELK (WT or D150A) and Flag-tagged Bcl-GL into COS7 cells, and then performed a TUNEL assay and FACS analysis to measure the proportions of apoptotic cells (see Material and methods). We first confirmed the exogenous expression of MELK and Bcl-GL in COS7 cells by western blot analysis (Figure 5a). As indicated in Figure 5b,c, the overexpression of full-length Bcl-GL (HA-Mock+Flag-Bcl-GL) significantly increased the proportion of TUNEL-positive cells compared with the cells transfected with the mock plasmids (HA-Mock+Flag-Mock), indicating that Bcl-GL induces apoptosis, as described previously [14]. In contrast, the co-overexpression of WT-MELK with Bcl-GL (HA-WT-MELK+Flag-Bcl-GL) reduced the proportion of TUNEL-positive cells compared with over-expression of Bcl-GL alone (HA-Mock+Flag-Bcl-GL) (P = 0.0001, unpaired t-test). However, the co-overexpression of D150A-MELK with Bcl-GL (HA-D150A-MELK+Flag-Bcl-GL) did not affect the proportion of TUNEL-positive cells. As shown in Figure 5d, FACS analysis of the cells under the same conditions also confirmed that the overexpression of Bcl-GL increased the sub-G1 population of cells compared with the mock-transfected cells. Similarly to the TUNEL analysis, the overexpression of WT-MELK with Bcl-GL reduced the proportion of sub-G1 cells (P = 0.03, unpaired t-test), while D150A-MELK increased the sub-G1 population. Our results imply that the kinase activity of MELK may play a critical role in the regulation of the pro-apoptotic function of Bcl-GL.

Bottom Line: Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney).We also found that MELK physically interacted with Bcl-GL through its amino-terminal region.Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Introduction: Cancer therapies directed at specific molecular targets in signaling pathways of cancer cells, such as tamoxifen, aromatase inhibitors and trastuzumab, have proven useful for treatment of advanced breast cancers. However, increased risk of endometrial cancer with long-term tamoxifen administration and of bone fracture due to osteoporosis in postmenopausal women undergoing aromatase inhibitor therapy are recognized side effects. These side effects as well as drug resistance make it necessary to search for novel molecular targets for drugs on the basis of well-characterized mechanisms of action.

Methods: Using accurate genome-wide expression profiles of breast cancers, we found maternal embryonic leucine-zipper kinase (MELK) to be significantly overexpressed in the great majority of breast cancer cells. To assess whether MELK has a role in mammary carcinogenesis, we knocked down the expression of endogenous MELK in breast cancer cell lines using mammalian vector-based RNA interference. Furthermore, we identified a long isoform of Bcl-G (Bcl-GL), a pro-apoptotic member of the Bcl-2 family, as a possible substrate for MELK by pull-down assay with recombinant wild-type and kinase-dead MELK. Finally, we performed TUNEL assays and FACS analysis, measuring proportions of apoptotic cells, to investigate whether MELK is involved in the apoptosis cascade through the Bcl-GL-related pathway.

Results: Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney). Suppression of MELK expression by small interfering RNA significantly inhibited growth of human breast cancer cells. We also found that MELK physically interacted with Bcl-GL through its amino-terminal region. Immunocomplex kinase assay showed that Bcl-GL was specifically phosphorylated by MELK in vitro. TUNEL assays and FACS analysis revealed that overexpression of wild-type MELK suppressed Bcl-GL-induced apoptosis, while that of D150A-MELK did not.

Conclusion: Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL. The kinase activity of MELK could be a promising molecular target for development of therapy for patients with breast cancers.

Show MeSH
Related in: MedlinePlus