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The cell competition-based high-throughput screening identifies small compounds that promote the elimination of RasV12-transformed cells from epithelia.

Yamauchi H, Matsumaru T, Morita T, Ishikawa S, Maenaka K, Takigawa I, Semba K, Kon S, Fujita Y - Sci Rep (2015)

Bottom Line: Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells.This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo.These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University Graduate School of Chemical Sciences and Engineering, Sapporo, Japan.

ABSTRACT
Recent studies have revealed that cell competition can occur between normal and transformed epithelial cells; normal epithelial cells recognize the presence of the neighboring transformed cells and actively eliminate them from epithelial tissues. Here, we have established a brand-new high-throughput screening platform that targets cell competition. By using this platform, we have identified Rebeccamycin as a hit compound that specifically promotes elimination of RasV12-transformed cells from the epithelium, though after longer treatment it shows substantial cytotoxic effect against normal epithelial cells. Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells. This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo. These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

No MeSH data available.


Related in: MedlinePlus

A novel type of high-throughput screening has lead to the identification of Rebeccamycin as a chemical compound that enhances cell competition between normal and transformed epithelial cells.(a) A scheme for the high-throughput screening platform we have developed. Normal MDCK cells were mixed with MDCK-pTR GFP-RasV12 cells at a ratio of 10:1 and seeded into a 96-well plate. The mixed cells were incubated for 16 h until a monolayer was formed. Then, the culture medium was exchanged for a new medium containing tetracycline and each small chemical compound, followed by incubation for 16 h. Finally, the GFP intensity of the remaining GFP-RasV12-transformed cells was quantified. (b) The secondary screening for small chemical compounds that specifically induces the elimination of RasV12-transformed cells that are surrounded by normal cells. (c) A structural formula of Rebeccamycin. (d) An example of the primary screening result including Rebeccamycin (orange circle). The blue dot shows the fluorescence intensity of MDCK-pTR GFP-RasV12 cells after treatment with each compound. The red line and black dot lines indicate the average value and the 3-fold standard deviation (SD) above or below the average, respectively. (e) Fluorescence images of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells (upper panels) or cultured alone (lower panels) with DMSO (left) or 2 μM Rebeccamycin (right). Arrows and arrowheads indicate cells that have become round and small, and been broken into pieces, respectively. Scale bar: 50 μm. (f) Quantification of the GFP intensity of MDCK-pTR GFP-RasV12 cells mixed with MDCK cells (white bar) or cultured alone (gray bar) with the indicated concentration of Rebeccamycin. Values are expressed as a ratio relative to DMSO treatment. Data are mean ± SD from three independent experiments. ***P < 0.001.
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f1: A novel type of high-throughput screening has lead to the identification of Rebeccamycin as a chemical compound that enhances cell competition between normal and transformed epithelial cells.(a) A scheme for the high-throughput screening platform we have developed. Normal MDCK cells were mixed with MDCK-pTR GFP-RasV12 cells at a ratio of 10:1 and seeded into a 96-well plate. The mixed cells were incubated for 16 h until a monolayer was formed. Then, the culture medium was exchanged for a new medium containing tetracycline and each small chemical compound, followed by incubation for 16 h. Finally, the GFP intensity of the remaining GFP-RasV12-transformed cells was quantified. (b) The secondary screening for small chemical compounds that specifically induces the elimination of RasV12-transformed cells that are surrounded by normal cells. (c) A structural formula of Rebeccamycin. (d) An example of the primary screening result including Rebeccamycin (orange circle). The blue dot shows the fluorescence intensity of MDCK-pTR GFP-RasV12 cells after treatment with each compound. The red line and black dot lines indicate the average value and the 3-fold standard deviation (SD) above or below the average, respectively. (e) Fluorescence images of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells (upper panels) or cultured alone (lower panels) with DMSO (left) or 2 μM Rebeccamycin (right). Arrows and arrowheads indicate cells that have become round and small, and been broken into pieces, respectively. Scale bar: 50 μm. (f) Quantification of the GFP intensity of MDCK-pTR GFP-RasV12 cells mixed with MDCK cells (white bar) or cultured alone (gray bar) with the indicated concentration of Rebeccamycin. Values are expressed as a ratio relative to DMSO treatment. Data are mean ± SD from three independent experiments. ***P < 0.001.

Mentions: Madin-Darby canine kidney (MDCK) cells are non-transformed epithelial cells that have been widely used for studying epithelial biology, because they retain characteristics of functional epithelia15. We have previously established MDCK cells stably expressing a constitutively active mutant of Ras (RasV12) in a tetracycline-inducible manner (MDCK-pTR GFP-RasV12 cells)10. Ras is a prototype-oncoprotein that is frequently mutated to its active forms in various types of human cancers16. Using this cell line, we have demonstrated that when RasV12-transformed cells are surrounded by normal epithelial cells, the transformed cells are occasionally extruded from the apical surface of an epithelial monolayer10 and that the surrounding normal epithelial cells are actively involved in the elimination of transformed cells14. By applying this cell competition between normal and RasV12-transformed cells, we have developed the following screening platform (Fig. 1a). First, normal MDCK cells and MDCK-pTR GFP-RasV12 cells are mixed at a ratio of 10:1 and cultured in a 96-well plate until they form an epithelial monolayer. Then, a small chemical compound is added in each well, together with tetracycline to induce expression of GFP-RasV12. After several hours, the GFP fluorescence intensity is measured to quantify the remaining GFP-RasV12-expressing cells. We have optimized the cell numbers and incubation times, thereby creating a condition where transformed cells are surrounded by normal cells at high cell density. In the primary screening, we selected small compounds that enhance the exclusion of GFP-RasV12-positive cells from epithelia. In the following secondary screening, we compared the effect on RasV12-transformed cells surrounded by normal cells with that on RasV12 cells cultured alone, and selected small compounds that specifically eliminate the former cells (Fig. 1b). After screening with 2,607 small chemical compounds, we identified Rebeccamycin as a hit compound (Fig. 1c,d and Supplementary Table 1). Rebeccamycin is an antibiotic isolated from the actinomycete strain, Saccharothrix aerocolonigenes, which inhibits topoisomerase I and induces anti-tumor activity against various cancer cell lines1718. For RasV12-transformed cells that were surrounded by normal cells, addition of Rebeccamycin induced morphological changes into round and small ‘stressed’ shapes (Fig. 1e; arrows), often leading to fragmentation (Fig. 1e; arrowheads). In contrast, this effect was not observed for RasV12 cells that were cultured alone (Fig. 1e) or normal cells (data not shown). This specific effect against RasV12 cells surrounded by normal cells was observed in a dose-dependent manner with a minimum effective dose at 0.5 μΜ (Fig. 1f). These data demonstrate that Rebeccamycin selectively affects RasV12-transformed cells that are surrounded by normal cells and enhances their elimination from the epithelium.


The cell competition-based high-throughput screening identifies small compounds that promote the elimination of RasV12-transformed cells from epithelia.

Yamauchi H, Matsumaru T, Morita T, Ishikawa S, Maenaka K, Takigawa I, Semba K, Kon S, Fujita Y - Sci Rep (2015)

A novel type of high-throughput screening has lead to the identification of Rebeccamycin as a chemical compound that enhances cell competition between normal and transformed epithelial cells.(a) A scheme for the high-throughput screening platform we have developed. Normal MDCK cells were mixed with MDCK-pTR GFP-RasV12 cells at a ratio of 10:1 and seeded into a 96-well plate. The mixed cells were incubated for 16 h until a monolayer was formed. Then, the culture medium was exchanged for a new medium containing tetracycline and each small chemical compound, followed by incubation for 16 h. Finally, the GFP intensity of the remaining GFP-RasV12-transformed cells was quantified. (b) The secondary screening for small chemical compounds that specifically induces the elimination of RasV12-transformed cells that are surrounded by normal cells. (c) A structural formula of Rebeccamycin. (d) An example of the primary screening result including Rebeccamycin (orange circle). The blue dot shows the fluorescence intensity of MDCK-pTR GFP-RasV12 cells after treatment with each compound. The red line and black dot lines indicate the average value and the 3-fold standard deviation (SD) above or below the average, respectively. (e) Fluorescence images of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells (upper panels) or cultured alone (lower panels) with DMSO (left) or 2 μM Rebeccamycin (right). Arrows and arrowheads indicate cells that have become round and small, and been broken into pieces, respectively. Scale bar: 50 μm. (f) Quantification of the GFP intensity of MDCK-pTR GFP-RasV12 cells mixed with MDCK cells (white bar) or cultured alone (gray bar) with the indicated concentration of Rebeccamycin. Values are expressed as a ratio relative to DMSO treatment. Data are mean ± SD from three independent experiments. ***P < 0.001.
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Related In: Results  -  Collection

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f1: A novel type of high-throughput screening has lead to the identification of Rebeccamycin as a chemical compound that enhances cell competition between normal and transformed epithelial cells.(a) A scheme for the high-throughput screening platform we have developed. Normal MDCK cells were mixed with MDCK-pTR GFP-RasV12 cells at a ratio of 10:1 and seeded into a 96-well plate. The mixed cells were incubated for 16 h until a monolayer was formed. Then, the culture medium was exchanged for a new medium containing tetracycline and each small chemical compound, followed by incubation for 16 h. Finally, the GFP intensity of the remaining GFP-RasV12-transformed cells was quantified. (b) The secondary screening for small chemical compounds that specifically induces the elimination of RasV12-transformed cells that are surrounded by normal cells. (c) A structural formula of Rebeccamycin. (d) An example of the primary screening result including Rebeccamycin (orange circle). The blue dot shows the fluorescence intensity of MDCK-pTR GFP-RasV12 cells after treatment with each compound. The red line and black dot lines indicate the average value and the 3-fold standard deviation (SD) above or below the average, respectively. (e) Fluorescence images of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells (upper panels) or cultured alone (lower panels) with DMSO (left) or 2 μM Rebeccamycin (right). Arrows and arrowheads indicate cells that have become round and small, and been broken into pieces, respectively. Scale bar: 50 μm. (f) Quantification of the GFP intensity of MDCK-pTR GFP-RasV12 cells mixed with MDCK cells (white bar) or cultured alone (gray bar) with the indicated concentration of Rebeccamycin. Values are expressed as a ratio relative to DMSO treatment. Data are mean ± SD from three independent experiments. ***P < 0.001.
Mentions: Madin-Darby canine kidney (MDCK) cells are non-transformed epithelial cells that have been widely used for studying epithelial biology, because they retain characteristics of functional epithelia15. We have previously established MDCK cells stably expressing a constitutively active mutant of Ras (RasV12) in a tetracycline-inducible manner (MDCK-pTR GFP-RasV12 cells)10. Ras is a prototype-oncoprotein that is frequently mutated to its active forms in various types of human cancers16. Using this cell line, we have demonstrated that when RasV12-transformed cells are surrounded by normal epithelial cells, the transformed cells are occasionally extruded from the apical surface of an epithelial monolayer10 and that the surrounding normal epithelial cells are actively involved in the elimination of transformed cells14. By applying this cell competition between normal and RasV12-transformed cells, we have developed the following screening platform (Fig. 1a). First, normal MDCK cells and MDCK-pTR GFP-RasV12 cells are mixed at a ratio of 10:1 and cultured in a 96-well plate until they form an epithelial monolayer. Then, a small chemical compound is added in each well, together with tetracycline to induce expression of GFP-RasV12. After several hours, the GFP fluorescence intensity is measured to quantify the remaining GFP-RasV12-expressing cells. We have optimized the cell numbers and incubation times, thereby creating a condition where transformed cells are surrounded by normal cells at high cell density. In the primary screening, we selected small compounds that enhance the exclusion of GFP-RasV12-positive cells from epithelia. In the following secondary screening, we compared the effect on RasV12-transformed cells surrounded by normal cells with that on RasV12 cells cultured alone, and selected small compounds that specifically eliminate the former cells (Fig. 1b). After screening with 2,607 small chemical compounds, we identified Rebeccamycin as a hit compound (Fig. 1c,d and Supplementary Table 1). Rebeccamycin is an antibiotic isolated from the actinomycete strain, Saccharothrix aerocolonigenes, which inhibits topoisomerase I and induces anti-tumor activity against various cancer cell lines1718. For RasV12-transformed cells that were surrounded by normal cells, addition of Rebeccamycin induced morphological changes into round and small ‘stressed’ shapes (Fig. 1e; arrows), often leading to fragmentation (Fig. 1e; arrowheads). In contrast, this effect was not observed for RasV12 cells that were cultured alone (Fig. 1e) or normal cells (data not shown). This specific effect against RasV12 cells surrounded by normal cells was observed in a dose-dependent manner with a minimum effective dose at 0.5 μΜ (Fig. 1f). These data demonstrate that Rebeccamycin selectively affects RasV12-transformed cells that are surrounded by normal cells and enhances their elimination from the epithelium.

Bottom Line: Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells.This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo.These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University Graduate School of Chemical Sciences and Engineering, Sapporo, Japan.

ABSTRACT
Recent studies have revealed that cell competition can occur between normal and transformed epithelial cells; normal epithelial cells recognize the presence of the neighboring transformed cells and actively eliminate them from epithelial tissues. Here, we have established a brand-new high-throughput screening platform that targets cell competition. By using this platform, we have identified Rebeccamycin as a hit compound that specifically promotes elimination of RasV12-transformed cells from the epithelium, though after longer treatment it shows substantial cytotoxic effect against normal epithelial cells. Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells. This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo. These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

No MeSH data available.


Related in: MedlinePlus