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p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance.

Yang JY, Ha SA, Yang YS, Kim JW - BMC Cancer (2010)

Bottom Line: The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms.YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype.Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetic Laboratory, College of Medicine, The Catholic University of Korea, Seoul 137-040, Republic of Korea.

ABSTRACT

Background: Cancer cells recurrently develop into acquired resistance to the administered drugs. The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms. Our aim is to define the potential targets, that makes drug-sensitive MCF-7 breast cancer cells turn to drug-resistant, for the anti-cancer drug development against drug resistant breast cancer cells.

Methods: Doxorubicin resistant human breast MCF-7 clones were generated. The doxorubicin-induced cell fusion events were examined. Heterokaryons were identified and sorted by FACS. In the development of doxorubicin resistance, cell-fusion associated genes, from the previous results of microarray, were verified using dot blot array and quantitative RT-PCR. The doxorubicin-induced expression patterns of pro-survival and pro-apoptotic genes were validated.

Results: YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype. Cell fusion increased diversity within the cell population and doxorubicin resistant MCF-7 cells emerged probably through clonal selection. Most of the drug resistant hybrid cells were anchorage independent. But some of the anchorage dependent MCF-7 cells exhibited several unique morphological appearances suggesting minor population of the fused cells maybe de-differentiated and have progenitor cell like characteristics.

Conclusion: Our work provides valuable insight into the drug induced cell fusion event and outcome, and suggests YB-1, GST, ABCB5 and ERK3 could be potential targets for the anti-cancer drug development against drug resistant breast cancer cells. Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

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Related in: MedlinePlus

Schema of cell fusion and chromosomal instability. A: Cytosolic cell fusion of two subtype of cells (homotype or heterotype). B and D: Most of cells, at the stage of nuclear cell fusion, revealed no further propagation and subsequently vanished. C: Viable progeny cells propagated again in culture. Giemsa-stained chromosomes of MCF-7 cells were examined. D: Abnormal sets of chromosomes as a consequence of cell fusion and (E) the cells containing giant nuclei (polyploid) were died out and the diverse populations were analogously converged into doxorubicin resistant clones. Table insert, fusion was quantitated by counting the number of cells (%) and nuclei present in a microscope field.
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Figure 3: Schema of cell fusion and chromosomal instability. A: Cytosolic cell fusion of two subtype of cells (homotype or heterotype). B and D: Most of cells, at the stage of nuclear cell fusion, revealed no further propagation and subsequently vanished. C: Viable progeny cells propagated again in culture. Giemsa-stained chromosomes of MCF-7 cells were examined. D: Abnormal sets of chromosomes as a consequence of cell fusion and (E) the cells containing giant nuclei (polyploid) were died out and the diverse populations were analogously converged into doxorubicin resistant clones. Table insert, fusion was quantitated by counting the number of cells (%) and nuclei present in a microscope field.

Mentions: The appearance of polyploidy cells may be caused by cell-to-cell fusion, we employed flow cytometry to test this assumption and to quantify the cell fusion event occurring after drug treatment. Each MCF-7 subtype, labeled with either EGFP/YB-1 or DsRed/YB-1, were co-cultured as a single monolayer. Figures 3A, B, figure 4A and 4B show that some of these cells were fused and yielded large multicellular syncytia after treatment with 10 nM doxorubicin for 6 days. Fractions of MCF-7 cells containing the multiple nuclei were increased after doxorubicin treatment (inserted table in Figure 3). The polyploidy cells having heterokaryons (Figure 4B) were identified and quantified by FACS sorting (Figures 4D and 4E). In order to increase cell to cell fusion ratio, 20 nM doxorubicin applied (Figure 4E and figure 5C). Initially, 66% out of R2 fraction containing fused cells was polyploidy, however, its ratio was decreased down to 31%, suggesting that chromosomes were eventually lost or re-arranged (Figure 3C and 3E) while acquiring the doxorubicin resistance (Table insert, Figure 3). The results demonstrate cell fusion events, followed by genome re-arrangement. The origin of cancer-initiating cell (cancer stem cell) remains elusive and the fusion of genetic and cytoplasmic material between cells could be important in the development of the cancer stem cell [26].


p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance.

Yang JY, Ha SA, Yang YS, Kim JW - BMC Cancer (2010)

Schema of cell fusion and chromosomal instability. A: Cytosolic cell fusion of two subtype of cells (homotype or heterotype). B and D: Most of cells, at the stage of nuclear cell fusion, revealed no further propagation and subsequently vanished. C: Viable progeny cells propagated again in culture. Giemsa-stained chromosomes of MCF-7 cells were examined. D: Abnormal sets of chromosomes as a consequence of cell fusion and (E) the cells containing giant nuclei (polyploid) were died out and the diverse populations were analogously converged into doxorubicin resistant clones. Table insert, fusion was quantitated by counting the number of cells (%) and nuclei present in a microscope field.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913965&req=5

Figure 3: Schema of cell fusion and chromosomal instability. A: Cytosolic cell fusion of two subtype of cells (homotype or heterotype). B and D: Most of cells, at the stage of nuclear cell fusion, revealed no further propagation and subsequently vanished. C: Viable progeny cells propagated again in culture. Giemsa-stained chromosomes of MCF-7 cells were examined. D: Abnormal sets of chromosomes as a consequence of cell fusion and (E) the cells containing giant nuclei (polyploid) were died out and the diverse populations were analogously converged into doxorubicin resistant clones. Table insert, fusion was quantitated by counting the number of cells (%) and nuclei present in a microscope field.
Mentions: The appearance of polyploidy cells may be caused by cell-to-cell fusion, we employed flow cytometry to test this assumption and to quantify the cell fusion event occurring after drug treatment. Each MCF-7 subtype, labeled with either EGFP/YB-1 or DsRed/YB-1, were co-cultured as a single monolayer. Figures 3A, B, figure 4A and 4B show that some of these cells were fused and yielded large multicellular syncytia after treatment with 10 nM doxorubicin for 6 days. Fractions of MCF-7 cells containing the multiple nuclei were increased after doxorubicin treatment (inserted table in Figure 3). The polyploidy cells having heterokaryons (Figure 4B) were identified and quantified by FACS sorting (Figures 4D and 4E). In order to increase cell to cell fusion ratio, 20 nM doxorubicin applied (Figure 4E and figure 5C). Initially, 66% out of R2 fraction containing fused cells was polyploidy, however, its ratio was decreased down to 31%, suggesting that chromosomes were eventually lost or re-arranged (Figure 3C and 3E) while acquiring the doxorubicin resistance (Table insert, Figure 3). The results demonstrate cell fusion events, followed by genome re-arrangement. The origin of cancer-initiating cell (cancer stem cell) remains elusive and the fusion of genetic and cytoplasmic material between cells could be important in the development of the cancer stem cell [26].

Bottom Line: The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms.YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype.Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetic Laboratory, College of Medicine, The Catholic University of Korea, Seoul 137-040, Republic of Korea.

ABSTRACT

Background: Cancer cells recurrently develop into acquired resistance to the administered drugs. The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms. Our aim is to define the potential targets, that makes drug-sensitive MCF-7 breast cancer cells turn to drug-resistant, for the anti-cancer drug development against drug resistant breast cancer cells.

Methods: Doxorubicin resistant human breast MCF-7 clones were generated. The doxorubicin-induced cell fusion events were examined. Heterokaryons were identified and sorted by FACS. In the development of doxorubicin resistance, cell-fusion associated genes, from the previous results of microarray, were verified using dot blot array and quantitative RT-PCR. The doxorubicin-induced expression patterns of pro-survival and pro-apoptotic genes were validated.

Results: YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype. Cell fusion increased diversity within the cell population and doxorubicin resistant MCF-7 cells emerged probably through clonal selection. Most of the drug resistant hybrid cells were anchorage independent. But some of the anchorage dependent MCF-7 cells exhibited several unique morphological appearances suggesting minor population of the fused cells maybe de-differentiated and have progenitor cell like characteristics.

Conclusion: Our work provides valuable insight into the drug induced cell fusion event and outcome, and suggests YB-1, GST, ABCB5 and ERK3 could be potential targets for the anti-cancer drug development against drug resistant breast cancer cells. Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

Show MeSH
Related in: MedlinePlus