IADR Abstract Archives
Changes in MCF-7 and HFF cells induced by ectopically expressed vimentin
Background: VIM gene in man encodes vimentin polypeptide. The emergent functions of vimentin include sustaining the cell shape and taking part in cytoskeletal interactions. Vimentin is important in lysosomes for transporting of low-density lipoprotein (LDL) cholesterol to the site of esterification. Vimentin also helps in migration of leukocyte to the site of inflammation eliciting an immune response. Methods: The expression of vimentin was observed by transducing MCF7 and HFF with recombinant retroviruses expressing AcGFP-vimentin and 3 x Flag-vimentin followed by G418 selection for 10 days. The stably expressing cells were visualized by using an epi-fluorescence microscope. The MCF-7 cells stably expressing vimentin were used for cell migration using scratch assay. The association of vimentin with microfilaments and microtubules was investigated by adding Cytochalasin B and Nocodazole, respectively.
Results: Transduction of MCF7 with AcGFP-Vimentin construct produced globular structures spread all over the cytoplasm. There was no vimentin filament assembly in the transduced cells. Strong staining of vimentin was seen at the intercellular junction which was never been reported in the literature. To investigate if the vimentin expression would affect cell migration, scratch wound healing assay was carried out using vimentin transduced MCF7 cells. The presence of globular vimentin in MCF-7 cells increased cell migration was observed. Although this construct did not produce vimentin filaments, yet the same construct when introduced in human foreskin fibroblasts (HFF), which had pre-existing vimentin filaments, the ectopically expressed vimentin was able to integrate into the pre-existing network suggesting that the synthesized vimentin was compatible with the in vivo filament network. To see if vimentin filaments were associated with actin filaments or microtubules, the HFF cells transduced with vimentin were treated with cytochalasin B, a microfilament disrupting drug, and with Nocodazole, a microtubule disrupting drug. None of these drugs have had any significant effect on vimentin filaments in HFF cells suggesting that these filaments are in direct association with other cytoskeletal elements in HFF cells. Conclusion: The vimentin polypeptide which was expressed did not form filaments in the cytoplasm. Although the molecular basis for the full-length vimentin not forming cytoplasmic filaments is not clear, it could be due to the AcGFP or Flag tags being placed at the N-terminus. As the vimentin polypeptide integrated into the existing filaments suggested that the constructs were not defective. The deposition of vimentin conjugate at the intercellular junction in epithelial cells is novel and might indicate its binding to a component of adherence junction. Vimentin expression in MCF-7 cells does not appear to have any significant impact on cell migration. The Nocodazole and Cytochalasin B did not affect the vimentin filaments suggesting that vimentin filaments in our system are not associated to neither microfilaments nor microtubules.
Results: Transduction of MCF7 with AcGFP-Vimentin construct produced globular structures spread all over the cytoplasm. There was no vimentin filament assembly in the transduced cells. Strong staining of vimentin was seen at the intercellular junction which was never been reported in the literature. To investigate if the vimentin expression would affect cell migration, scratch wound healing assay was carried out using vimentin transduced MCF7 cells. The presence of globular vimentin in MCF-7 cells increased cell migration was observed. Although this construct did not produce vimentin filaments, yet the same construct when introduced in human foreskin fibroblasts (HFF), which had pre-existing vimentin filaments, the ectopically expressed vimentin was able to integrate into the pre-existing network suggesting that the synthesized vimentin was compatible with the in vivo filament network. To see if vimentin filaments were associated with actin filaments or microtubules, the HFF cells transduced with vimentin were treated with cytochalasin B, a microfilament disrupting drug, and with Nocodazole, a microtubule disrupting drug. None of these drugs have had any significant effect on vimentin filaments in HFF cells suggesting that these filaments are in direct association with other cytoskeletal elements in HFF cells. Conclusion: The vimentin polypeptide which was expressed did not form filaments in the cytoplasm. Although the molecular basis for the full-length vimentin not forming cytoplasmic filaments is not clear, it could be due to the AcGFP or Flag tags being placed at the N-terminus. As the vimentin polypeptide integrated into the existing filaments suggested that the constructs were not defective. The deposition of vimentin conjugate at the intercellular junction in epithelial cells is novel and might indicate its binding to a component of adherence junction. Vimentin expression in MCF-7 cells does not appear to have any significant impact on cell migration. The Nocodazole and Cytochalasin B did not affect the vimentin filaments suggesting that vimentin filaments in our system are not associated to neither microfilaments nor microtubules.