In vitro mesenchymal-epithelial transition in NIH3T3 fibroblasts results in onset of low-dose radiation hypersensitivity coupled with attenuated connexin-43 response.

BACKGROUND: Mesenchymal-to-epithelial transition (MET) is associated with altered cell adhesion patterns. Independent studies showed that cellular adhesion regulates low-dose hyper-radiosensitivity (HRS), a phenomenon reported widely in tumour cells. Therefore, present study aimed to investigate whether MET and associated cellular adhesion alterations affect cellular radiosensitivity.

METHODS: We established multiple stages of MET by in vitro transformation of NIH3T3 mouse embryonic fibroblasts. Nutritional deprivation followed by repetitive treatment cycles of 3-methylcholanthrene and phorbol-12-myristate-13-acetate with frequent isolation of foci established three progressive strains (NIH3T3.1, NIH3T3x3, NIH3T3x8x3) depicting MET, and one strain (NIH3T3x12) with partial reversion. Alterations in morphology, cell adhesion properties, expression/intracellular localization of cell adhesion proteins, microRNA expression and cellular radiosensitivity were studied in these stably transformed cell strains.

RESULTS: All four transformants had increased proliferation rate, saturation density, bipolarity, E-cadherin expression; coupled with reduced cell size/spreading, pseudopodia/migration, and fibroblast marker protein and vimentin. The most aggressive trans-differentiated (phenotypically epithelial) cell strain, NIH3T3x8x3 acquired ~30% higher growth potential associated with more than two-fold reduction in cell size and migration. These phenotypic changes accompanied ~40% reduction in endogenous or radiation-induced connexin-43 expression/mitochondrial translocation. Incidentally, all three progressive strains displayed prominent HRS (αs /αr : 7.95-37.29) whereas parental (NIH3T3) and reverting (NIH3T3x12) strains lacked HRS and had distinct radiation-induced Cx43 translocation into mitochondria.

CONCLUSION: Our study shows that trans-differentiating fibroblasts progressively acquiring epithelial features during MET process, display low-dose hyper-radiosensitivity associated with altered Cx43 behaviour.

GENERAL SIGNIFICANCE: This study demonstrates that MET progression triggers low-dose hyper-radiosensitivity in trans-differentiating cells, which has significant therapeutic implications.