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 (α_s/α_r: 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.