Insoluble Ni compound-induced gene amplification/gene silencing causes over-expression of microtubules/microfilaments, cell shape changes, and de-regulation of global gene expression/Ca+2 gradients, inducing morphological/ neoplastic transformation of 10T1/2 mouse embryo cells

Abstract

Inhalation of Ni-containing sulfidic ore dusts and smoking cigarettes in Ni refineries causes human respiratory cancer. Inhalation of Ni3S2 or green NiO induces respiratory cancer in rats. Ni3S2 and green NiO were phagocytosed into and induced morphological/A. I./neoplastic transformation in 10T1/2 mouse embryo cells. mRNA differential display showed that 130 genes were differentially expressed between non-transformed and two MCA/four Ni-transformed, 10T1/2 cell lines. Ni/MCA-transformed cell lines displayed a) ect-2 gene amplification/higher levels of ect-2 mRNA/protein; b) increased levels of calnexin mRNA/protein; and c) absence of DRIP80 and β-centaurin-2 mRNAs. We hypothesized amplification of the ect-2 gene led to higher steady-state levels of rhoA-GTP, inducing higher steady-state levels of microtubules (MTs) in Ni/-MCA transformed cell lines. We stained non-transformed/transformed 10T- 1/2 cell lines with fluorescent antibody to α-tubulin/β-tubulin, to stain MTs. MTs were distributed homogeneously in long, thin fibers in non-transformed 10T1/2 cells, but present at higher levels/aggregated in some areas/missing in other areas of transformed cells, confirming hypothesis #1. We next hypothesized transcriptional silencing of the β-centaurin-2 gene led to increased steady-state levels of microfilaments (MFs) and altered their intracellular distribution, in Ni/MCA transformed cell lines. We stained cells with fluorescent phalloidin, a fungal toxin that binds to MFs, and demonstrated MFs were distributed homogeneously in long, thin fibers in non-transformed 10T1/2 cells, but present at higher levels/aggregated, in some areas and missing in other areas of transformed cells, confirming hypothesis #2. Over-expression/clumping of MFs/MTs rounded transformed cells, altering their contact with extra-cellular matrix. Third, we hypothesized that silencing the DRIP80 gene led to alterations in Ca+2 ion gradients in transformed cell lines, and confirmed this by staining cells with the Ca+2 fluorophore, FLUO 3AM. This would alter activities of Ca+2-dependent enzymes in transformed cell lines, contributing to aberrant physiologies. Ni+2 ion therefore induced ect-2 gene amplification/over-expression and caused silencing of the β-centaurin-2 and DRIP80 genes, leading to over-expression of MTs/ MFs, respectively, hence cell shape changes and altered gene expression, and to aberrant Ca+2 ion gradients, which would cause changes in activities of Ca+2-dependent enzymes. Changes in MTs, MFs, and Ca+2 ion gradients would de-regulate expression of 130 genes/alter Ca+2-dependent enzyme activities, contributing to induction of cell transformation.