Purpose: Growth of Chinese hamster V79 cells as multicell spheroids leads to an increase in resistance to killing by ionizing radiation and etoposide. Differential display was used to identify changes in gene expression that occur when cells are grown as spheroids.
Materials and methods: Differential display was performed using exponentially growing Chinese hamster V79 cells and the outer cell layer of V79 spheroids. Using six different pairs of primers, 20 altered bands were selected. Eight genes, confirmed using reverse Northerns, showed a match in a GenBank search. Antibodies against a calcium-binding protein, mts1, confirmed differential expression of this protein. Intracellular free calcium levels were measured using fluo-3 fluorescence, and the effect of a calcium-binding agent on etoposide resistance was examined using the comet assay.
Results: Genes upregulated in the outer cell layer of spheroids relative to monolayers included: (1) mts1 (S100A4), a calcium binding protein implicated in proliferation, metastasis, cell adhesion, and angiogenesis; (2) cytochrome c oxidase II; (3) B-ind1, a mediator of Rac-1 signaling; (4) TRAM, an endoplasmic reticulum protein. Genes downregulated in spheroids were: (5) phosphoglycerate kinase; (6) ARL-3, a ras-related GTP binding protein; (7) MHC class III complement 4A; and (8) 2,4-dienoyl-CoA. Immunohistochemistry confirmed overexpression of mts1 and another calcium-binding protein, calreticulin, in V79 outer spheroid cells relative to monolayers. C6 rat glioma and SiHa human cervical carcinoma cells that demonstrate a contact effect also showed upregulation of mts1 or calreticulin, while WiDr colon carcinoma cells that lack contact resistance showed no change in expression of either calcium binding protein. Intracellular free calcium levels were found to be almost two times lower in the outer cells of V79 spheroids compared to monolayers. V79 monolayer and outer spheroid cells treated with the calcium chelating agent BAPTA-AM showed a similar level of DNA damage by etoposide.
Conclusions: Expression of genes involved in calcium binding, signaling and metabolism are differentially expressed when V79 cells are grown as spheroids. Differences in the levels of intracellular calcium may underlie the contact effect.