[Epithelial to Mesenchymal Transition Marker in 2D and 3D Colon Cancer Cell Cultures in the Presence of Laminin 332 and 411].

Received August 28, 2018; revised October 10, 2018; accepted November 6, 2018 The loss of apical-basal cell polarity is a necessary stage of the epithelial-mesenchymal transition (EMT). Polarized epithelial cells interact with the basement membrane (BM) and, in particular, with laminins, the major components of BM. Here, we examined the effect of the transition of colon cancer cells from 2D polarized state to non-polarized 3D state on the expression of EMT associated genes, as well as the role of laminins 332 and 411 (LM-332 and LM-411) in this process. The three studied cell lines, HT-29, HCT-116 and RKO, were found to have different sensitivity to cultivation conditions (2D to 3D changes) and to addition of laminins. One of the possible reasons for this maybe a difference in the initial 2D state of the cells. In particular, it was shown that the cell lines were at different EMT stages. HT-29 exhibited more epithelial expression profile, RKO was more mesenchymal, and HCT-116 was in an intermediate state. The most laminin-sensitive cell line was HCT-116. The magnitude and the specificity of cell response to LM-332 and LM-411 depended on the expression pattern of laminins' receptors. EMT gene expression profile was not substantially changed neither during the transition from 2D to 3D state, nor the presence of laminins' isoforms. However, we detected changes in expression of SNAI1 and ZEB1 genes encoding transcription factors that control the EMT process. Notably, in all three studied cell lines, the expression of SNAI1 was enhanced in response to laminin treatment.

Enzyme-Substrate Reporters for Evaluation of Substrate Specificity of HIF Prolyl Hydroxylase Isoforms.

An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD isoform preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of "paired" enzyme-substrate reporters to evaluate the potency of "branched tail" oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform.