Kidney‐specific enzyme expression by human kidney cell lines generated through oncogene transfection

Abstract

The human kidney cell line 293 was generated by transfection of adenovirus DNA into normal human embryonic kidney (HEK) cells (Graham et al., 1977), whereas the human kidney cell lines ST-1i and STt-4i were generated by transfection of HEK cells with plasmids encoding SV40 viral oncogenes (Abcouwer et al., 1989). In this study, we examined kidney-specific enzyme activity levels in 293, ST-1i, and STt-4i cells to determine their ability to exhibit kidney-specific gene expression. Enzymes examined were leucine aminopeptidase (LAP), gamma-glutamyl transpeptidase (gamma-GTP), and the disaccharidases trehalase and maltase. Enzymatic activity levels were compared to three other kidney cell lines (MDCK, OK, and LLC-PK1) as well as to normal human embryonic kidney (HEK) cells and the human hepatoma cell line, Hep G2. Modulation of kidney-specific enzyme activities was assessed in response to several differentiation-inducing agents (adenosine, n-butyric acid, hexamethylene bisacetamide (HMBA), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), isobutyl methyl xanthine (IBMX), di butyryl cAMP, and retinoic acid). ST-1i and STt-4i exhibit elevated levels of LAP, gamma-GTP, trehalase, and maltase, consistent with their kidney cell origin, whereas 293 cells exhibit elevated levels of just gamma-GTP and maltase. Maltase and gamma-GTP enzyme activities in ST-1i and STt-4i cells were very responsive to the various inducing agents; 293 cells were less responsive at the inducer concentrations examined. None of the three human cell lines formed domes under any of the experimental conditions. In summary, ST-1i and STt-4i are comparable to normal HEK cells in expression of kidney-specific enzymes and in responsiveness to differentiation-inducing agents, in spite of continued expression of SV40 oncogenes.

Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress

“…While LLC-PK1 is a spontaneously immortalized cell line, HK-2 is immortalized by transduction with human papilloma virus 16 (HPV-16) E6/E7 genes. The E6/E7 genes of HPV-16 have been reported to immortalize epithelial cells of diverse origin without significantly changing its phenotype or function [54][55][56][57][58][59]; however, most of immortalized cell lines have potentially limited their utility with derangements in cell function and structural integrity as a result of altered gene expression [60][61][62][63]. In addition, differences in OTA binding to intracellular proteins have been observed among several species, where human kidney homogenates showed a higher OTA protein binding than pig kidney homogenates [64][65][66].…”

Intracellular Trafficking of Thyroid Peroxidase to the Cell Surface

“…A postnuclear supernatant (0.5 ml) was sedimented at 14,000 ϫ g for 4 min to generate supernatant (S) and pellet (P) fractions. ␥-Glutamyl transpeptidase (GGT), a plasma membrane marker of 293 cells (35), was located primarily in the S fraction, whereas the ER marker calnexin (CNX) was located primarily in the P fraction (Fig. 5A).…”

FRET sensor-based quantification of intracellular trehalose in mammalian cells

“…9) Moreover, human kidney cells such as HEK293T express trehalase activity. 20) We therefore tested whether the trehalose accumulation was reversible. Removal of substrate after addition showed clear reversibility, which can be due to either metabolism or efflux ( Fig.…”