Gene expression profiles in human peripheral blood mononuclear cells as biomarkers for nutritional in vitro and in vivo investigations

Abstract

Identification of chemopreventive substances may be achieved by measuring biological endpoints in human cells in vitro. Since generally only tumour cells are available for such investigations, our aim was to test the applicability of peripheral blood mononuclear cells (PBMC) as an in vitro primary cell model since they mimic the human in vivo situation and are relatively easily available. Cell culture conditions were refined, and the basal variation of gene expression related to drug metabolism and stress response was determined. Results were compared with profiles of an established human colon cell line (HT29) as standard. For biomarker development of nutritional effects, PBMC and HT29 cells were treated with potentially chemopreventive substances (chrysin and butyrate), and gene expression was determined. Key results were that relevant stress response genes, such as glutathione S-transferase T2 (GSTT2) and GSTM2, were modulated by butyrate in PBMC as in HT29 cells, but the blood cells were less sensitive and responded with high individual differences. We conclude that these cells may serve as a surrogate tissue in dietary investigations and the identified differentially expressed genes have the potential to become marker genes for population studies on biological effects.

Keywords: Biomarker; Butyrate; Gene expression; HT29 cells; Human peripheral blood mononuclear cells.

Fig. 1

Relative gene expression (based on expression of housekeeping gene GAPDH [ppm]) of target genes in untreated cells in two different platforms: a cDNA array, b Real-time PCR. Basal gene expression in PBMC (filled characters) was characterised by high individual variation, whereas HT29 cells (open characters) demonstrated low variability. Variation was detected more sensitive in real-time PCR experiments. GSTM2 was not detectable in HT29 cells using cDNA array

Fig. 2

Real-time PCR analysis of selected genes that were investigated in PBMC. Shown are the basal gene expression profiles for 10 subjects after three different time points, namely at a starting point (week zero), after 8 weeks, and again after 15 weeks. GAPDH (housekeeping gene) was used as internal reference control (gene expression related to GAPDH [ppm])

Fig. 3

a and b Real-time PCR analysis of selected genes that were investigated in PBMC (left side) and HT29 cells (right side). Shown are effects of butyrate and chrysin after 24-h incubation on the expression levels of CAT, SOD2, COX-2, UGT1A1, GSTP1, GSTT2, and GSTM2. The modulations of mRNA levels were obtained by comparing the treatment groups to the medium controls and calculating the fold changes. GAPDH (housekeeping gene) was used as internal reference control (gene expression related to GAPDH [ppm]). Significant differences to the controls were calculated by a one-way ANOVA with Bonferroni’s post-test (***P < 0.001, **P < 0.01, *P < 0.05, n = 4–7 for PBMC and n = 3–4 for HT29)