Biochimica et Biophysica Acta (BBA) - General Subjects
NMR-based metabolic profiling of human hepatoma cells in relation to cell growth by culture media analysis
Introduction
Many cell types in culture are subjected to contact inhibition of proliferation dependent on cell–cell and cell–matrix interactions, adhesion-dependent signaling, cytoskeletal dynamics and regulated by substrates and growth factor availability. This behaviour, typical of primary cultures, is also common in immortalized cell lines.
In this context, the impact of cell density in culture may have important consequences when testing new drugs or biologically active molecules, in particular those believed to influence proliferation and growth.
For instance, it has been shown that cell density influences energy metabolism [1], the activation of p53 tumor suppressor [2], the de novo synthesis of sphingolipids [3] and sensitivity to drugs [4], and recently the “critical cell density” of a cultured population was thought to be involved in the adaptive drug resistant mechanism [5].
However, the underlying metabolism that is characteristic of sparse and high density cultures has not been deeply investigated nor the relationship between proliferative rate and metabolism in cells at different phases of cell growth in culture has not been fully clarified. Recently, results obtained by 1H-NMR spectroscopy on extracts of a glioma cell line have shown changes in metabolic patterns in relation to log-, confluent- and post-confluent growth stages [6].
Cell growth rate is closely linked to the phases of the cell-cycle. A number of different enzyme activities involved in nucleotide synthesis [7], different phospholipid metabolisms [8], differential response to drugs [9], morpho-structural variations and variations in mitochondria intracellular distribution have been associated with cells in different phases of the cycle [10], [11]. In a previous work, we demonstrated a different metabolic profile of cell subpopulations in relation to cell cycle phases by 13C-NMR spectroscopy using [1,2-13C2] glucose as a stable-isotope tracer [12].
Metabolic profiling allows the analysis of the cell metabolome that is defined as the quantitative complement of low-molecular weight metabolites present in a cell under a given set of physiological activities [13], [14].
As changes in medium metabolites, substrate utilization and production and substrate flux distribution reflect the cell physiological state and phenotype and thus the cell metabolome [15], we investigated if the monitoring of metabolites consumed by and released into the medium by cells cultivated at different cell densities provide sufficient information on the metabolic pathways involved. In this respect, NMR spectroscopy, in combination with multivariate data analysis, is a suitable technique allowing the simultaneous quantification of a large number of metabolites without any a priori hypothesis regarding the involved biochemical pathways and leading to the identification of a specific metabolic fingerprint.
In this study we utilized a hepatoblastoma cell line (HepG2) which retains the expression of most liver-specific genes [16]. This cell line has been widely used for toxicological and pharmacological studies. In order to obtain the metabolic profiling of cell cultures in relation to the confluence or subconfluence growing state, changes in the exometabolome were evaluated by using 1H-NMR spectroscopy and Multivariate Data Analysis (MVDA) on medium data. This approach allowed us to discriminate two different metabolic profiles in relation to different phases of cell growth in culture and the results were interpreted on the basis of the comparison of the pattern of correlations obtained in the two physiological cell states. The pattern of correlations represents the interconnectivity of the fluxes of metabolites with respect to their pair-wise correlations and can be interpreted as a fingerprint of the underlying system that provides information about the specific physiological state of the cells at a given point in time which can be interpreted as changes in the regulation of metabolic fluxes among the different intracellular pathways [17], [18].
Section snippets
Materials
Minimum Essential Medium (MEM), l-glutamine and fetal bovine serum (FBS) were purchased from GIBCO (Auckland, NZ). Sodium pyruvate, sodium bicarbonate, penicillin and streptomycin, phosphate buffer saline (PBS), Trypan Blue, Trypsin-EDTA 0.25% solution, Propidium Iodide, Triton X100, bovine serum albumin were all from Sigma Aldrich (St. Louis, MO). Methanol, ethanol and chloroform were from Carlo Erba Reagenti (Rodano, Milan, Italy). 3-(trimethylsilyl)-tetradeutero-sodium propionate (TSP) was
Cell proliferation and phase distribution
Cells, seeded at 4 × 104 and 8 × 104 cells/cm2, were cultured for 24 h to allow the attachment. Then the medium was changed and the cells were cultured for a further 24 h to reach subconfluence or confluence respectively (< 40% and > 90% of the culture dishes occupied, respectively). In separate experiments cells showed the same proliferation rate as evaluated by direct cell counts during the first 48 h of culture regardless of the initial seeding density. A decrease in the growth rate was evidenced
Discussion
Proliferation rate is a widely used quantitative parameter to evaluate in vitro the efficacy of proliferative and anti-proliferative drugs in cancer cells. However, culture conditions such as the adhesion matrix, medium composition and supplementation, the presence of growth factors and cell density in particular may induce variations in the proliferation rate as well as in the distribution within the different phases of the cell cycle.
In this context, a particular care in adherent cell
Acknowledgement
This study was supported by a “La Sapienza” University Grant (2005).
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