Abstract
Dermal fibroblast cells can adopt different cell states such as proliferation, quiescence, apoptosis or senescence, in order to ensure tissue homeostasis. Proliferating (dividing) cells pass through the phases of the cell cycle, while quiescent and senescent cells exist in a non-proliferating cell cycle-arrested state. However, the reversible quiescence state is in contrast to the irreversible senescence state. Long-term quiescent cells transit into senescence indicating that cells age also when not passing through the cell cycle. Here, by label-free in vitro vibrational spectroscopy, we studied the biomolecular composition of quiescent dermal fibroblast cells and compared them with those of proliferating and senescent cells. Spectra were examined by multivariate statistical analysis using a PLS-LDA classification model, revealing differences in the biomolecular composition between the cell states mainly associated with protein alterations (variations in the side chain residues of amino acids and protein secondary structure), but also within nucleic acids and lipids. We observed spectral changes in quiescent compared to proliferating cells, which increased with quiescence cultivation time. Raman and infrared spectroscopy, which yield complementary biochemical information, clearly distinguished contact-inhibited from serum-starved quiescent cells. Furthermore, the spectra displayed spectral differences between quiescent cells and proliferating cells, which had recovered from quiescence. This became more distinct with increasing quiescence cultivation time. When comparing proliferating, (in particular long-term) quiescent and senescent cells, we found that Raman as well as infrared spectroscopy can separate these three cellular states from each other due to differences in their biomolecular composition. Our spectroscopic analysis shows that proliferating and quiescent fibroblast cells age by similar but biochemically not identical processes. Despite their aging induced changes, over long time periods quiescent cells can return into the cell cycle. Finally however, the cell cycle arrest becomes irreversible indicating senescence.
Highlights
Dermal fibroblasts are quiescent, a state of reversible cell cycle arrest [1]
Cellular quiescence has been studied in detail by various biomolecular methods such as flow cytometry to analyze for cell cycle distributions, immunoblotting for monitoring of specific proteins or microarray analysis to compare gene expression profiles in proliferating and quiescent cells [4, 8,9,10, 46]
In the FT-IR data, protein bands decreased and a lipid band (1740 cm-1) increased with senescence, which is in the agreement with previous experiments noticeably matching the partial least squares linear discriminant analysis (PLS-LDA) results [36]
Summary
Dermal fibroblasts are quiescent, a state of reversible cell cycle arrest [1]. Wound-activated fibroblasts proliferate and migrate to wounds, coordinate the healing response by secretion of molecules and recruiting endothelial cells Specific molecular mechanisms trigger quiescence actively [7], as for instance contact inhibition, presence or absence of nutrients, adhesion loss or mitogen withdrawal All of these are associated with comprehensive changes in gene expressions or regulations of cell division genes [2]. Preventive protection from radical damages or transition into terminal differentiation constrain the reversible non-dividing state [8]. These processes result in slightly different quiescent states which share a common genetic program [8,9,10]. Cells protect themselves by short-term quiescence from a transition into senescence [10], whereas long-term quiescence induces a senescent-associated cell cycle arrest in order to prevent malignant transformations [9]
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