Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts

Abstract

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.

Figure 1. Fit of Eq. 1 to constant growth for HeLa (own data: green squares, fit: blue line) and rat fibroblast cells (data: blue circles , fit: red dashed line).

See also Table 1.

Figure 2. Model scheme.

a, proliferation; b, extension with second cell state species; c, complete final model (upper part: P-C-S transitions; lower part: stress induction and cell response function F).

Figure 3. Relative number MRC-5 fibroblast cells positive for cellular markers after irradiation by the doses 0, 0.5 and 20 Gy.

MRC-5. a, DNA damage marker γH2AX; b, cell cycle arrest marker p21; c, cycle arrest marker p16; d, senescence marker SA-β-Gal. The experimental error in such experiments is less than ±5%.

Figure 4. PD curves of human fibroblasts.

Model fitting of different radiation doses and experimentally derived PDs. The data were fitted using the same parameter set for all radiation doses (differing only in the applied amount of irradiation time) applying the model described by Eq. 3a–d: a, MRC-5 fibroblast, different radiation doses 0, 0.5 and 20 Gy; b, WI-38 fibroblast, radiation doses 0, 2 and 15 Gy.

Figure 5. Simulation of WI-38 fibroblast data.

a, Experimental growth data (circled) were fitted by model Eq. 3a–d using Eq. 4 as an expression for monotonically increasing stress γ; b, the fraction of proliferating cells P, cells showing a cell cycle arrest or a senescent phenotype (C+S) and solely the fraction of senescent cells S are shown together with the appearance of biomarkers. Biomarker values (p16, p21, SA-β-Gal and SAHF) were measured by immune-fluorescence as number of positive cells .

Figure 6. Simulation of BJ fibroblast data.

a, experimental data (circled) were fitted by model Eq. 3a–d using Eq. 4 as an expression for monotonically increasing stress γ; b, the fraction of proliferating cells P, cells showing a cell cycle arrest or a senescent phenotype (C+S) and solely the fraction of senescent cells S are shown together with the appearance of biomarkers. Biomarker values (p16, p21, SA-β-Gal and SAHF) were measured by immune-fluorescence as number of positive cells .

Figure 7. Simulation of MRC-5 fibroblast data.

a, experimental data (circled) were fitted by model Eq. 3a–d using Eq. 4 as an expression for monotonically increasing stress γ; b, the fraction of proliferating cells P, cells showing a cell cycle arrest or a senescent phenotype (C+S) and solely the fraction of senescent cells S are shown together with the appearance of biomarkers. Biomarker values (p16, p21, SA-β-Gal and SAHF) were measured by immune-fluorescence as number of positive cells .