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. Author manuscript; available in PMC: 2019 Sep 27.
Published in final edited form as: Methods Mol Biol. 2017;1534:31–39. doi: 10.1007/978-1-4939-6670-7_3

Cellular Model of p21-Induced Senescence

Michael Shtutman 1, Bey-Dih Chang 2, Gary P Schools 1, Eugenia V Broude 3
PMCID: PMC6764449  NIHMSID: NIHMS1051529  PMID: 27812865

Abstract

Cellular senescence is a unique process of normal physiology, from embryonic development to aging, also known for its association with a broad range of pathological conditions. Therefore a reliable model of cellular senescence remains an indispensable tool for the investigation of senescence-associated changes and human disease. Here we describe a model of HT1080 fibrosarcoma cells with an inducible senescence phenotype. These cells are equipped with the lac repressor and exogenous p21 under the control of a lac repressor regulated promoter. The senescent phenotype is induced in these cells by isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression of senescence-associated cell cycle inhibitor p21Wafl/Clp1/Sdi1.

Keywords: Senescence, p21Waf1/Cip1/Sdi1, Lac-repressor, IPTG, HT1080-p21–9

1. Introduction

Cell senescence, originally defined as irreversible proliferative arrest that occurs in normal cells after a limited number of cell divisions, is now viewed more broadly as a general biological program of terminal growth arrest. Cellular senescence is associated with normal development and pathological conditions [1, 2]. Cells that underwent senescence cannot divide even if stimulated by mitogens, but they remain metabolically and synthetically active and show characteristic changes in morphology, such as enlarged and flattened cell shape and increased granularity [3]. The most widely used surrogate marker of senescent cells is the senescence-associated β-galactosidase activity (SA-β-gal), which is detectable by X-gal staining at pH 6.0 [4]. SA-β-gal appears to reflect increased activity of lysosomal acid β-galactosidase [5]. As elucidated primarily in the normal fibroblast models, growth arrest of senescent cells is initiated with the activation of p53. In the case of replicative senescence, p53 protein is stabilized through the involvement of p14ARF, a tumor suppressor that sequesters the Mdm2 protein, which promotes p53 degradation. The activated p53 has multiple effects on gene expression, the most relevant of which in regard to senescence is transcriptional activation of p21Wai1/CiP1/Sdi1, a pleiotropic inhibitor of cyclin/cyclin-dependent kinase (CDK) complexes that mediate cell cycle progression [6]. p21 induction causes cell cycle arrest in senescent cells. The activation of p53 and p21 in senescent cells is only transient; protein levels of p53 and p21 decrease after the establishment of growth arrest. While p21 expression goes down, another CDK inhibitor, p16Ink4A, becomes constitutively upregulated and maintains growth arrest in senescent cells [7, 8].

To examine the transition from growth arrest to senescence and the properties of senescent cells, we have used a cellular system based on inducible expression of p21WAF1/CIP1, in human HT1080 fibrosarcoma cells. p21 was cloned in an IPTG-inducible retroviral vector (see Fig. 1a) and transduced into HT1080 cells expressing the lac I repressor. IPTG dose-dependent induction of p21 (see Fig. 2a, b) resulted in growth arrest in G1 (at low p21 levels) or in G1, S, and G2 (at high p21 levels) (see Fig. 2c). p21 induction also led to time- and dose-dependent expression of senescence-associated β-galactosidase (SA-β-gal) and morphologic features of senescent cells (see Fig. 2). After the removal of IPTG, most of the cells reentered the cell cycle, but many of them died or stopped growing after a small number of cell divisions. The dead or growth-arrested cells were predominantly in G2/M or polyploid. The failure to recover was directly correlated with the induced levels of p21 and the duration of p21 induction. Cells that were released from IPTG after 5 days of p21 induction (poor recovery) showed predominantly abnormal mitoses, in contrast to a high frequency of normal mitoses in cells that were released after 1 day of induction (significant recovery) [9]. Analysis of the effects of transient p21 induction on the expression of genes involved in the control of cell division suggests that p21-mediated inhibition of specific genes involved in mitosis control (such as CDC2 or cyclin A) may be responsible for abnormal mitosis after release from p21-induced growth arrest (see Fig. 3) [9, 10]. Hence, the failure of HT1080 p21–9 cells to recover after p21-induced growth arrest is due to mitotic catastrophe [9].

Fig. 1.

Fig. 1

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induction of p21 expression and cell senescence by IPTG. (a) Scheme of LNp21C03 retroviral vector. (b) HT1080-p21–9 cells were treated with 50 μM of IPTG for 8–72 h. Merged image of DIC and staining with p21 antibodies is shown. (c) Cells were treated with 50 μM IPTG for 72 h. Senescent phenotype was determined by staining for SA-β-gal activity with X-gal at pH 6.0. SA-β-gal expression in untreated (left) and IPTG-treated (right) p21–9 cells

Fig. 2.

Fig. 2

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Dose-dependent induction of cell cycle arrest (a), p21 protein, and SA-β-gal by IPTG (b, c). HT1080-p21–9 cells were treated for 48 h with IPTG. ELISA measurement using WAF1 ELlSA kit (Oncogene Research) (b) and western blot (c) of p21 protein expression. The percentage of sA-β-gal-positive cells was determined by X-gal staining at pH 6.0 following scoring of 100–400 cells per sample.

Fig. 3.

Fig. 3

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Release from p21. (a) FACS profile of DNA content of attached cells at different time points after release from 1-day treatment with 50 μm of IPTG. (b) Examples of normal (left) and abnormal (right) mitotic figures observed in p21–9 cells after release from IPTG (visualized with DAPI staining).

1.1. Applications of p21-Inducible HT1080-p21–9 Cell Line

Initially, p21-inducible cell system had been developed to recapitulate effects of DNA damaging drugs and to investigate the mechanisms of drug-induced senescence [11]. However, this system turned out to be very suitable for the analysis of many aspects of cellular senescence. These cells were extensively used for understanding p21-dependent transcriptional regulation and regulation of stability of tumor suppressors [12–15]. p21–9 cells were utilized for the investigation of the function of cell cycle controlling proteins in the regulation of intracellular localization of human papillomaviruses and mechanisms of regulation of cellular senescence by mTOR pathway and hypoxia [15–18]. The cells were used as a model for studies of abnormal mitosis and of p21-dependent regulation of ROS production [9, 19]. The inducible cells allow for easy comparison between proliferating and arrested (p21-induced) states. The system was also used for comparative analysis of the effects of growth inhibitory siRNAs on dividing and arrested cells and kinetics of shRNA silencing in proliferating and halted cells [20]. Additionally, it was suggested that protein biosynthesis is increased in senescent cells; therefore, p21-induced cells were used for enhanced production of exogenous proteins [21]. p21–9 cells were utilized for developing high-throughput screening systems used for identification of chemical inhibitors of cyclin-dependent kinases [22–24].

1.2. Development of p21-Inducible Cellular System

The inducible system is based on a derivative of HT1080 human fibrosarcoma cells. HT1080 cells containing ecotropic retroviral receptor were transfected with p3’SS plasmid, expressing a modified lac I repressor and carrying a hygromycin-resistance gene [25, 26]. Transfected cells were selected with 100–120 μg/mL of hygromycin, and individual colonies were picked and screened for optimal repressor activity. Modified LNCX vector, containing tri-meric lac operator downstream of CMV early promoter (LNXCO3) and carrying firefly luciferase, was transduced into the colonies and induction of luciferase activity by treatment with IPTG was measured. Based on the screening, the best clone (HT1080 3’SS6) was identified, where luciferase activity was induced up to 15-fold with 1.25 mM of IPTG [27].

HT1080-p21–9 cells were generated by transduction of a p21-expressing retrovirus into HT1080 3’Ss6 cells [11]. The inducible retroviral vector LNp21CO3 was constructed by cloning of 492 bp p21 coding sequence into the IPTG-inducible retroviral vector LNXCO3 [28]. LNp21CO3 retroviral vector was transduced into HT1080 3’SS6 cells, and the transduced population was selected with 200 μg/mL G418. Clonal line p21–9 was derived from the LNp21CO3-transduced population of HT1080 3’SS6 cells by end-point dilution followed by screening of individual colonies for the strongest induction of p21 expression by IPTG. Like the parental HT1080 cell line, p21–9 cells express the wild-type pRb and p53 [11, 29].

2. Materials

Prepare all solutions using ultrapure deionized water (18 MΩ at 25 °C) and cell culture grade reagents. All solutions applied to cultured cells have to be filtered through sterile 0.22 μM filter (EMD-Millipore Polyethersulfone (PES) filter or equivalent) and kept sterile.

  1. DMEM tissue culture medium: 200 mM L-glutamine, fetal bovine serum.

  2. G418 (Thermo Fisher Scientific or equivalent). To prepare 50 mg/mL stock solution, weigh out 500 mg G418 and transfer to 10 mL of deionized water and mix until fully dissolved. Sterilize by filtration and store in 1 mL aliquots at –20 °C.

  3. IPTG (isopropyl-β-d-thiogalactopyranoside, EMD-Millipore #5800 or equivalent). To prepare 10 mM stock solution, transfer 238 mg of IPTG to 10 mL of deionized water and mix until fully dissolved. Sterilize by filtration and store in 1 mL aliquots at –20 °C.

  4. Anti-p21 antibodies: Mouse antihuman CIP1 monoclonal antibody (Clone 70/CIP1/WAF1).

3. Methods

3.1. Induction of p21 and Monitoring of p21-Induced Senescence

  1. Cells need to be plated at a relatively low density, approximately 40,000 cells per 60 mm tissue culture plate (see Notes 1 and 2).

  2. At least 12 h cell reattachment should be allowed before treatment.

  3. IPTG at 50 μM should be applied to achieve full induction of p21 expression. There is direct dose dependence of p21 induction by IPTG up to 50 μM. Higher concentrations do not affect the level of p21 expression.

  4. Cells are undergoing growth arrest and fully develop the senescent phenotype 72 h post induction. Example of induction of p21 expression and accumulation of senescent phenotype (see Fig. 1b).

  5. Senescent phenotype can be verified by SA-β-gal staining at pH 6.0 (see refs. 4, 30) (see Fig. 1c).

3.2. Release from p21

  1. Cells need to be plated at a relatively low density, approximately 40,000 cells per 60 mm tissue culture plate or 5000 cells per cover slip (for microscopy).

  2. At least 12 h cell reattachment should be allowed before treatment.

  3. IPTG at 50 μM should be applied to achieve full induction of p21 expression.

  4. Cells are undergoing growth arrest and fully develop the senescent phenotype 72 h post induction. There is direct correlation between the severity of p21-related recovery problems and the length of p21 induction and IPTG concentration.

  5. To release cells from p21 induction, IPTG-containing medium has to be thoroughly removed, cells washed twice with PBS, and fresh medium added.

  6. Senescent phenotype can be verified by SA-β-gal staining; at pH 6.0, mitotic abnormalities and cell death can be followed by time-lapse video microscopy and FACS analysis (see refs. 4, 9, 30) (see Fig. 3).

4. Notes

  1. Cells need to be propagated in standard DMEM medium supplemented with 10 % FBS. Due to negative selection against p21, cells shouldn’t be propagated in culture more than 2 weeks.

  2. If p21 inducibility is diminished, cells could be reselected with G418 (200 μg/mL) followed by subclone analysis. For each selected subclone, p21 expression has to be shown by western blot with anti-p21 antibodies, and the presence of senescence phenotype has to be confirmed.

Acknowledgments

This work was supported by NIH grant P20GM109091 (M.S., G.S., E.V.B.) and ACS grant IRG-13-043-01 (E.V.B.).

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