Biomarkers of Cell Senescence Assessed by Imaging Cytometry

Abstract

The characteristic features of senescent cells such as their “flattened” appearance, enlarged nuclei and low saturation density at the plateau phase of cell growth, can be conveniently measured by image-assisted d cytometry such as provided by the laser scanning cytometry (LSC). The “flattening” of senescent cells is reflected by the decline in local density of staining (intensity of maximal pixel) of DNA-associated fluorescence [4,6-diamidino-2- phenylindole (DAPI)] paralleled by an increase in nuclear size (area). Thus, the ratio of the maximal pixel of DAPI fluorescence per nucleus to the nuclear area provides a very sensitive morphometric biomarker of “depth” of senescence, which progressively declines during induction of senescence. Also recorded is cellular DNA content revealing cell cycle phase, as well as the saturation cell density at plateau phase of growth, which is dramatically decreased in cultures of senescent cells. Concurrent immunocytochemical analysis of expression of p21^WAF1, p16^INK4a or p27^KIP1 cyclin kinase inhibitor provides additional markers of senescence. These biomarker indices can be expressed in quantitative terms (“senescence indices”) as a fraction of the same markers of the exponentially growing cells in control cultures.

1. Introduction

Cellular senescence can be subdivided into two distinct categories. The first is replicative senescence, observed after certain rounds of division as a loss of replicative capacity of normal proliferating diploid cells in culture (“Hayflick’s limit”) (1). This type of senescence is a consequence of a progressive shortening of telomeres with each division until a cell reaches the state of telomere dysfunction (2). The second category is independent on telomere shortening or dysfunction and is referred to as premature cell senescence (review, 3). Persistent cellular stress including oxidative DNA damage leading to constitutive replication stress (4,5), activation of oncogenes (6) and loss of tumor suppressor genes (7) are among the factors and mechanisms inducing premature cells senescence. Although during chemo- or radio- therapy apoptosis is preferred mode of cell death in certain malignancies such as acute leukemias, in solid tumors the mechanism of growth suppression often relies on irreversible impairment of cell reproductive capacity defined as a drug- or radiation-induced senescence, which also belongs to the category of premature senescence (8,9). There is growing body of evidence that induction of premature senescence in vivo plays also a critical role as barrier in tumor development, including prevention of tumorigenesis of the induced pluripotent stem cells ( iPSCs; 10).

Regardless of the category and the mechanism of induction of senescence there are common markers to identify senescent cells. The most characteristic are morphological features (11). Senescent cells are characterized by much “flattened” appearance, enlarged and often irregular-shape nuclei, cytoplasmic granularities and low saturation density at the plateau phase of growth. An increase in cellular size is accompanied by an increase in nuclear and nucleolar size. There are numerous vacuoles in the cytoplasm, increased number of cytoplasmic microfilaments, the presence of enlarged lysosomal bodies and prominent Golgi apparatus (11–13). Also apparent in some types of cells are abnormalities of nuclear chromatin presented as senescence-associated heterochromatic foci (SAHF). SAHF are abundant in histone H3 modified at lysine 9 (K9M H3) and its binding partner heterochromatin protein 1 (HP1) (13–16).

Several molecular biomarkers also characterize senescent cells. Suppression of proliferation of these cells is to a large degree mediated by over-expression of inhibitors of cyclin-dependent kinases (CKIs) p21^WAF1, p16^INK4A and/or p27^KIP1 associated with activation of tumor suppressor p53 pathways (17–20). Persistent expression of markers of DNA damage response, particularly of Ser139-phosphorylated histone H2AX (γH2AX), is an additional feature of senescent cells (21,22). These molecular markers, however, are neither unique nor highly specific to senescent cells. Among all biomarkers the most specific are: (i) the characteristic changes in cell morphology, and (ii) the induction of senescence-associated β-galactosidase activity, the latter considered to be the hallmark of cell senescence (23,24).

We have recently reported that one of the most specific morphological features of senescent cells, namely their “flattening” and nuclear enlargement, can be quantitatively assessed using imaging cytometry, such as provided by laser scanning cytometer (LSC) (25,26). LSC is the microscope-based quantitative-image analyzer that offers the combined advantages of flow cytometry and image analysis (27–29) (Fig. 1). It can analyze over 100 cells per second, record a large number of the measured parameters including cell morphometric data, and preserve images of the analyzed cells. We have recently shown that a simple assay based on measurement of nuclear size (area) combined with the intensity of maximal pixel of DNA-associated fluorescence per nucleus, after DNA staining with fluorescent dyes [(e.g. 4,6-diamidino-2-phenylindole (DAPI)], provides a convenient marker of the degree (“depth”) of cell senescence (25). The protocol for identification of senescent cells and for quantitative assessment of a “depth” of cell senescence is given below. This protocol includes also immunocytochemical detection of CKIs p21^WAF1, p27^KIP1 or p16^INK4a which are additional, although as mentioned, not highly specific markers of senescent cells (17–20).

Fig. 1.

Schematic illustration of iCys^® Research Imaging Cytometer outfitted with 405, 488, 532, and 633 nm lasers.

2. Materials

2.0. Common materials

1. Chambered slides, available from Nunc Lab-Tek II (Fisher Scientific, Pittsburgh, PA): see Subheading 3.1.

2. Phosphate-buffered saline (PBS), pH 7.4.

3. Formaldehyde (methanol-free, “ultrapure”) (Polysciences, Warrington, PA), 1% (v/v) in PBS (See note 1).

4. Ethanol (70%).

5. Coplin jars

6. Parafilm “M” (American National Can, Greenwich, CT)

7. Solution of 0.1%Triton X-100 (v/v) (Sigma/Aldrich, St. Louis, MO) in PBS

8. Antifade coverslips mounting medium (Invitrogen/Molecular Probes).

2.1. Immunocytochemical and DNA staining reagents

1. Bovine serum albumin (BSA; Sigma/Aldrich) dissolved (1%; w/v) in PBS (BSA solution)

2. p21^WAF1 and/or p16^INK4a and/or p27^KIP1Ab (Santa Cruz Biotechnology, Santa Cruz, CA) (See subheading 3)

3. The secondary fluorochrome-tagged Abs: AlexaFluor 488 or AlexaFluor 633 Ab (Invitrogen/Molecular Probes, Eugene, OR).

4. Stock solution of 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen/Molecural Probes). Dissolve 1 mg DAPI dihydrochloride in 1 mL of deionized water (2.66 mM concentration) (See note 2);

5. Staining solution of DAPI prepared freshly before the use: Add 5 uL of the stock DAPI solution to 2 mL of PBS (final DAPI concentration 2.5 ug/mL)

2.2. Equipment to measure cell fluorescence

1. Laser scanning microscope (LSC), preferentially new model of LSC (iGeneration; Research Imaging Cytometer; iCysR) manufactured by CompuCyte, Westwood, MA) (See Fig. 1) (See note 3).

3. Methods

3.1. Induction of cell senescence

The methodology is applicable to cells growing attached to surface of culture vessel and not to cells growing in suspension. Because morphology of cells undergoing replicative senescence (e.g. due to telomere dysfunction) is similar to that of premature senescence such as that of stress- or drug-induced, this protocol can be used to assess depth of senescence in either case. Prepare cell cultures by seeding cells on single- or multi- chambered incubation slides. (See notes 4, 5 and 6 for approaches to induce premature cell senescence in these chambered cultures).

3.2. Cell fixation and staining

1. Remove the culture medium from each chamber on the slide and replace it with PBS, to fill up the chamber

2. Remove PBS and replace it with ice-cold 1% formaldehyde solution in PBS, to fill up the chamber, close chambers caps. Keep cells in formaldehyde on ice for 15 min.

3. Remove formaldehyde and replace it with ice-cold 70% ethanol, close chamber caps and transfer slides to freezer at −20 °C. The slides can be stored in 70% ethanol at −20 °C for several days.

4. After storage in ethanol, carefully remove the chamber’s walls from the slides and incubate the slides in PBS in Coplin jars for 5min, then repeat incubation in fresh PBS for another 5 min.

5. Remove the slides from Coplin jar and place them horizontally in a moisturized chamber.

6. Carefully layer a small volume (approximately100 μl) of 0.1% Triton X-100 in PBS for 15min, then 1% BSA solution for 30min.

7. After 30 min replace the BSA solution with the solution of BSA containing primary p21 or p16 Ab at 1:100 dilution (See notes 7 and 8), incubate at room temperature for 1.5 h or overnight at 4°C.

8. Rinse the cells with 1% BSA solution in PBS and layer approximately 100 μl of BSA solution containing AlexaFluor 488 or AlexaFluor 633- tagged secondary Ab at 1:100 dilution (See note 7), incubate for 45min at room temperature.

9. Rinse the slides with PBS and layer approximately 100 μl of DAPI staining solution.

10. After 15 min, rinse away the DAPI staining solution with PBS, then add a drop of anti-fade mounting medium and mount under the coverslip (See note 9).

3.3. Fluorescence measurement and data analysis

1. Measure cell fluorescence on LSC. Excite DAPI fluorescence with 405 nm diode laser, AlexaFluor 488 with 488 nm diode laser and AlexaFluor 633 with 633 nm Helium-Neon laser. Collect blue (DAPI), green (AlexaFluor 488) and far red (AlexaFluor 633) fluorescence emission with the respective photomultipliers (See notes 10, 11, 12, and 13).

Fig. 2.

Analysis of nuclear changes and expression of p21^WAF1 of A549 cells in cultures treated with Mxt to induce cell senescence.

The cells were untreated (Ctrl) or treated with 2 nM Mxt for 24, 48, or 72 h, their DNA was stained with DAPI and expression of p21^WAF1 was detected immunocytochemically (AlexaFluor 633 nm indirect Ab). The intensity of maximal (max) pixel of DNA-bound DAPI fluorescence reveals highest local level of chromatin condensation per nucleus. In the untreated cultures mitotic (M) and immediately postmitotic (pM) cells have the highest value of maximal pixel, reflecting their highly condensed chromatin. In senescing cells due to “flattening” of the cell and nucleus, the nuclear area is increased and intensity of maximal pixel of DAPI fluorescence is decreased (25). The insets in the top left panels show DNA frequency histograms of cells from the respective cultures. The bar plots at the bottom panels show mean values (+standard deviation) of nuclear DNA(DAPI) area, mean value of DNA(DAPI) fluorescence intensity of maximal pixel per nucleus, and the ratio of maximal pixel to nuclear area. The ratio of maximal pixel to nuclear area of the Mxt-treated cells (“cell senescence index”; SI) is expressed as a fraction of such ratio of the respective control (untreated cells, cells growing exponentially) (SI = 1.0). The lowest is the SI value the greater is “depth’ of senescence.

Fig. 3.

Decreased cell saturation density at confluence as another marker (senescence index, SI) reflecting “depth” of cell senescence.

The A6549 cells were untreated (Ctrl) or grown in the presence of 2 nM Mxt for 48 or 72 h in chamber slide-cultures. The cells were fixed and stained with DAPI as described in the protocol. Their presence and location on the slides concurrently with other parameters (measured as shown in Fig 2) was recorded by LSC (25,27–29). The observed decrease in cell density in the Mxt-treated cultures is presented as a fraction (senescence index, SI) of cell density in the untreated exponentially growing culture (SI = 1.00).

Abbreviations

Ab

Antibody

ATM

Ataxia Telangiectasia mutated protein kinase

BSA

Bovine serum albumin

CKIs

Inhibitors of cyclin-dependent kinases

DAPI

4,6-diamidino-2-phenylindole

γH2AX

Ser139-phosphorylated histone H2AX

HP1

heterochromatin protein 1

iCys

Research Imaging Cytometer®

Mtx

Mitoxantrone

iPSCs

Induced pluripotent stem cells

LSC

Laser scanning cytometry

PBS

Phosphate-buffered saline

PI

Propidium iodide

SI

Senescence Index

SAHF

Senescence-associated heterochromatic foci

TSA

Trichosporin A