Tetracycline-mediated fluorescence correlates with passage number and β-galactosidase activity in HeLa and HEK293T cells

Tetracycline (Chemical Abstracts Service number 60-54-8) is a fluorescently active molecule used widely as an antibiotic (Chopra and Roberts, 2001), a clinically-approved fluorochrome reagent for labelling human bone osteocytes in vivo and as a label for identifying pathologically calcified tissues in histological samples (Pautke et al., 2010). To date no applications have been reported for tetracycline in the field of cellular senescence.

The genetic toolbox for engineering mammalian cells includes transgenes incorporating tetracycline-inducible promoters, referred to as ‘Tet-On’ systems (Wieland and Fussenegger, 2012). We initially sought to characterise a novel, plasmid-encoded, ‘Tet-On’ promoter controlling transcription of an open reading frame for green fluorescent protein (GFP). We tested the plasmid via transient transfection of the HeLa cell line, an adherent, immortal, tumour-derived line used commonly in clinical research and biotechnology applications (Kast et al., 2021).

Fig. 1 shows results from our initial attempt to transiently transfect HeLa cells with a plasmid incorporating a ‘Tet-on’ system for controlling GFP expression. Rows A-C of Fig. 1 show results from untreated cells, cells incubated with Superfect only, and cells incubated with Superfect and plasmid only, showing no marked increase in fluorescence. When a 24-h incubation with media supplemented with 2 μg/ml tetracycline was introduced, post-transfection, a marked increase in fluorescence was observed (Fig. 1, row D). In isolation, rows C and D of Fig. 1 would appear to evidence the expected ‘Tet-On’ phenotype, with the marginal increase in fluorescence on Day 4 in Row D due to leakiness of the Tet-On promoter system. However, control experiments showed that omitting plasmid (Fig. 1, row E) and omitting both plasmid and Superfect (Fig. 1, row F) still resulted in marked fluorescence when cells were incubated with 2 μg/ml tetracycline. To determine the repeatability of the observed fluorescence in the absence of plasmid, three further independent repeats of the experiments in the 1-day and 4-day columns (Fig. 1) were performed. All repeats showed the same pattern of fluorescence appearance in the absence of plasmid and transfection reagent and the presence of tetracycline.

The fluorescence observed in Fig. 1, Rows E and F, prevented confirmation of a plasmid-encoded ‘Tet-On’ function as they suggested all observed fluorescence, including Fig. 1, Row D, resulted from incubation with 2 μg/ml tetracycline alone. We considered that the observations in Fig. 1 may suggest a previously unreported property of senescent immortal cells; namely that of increased association with tetracycline. To test this hypothesis, we purchased new batches of the immortal cell lines HeLa and HEK293T, from the American Type Culture collection (ATCC).

We seeded ATCC-purchased HeLa and HEK cell lines the day before to give confluence of approximately 75% on the day of experimentation. Media was then replaced with fresh media either with or without 2 μg/ml tetracycline. Tetracycline-containing media was then left on the cells for up to 24 h before being removed prior to flow cytometry (Fig. 2A). This procedure (Fig. 2B-2E) was performed for cells that had undergone 4 (P4) or 23 passages (P23) since cryorevival.

P23 HeLa cells exhibited markedly greater fluorescence than P4 HeLa cells after 16 and 24 h in the presence of tetracycline (Fig. 2B,C). P23 HeLa cells also showed a clear difference in fluorescence between cells that had been incubated with unsupplemented media and those incubated with media containing 2 μg/ml tetracycline (Fig. 2C, compare grey and black data lines). This difference was greatest after 24 h incubation with tetracycline. No such difference was observed with the P4 Hela cells (Fig. 2B, compare grey and black data lines).

P23 HEK cells also showed greater fluorescence than P4 HEK cells after 16 and 24 h in the presence of tetracycline (Fig. 2D,E). P23 HEK cells showed the clearest difference in fluorescence between cells that had been incubated with and without tetracycline, with the greatest difference observed after 16 h incubation (Fig. 2E, compare grey and black data lines). Three further independent repeats of the experiments in the 4-h and 24-h columns (rows B-E) confirmed the replicability of the observed greater fluorescence with greater passage number.

Observations in Fig. 2B-E were consistent with tetracycline-mediated fluorescence increasing as passage number increased. This may be due senescence-mediated changes to the cell surface that increase net affinity for tetracycline, given that cellular senescence can remodel the surface of cells (Kim et al., 2017; Madsen et al., 2017).

We tested whether HeLa or HEK passaging correlates with β-galactosidase activity, a widely used marker of cellular senescence (Lee et al., 2006). We used the CellEvent Senescence Green Flow Cytometry Assay Kit from Thermo Fisher Scientific to measure β-galactosidase in HEK cells that had undergone 4 (P4), 14 (P14) or 23 passages (P23) since cryorevival and equivalent HeLa cells at P4, P17 and P23 (Fig. 2F). The kit features the ‘Green Probe’ substrate which fluoresces upon cleavage by β-galactosidase.

HeLa cell passage number showed a linear relationship with β-galactosidase activity (Fig. 2F, right hand data panel) with β-galactosidase activity increasing after 17 and again after 23 passages, compared to the level at 4 passages. For HEK cells β-galactosidase activity markedly increased after 23 passages but there was effectively zero increase from passage 4 to 14 (Fig. 2F, left hand data panel). These observations were consistent across n=4 independent experimental repeats.

We understand this observed property of tetracycline with respect to the senescence of immortalised cell lines to be novel. We anticipate tetracycline could be exploited to rapidly assess senescence of HeLa or HEK-based cell lines of uncertain provenance and passage number. Further characterisation and validation will be needed to establish whether senescence-based tetracycline-binding is broadly applicable to other biological settings.

We initially used HeLa cells from a historic cell bank, cultivated using standard techniques with 10% v/v foetal bovine serum in High-Glucose/GlutaMAX Dulbecco's Modified Eagle's Medium (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) in capped-vented T175 flasks (Corning Limited, Union City, CA, USA). Cells were plated in a media solution of typically 2×10⁵ cell/ml in static incubators at 37°C, 5% CO₂, and passaged upon reaching 80% confluence.

This initial HeLa cell line was estimated to have undergone more than 20 consecutive passages. Cells were seeded at 5×10⁵/ml and yielded a confluency of approximately 75% after overnight growth. Media was changed to serum-free Ultraculture media (Lonza, UK), and plasmid DNA plus Superfect (Qiagen, MD, USA) added to cells. Cells were prepared for analysis by flow cytometry 1, 2, 3 or 4 days after transfection. For cytometry cells were washed with 10 ml phosphate-buffered saline (PBS), incubated with 2 ml trypsin-EDTA (Merck-Millipore, Germany) at 37°C for 5 min then 2 ml PBS added addition for resuspension. Cells were then centrifuged and re-suspended in 1-2 ml PBS to remove residual trypsin. Fluorescence analysis was performed using the BD Accuri C6 Plus (BD Biosciences) device, counting 20,000 ungated events. Histogram data analysis was performed using absolute mean fluorescence intensity (MFI) with the FL1-A laser, which can detect both the emission peaks of tetracycline (529 nm) and GFP (509 nm).

The authors are pleased to acknowledge all staff and students of the UCL Biochemical Engineering Department, particularly Ludmila Ruban and Dhushy Stanislaus, who supported all tissue culture and and molecular biology work as part of the Department's core facilities team. Tania Rodin is acknowledged for making availlable the plasmid used in this study. The authors also wish to acknowledge all of their friends and families.