P14ARF-p53 induced senescence revisited: protector or precursor of latent breast cancer?


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Eileen McGowan (1), Daniel Yagoub (2), Nikki Alling (3)
Centenary, Sydney, Australia (1), University of Sydney, Sydney, Australia (2), Royal Prince Alfred Hospital, Sydney, Australia (3), Garvan Institute of Medical Research, Sydney, Australia (4)

Background

Many years of oncogenic insult and hormonal flux have important long-term implications for breast cancer emergence and progression. P14ARF-p53 induced premature senescence is proposed to be a protective response against oncogenic signals and cancer development associated with mitochondria dysfunction and oxidative DNA damage. Here we investigated the effects of reactivation of the p14ARF-p53 pathway in MCF-7 breast cancer cells focusing on mitochondria and lysosomal activity and metabolism.

Method

P14ARF was conditionally expressed in MCF-7 cells using the LacSwitch™ IPTG-inducible system. Cell cycle parameters were measured by cell counts, Syber-green DNA-labelling , Edu Click-iT™ and flow cytometric analysis. Senescence-like morphological changes were determined by imaging and senescence-associated-beta-galactosidase (SA-beta-gal) staining. Protein expression was analysed by Western blot and fluorescent imaging.  Mitochondria content was measured by high content image analysis and metabolic activity and membrane potentiality measured by MTS and TMRE assays. Acridine orange staining was used to assess lysosomal function and potentiality by flow cytometric analysis.

Results

Conditional expression of p14ARF in MCF-7 cells induces rapid arrest in G1/G2M phases of the cell cycle and increase in cell growth.  Cells show a senescence-like phenotype defined by enlarge flat cytoplasm with multinuclei, increase in p53, p21 and decrease in retinoblastoma proteins.  Additionally, cells stain for SA-beta-gal.  However, here we show that mitochondria content and functionality is increased with no overt impairment of mitochondria membrane potentiality. Furthermore lysosomal content and membrane potentiality is also increased.  No overt signs of oxidative DNA damage are observed.

Conclusion

Although reactivation of p14ARF-p53 induces cell cycle arrest with many of the hallmarks of senescence we show that this pathway induces a highly metabolically active, functional cell.  Arguably, these results suggest that induction of the p14ARF-p53 pathway does not necessarily remove or inactivate cancerous breast cells and in fact may lay the seeds for precursor, or latent, cancer cells.

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