Late radiation cystitis is an adverse event associated with irradiation of pelvic tumours that occurs months to years after the completion of therapy.  Symptomatic radiation cystitis appears in 5-10% of cases, presents with pain, incontinence and recurrent haematuria, may be life-threatening and there are currently no non-invasive or effective treatments.  Although pathophysiology of late cystitis remains largely unclear, urothelial as well as endothelial cells play an important role [1].  These cells are among the most sensitive to radiation insult and their obliteration leads to inflammation, hypoxia and, ultimately, bladder fibrosis.
Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodelling.  In general, it is a stress response (or survival strategy) due to different triggers including telomere shortening with aging, DNA damage due to irradiation therapy, or oxidative stress resulting in prevention of cell division and resistance to cell death.  Thus, senescence can be a consequence of both physiological and pathological processes that are largely dependent on the microenvironment created by the senescence-associated secretory phenotype (SASP).  The different paracrine activity of these SASPs determine whether there is continued decreased function as in aging, or cell survival as in cancer [2].
We have hypothesised that urothelial cell senescence can be a contributing factor for emergence of late radiation cystitis and compared nuclear localization of senescence marker, p21, expression in control and irradiated bladders.

Young male and female C57Bl/6 mice were anesthetized with Avertin (250 mg/kg, IP) and pelvic areas encompassing the bladders irradiated with 10 Gy (X-RAD320 Biological X-Ray Irradiator, PXI Inc).  Two weeks to nine months later, animals’ bladder function was tested with urine spot tests and tissues from irradiated and age-matched controls were saved in 10% PFA. Upon deparafinization, heat-mediated antigen retrieval and permeabilization, non-specific binding was blocked with 10% normal horse serum, sections (3 μm thick) incubated with p21 primary antibody (Abcam, ab188224, 1:200 dilution) and visualized with complementary fluorescent secondary antibody (Thermofisher, donkey anti-rabbit alexafluor 594, 1:500 dilution).  Nuclei were counterstained with DAPI (Sigma, 1:5,000) and sections coverslipped and mounted with Immu-Mount (Thermofisher).  Images were acquired and processed on a confocal microscope with associated software (FV3000, Olympus).

It has been earlier demonstrated that following a single 10 Gy dose of irradiation mice develop both acute (days to week) and late cystitis, the latter occurring as early as two to three months following radiation and presenting with recurrent inflammation, bladder fibrosis and decreased capacity.  These experiments confirmed that late cystitis persisted till at least nine months post radiation insult.  Immunohistochemical staining of the tissues demonstrated increased expression of p21 in irradiated bladders, especially in urothelial layer, with further significant increase at a later stage.  While the number of p21-positive urothelial cells in age-matched control tissues was 97 ± 20 cells/mm2, two weeks post irradiation it was 347 ± 57 cells/mm2 and nine months later – 834 ± 315 cells/mm2.

Cellular senescence plays an important role in tissue repair after short-term damage, however, insufficient elimination and/or excessive accumulation of senescent cells at the following stages is associated with increased tissue damage and fibrosis.  P21 is a cyclin-dependent kinase inhibitor that maintains the viability of DNA damage-induced senescent cells and is considered to be a reliable senescence marker.  Its’ increase in bladder wall at the later stages of radiation cystitis indicates a large number of urothelial cells, which may promote recurring inflammation and further tissue fibrosis.

We have demonstrated that cellular senescence is involved in urothelial response to radiation insult, is exacerbated overtime and may play an important role in the emergence of late radiation cystitis – the adverse event that often occurs years post therapy in humans. Thus, targeting cell senescence with emerging senotherapeutics may help prevent this condition.

Helissey C, Cavallero S, Brossard C et al. Chronic Inflammation and Radiation-Induced Cystitis: Molecular Background and Therapeutic Perspectives. Cells. 2021. 10(1):21. PMID: 33374374.
Yosef R, Pilpel N, Papismadov N et al. P21 Maintains Senescent Cell Viability under Persistent DNA Damage Response by Restraining JNK and Caspase Signalling. EMBO J. 2017. 36(15):2280-2295. PMID: 28607003.

Continence 7S1 (2023) 100807
DOI: 10.1016/j.cont.2023.100807