Radiotherapy-Induced Senescence and its Effects on Responses to Treatment.

Radiotherapy is still a treatment of choice for many malignancies, often in combination with other strategies. However, its efficacy is limited by the dose that can be safely administered without eliciting serious side-effects, as well as the fact that recurrence is common, particularly in large tumours. Combining radiotherapy with drugs that could sensitise cells to radiation and/or reduce the factors that promote the recovery of the surviving cancer cells is a promising approach. Ionising radiation has been shown to induce senescence and the accumulation of senescent cells creates a microenvironment that facilitates neoplastic growth. This provides a rationale to test the addition of anti-senescent drugs, some of which are already available in the clinic, to radiotherapy protocols. Here, we discuss the relevance of radiotherapy-induced senescent cell accumulation and the potential interventions to minimise its negative effects.

Fractionated radiation exposure amplifies the radioresistant nature of prostate cancer cells.

The risk of recurrence following radiation therapy remains high for a significant number of prostate cancer patients. The development of in vitro isogenic models of radioresistance through exposure to fractionated radiation is an increasingly used approach to investigate the mechanisms of radioresistance in cancer cells and help guide improvements in radiotherapy standards. We treated 22Rv1 prostate cancer cells with fractionated 2 Gy radiation to a cumulative total dose of 60 Gy. This process selected for 22Rv1-cells with increased clonogenic survival following subsequent radiation exposure but increased sensitivity to Docetaxel. This RR-22Rv1 cell line was enriched in S-phase cells, less susceptible to DNA damage, radiation-induced apoptosis and acquired enhanced migration potential, when compared to wild type and aged matched control 22Rv1 cells. The selection of radioresistant cancer cells during fractionated radiation therapy may have implications in the development and administration of future targeted therapy in conjunction with radiation therapy.

HDAC inhibitor confers radiosensitivity to prostate stem-like cells.

BACKGROUND: Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction. METHODS: Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α2ß1integrin(hi)/CD133(+)), transit-amplifying (TA, α2ß1integrin(hi)/CD133(-)) and committed basal (CB, α2ß1integrin(lo)) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser. RESULTS: Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation. INTERPRETATION: Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction.

Stra6, a retinoic acid-responsive gene, participates in p53-induced apoptosis after DNA damage.

Stra6 is the retinoic acid (RA)-inducible gene encoding the cellular receptor for holo-retinol binding protein. This transmembrane protein mediates the internalization of retinol, which then upregulates RA-responsive genes in target cells. Here, we show that Stra6 can be upregulated by DNA damage in a p53-dependent manner, and it has an important role in cell death responses. Stra6 expression induced significant amounts of apoptosis in normal and cancer cells, and it was also able to influence p53-mediated cell fate decisions by turning an initial arrest response into cell death. Moreover, inhibition of Stra6 severely compromised p53-induced apoptosis. We also found that Stra6 induced mitochondria depolarization and accumulation of reactive oxygen species, and that it was present not only at the cellular membrane but also in the cytosol. Finally, we show that these novel functions of Stra6 did not require downstream activation of RA signalling. Our results present a previously unknown link between the RA and p53 pathways and provide a rationale to use retinoids to upregulate Stra6, and thus enhance the tumour suppressor functions of p53. This may have implications for the role of vitamin A metabolites in cancer prevention and treatment.

Carbonic anhydrase IX expression and outcome after radiotherapy for muscle-invasive bladder cancer.

AIMS: Carbonic anhydrase IX (CA IX) expression has been described as an endogenous marker of hypoxia in solid neoplasms. Furthermore, CA IX expression has been associated with an aggressive phenotype and resistance to radiotherapy. We assessed the prognostic significance of CA IX expression in patients with muscle-invasive bladder cancer treated with radiotherapy. MATERIALS AND METHODS: A standard immunohistochemistry technique was used to show CA IX expression in 110 muscle-invasive bladder tumours treated with radiotherapy. Clinicopathological data were obtained from medical case notes. RESULTS: CA IX immunostaining was detected in 89 ( approximately 81%) patients. Staining was predominantly membranous, with areas of concurrent cytoplasmic and nuclear staining and was abundant in luminal and perinecrotic areas. No significant correlation was shown between the overall CA IX status and the initial response to radiotherapy, 5-year bladder cancer-specific survival or the time to local recurrence. CONCLUSIONS: The distribution of CA IX expression in paraffin-embedded tissue sections seen in this series is consistent with previous studies in bladder cancer, but does not provide significant prognostic information with respect to the response to radiotherapy at 3 months and disease-specific survival after radical radiotherapy.

Radiation-induced transgenerational alterations in genome stability and DNA damage.

Mutation induction in directly exposed cells is currently regarded as the main component of the genetic risk of ionizing radiation for humans. However, recent data on the transgenerational increases in mutation rates in the offspring of irradiated parents indicate that the genetic risk could be greater than predicted previously. Here, we have analysed transgenerational changes in mutation rates and DNA damage in the germline and somatic tissues of non-exposed first-generation offspring of irradiated inbred male CBA/Ca and BALB/c mice. Mutation rates at an expanded simple tandem repeat DNA locus and a protein-coding gene (hprt) were significantly elevated in both the germline (sperm) and somatic tissues of all the offspring of irradiated males. The transgenerational changes in mutation rates were attributed to the presence of a persistent subset of endogenous DNA lesions (double- and single-strand breaks), measured by the phosphorylated form of histone H2AX (gamma-H2AX) and alkaline Comet assays. Such remarkable transgenerational destabilization of the F(1) genome may have important implications for cancer aetiology and genetic risk estimates. Our data also provide important clues on the still unknown mechanisms of radiation-induced genomic instability.

Concomitant chemoradiotherapy for muscle-invasive bladder cancer: the way forward for bladder preservation?

Muscle-invasive bladder cancer is a common malignancy with a high mortality rate. Despite ongoing debates about the optimal primary intervention, radical cystectomy remains the cornerstone of first-line therapy in many institutions. Over the past decade, bladder-preserving strategies involving transurethral resection (TUR), chemotherapy and radiotherapy have evolved. However, the advantage of these approaches over radiation treatment as monotherapy has yet to be fully evaluated. In other tumour models, most notably cervical and anal cancer, radiation and chemotherapy delivered concomitantly have resulted in significant survival advantages. Here, we consider the potential value of this approach in the treatment of invasive bladder cancer. Concomitant chemoradiotherapy is currently the mainstay of several bladder-preserving programmes reported in the medical literature. Overall, local control and survival rates compare favourably with contemporary cystectomy series; however, difficulties in drawing valid conclusions are highlighted. Concomitant chemoradiotherapy may have a role in the management of certain patient subgroups, and the debate should remain open. Further large-scale randomised trials are needed, and information regarding bladder function and quality of life after treatment is lacking at present. The importance of close follow-up and prompt salvage cystectomy is emphasised.

Measurements using the alkaline comet assay predict bladder cancer cell radiosensitivity.

In the UK, the two main treatments of invasive bladder cancer are radiotherapy or cystectomy. However, approximately 50% of patients undergoing radiotherapy fail to respond. If tumour radiosensitivity could be predicted in advance, it may be possible to improve control rates significantly by selecting for radiotherapy those patients whose tumours are radiosensitive. Additionally, patients who would benefit from surgery would be identified earlier. The alkaline comet assay (ACA) is a sensitive method for the detection of DNA strand break damage in cells. In the present study, using six bladder cancer cell lines of differing radiosensitivities, cell survival was compared to the manifestation of radiogenic DNA damage as assessed by ACA. For all the cell lines, the extent of comet formation strongly correlates with cell killing (R2>0.96), with a greater response being noted in radiosensitive cells. In repair studies, measures of residual damage correlate with survival fraction at 2 Gy (R2>0.96), but for only five of the cell lines. Finally, cells from human bladder tumour biopsies reveal a wide range of predicted radiosensitivies as determined by ACA. Overall, these studies demonstrate ACA to be a good predictive measure of bladder cancer cell radiosensitivity at low dose, with potential clinical application.