Shahnaz T. Al Rashid

Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.

Targeting homologous recombination using imatinib results in enhanced tumor cell chemosensitivity and radiosensitivity

RAD51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. Because imatinib (Gleevec) has been reported to reduce RAD51 protein levels, we tested the clonogenic survival for RT112, H1299, PANC1, and PC3 tumor cell lines of varying p53 status and normal GM05757 normal fibroblasts after exposure to single agent imatinib (0–20 μmol/L; 0–72 hours). We also combined imatinib with DNA damaging agents that are toxic to RAD51-deficient cells, including ionizing radiation, gemcitabine, and mitomycin C. We observed decreased nuclear expression and chromatin binding of RAD51 protein following imatinib treatment. Imatinib also resulted in decreased error-free HR as determined by a flow cytometry–based integrated direct repeat-green fusion protein reporter system; this correlated to reduced RAD51 expression. Clonogenic survival experiments revealed increased cell kill for imatinib-treated cells in combination with ionizing radiation, gemcitabine, and mitomycin C, due in part to mitotic catastrophe. In experiments using imatinib and gemcitabine, tumor cell lines were sensitized to a greater extent than normal fibroblasts. This preservation of the therapeutic ratio was confirmed in vivo using PC3 xenograft growth delay and intestinal crypt cell clonogenic assays. HR inhibition may be an additional mechanism of action for the chemosensitization and radiosensitization of solid tumors with imatinib with preservation of the therapeutic ratio. [Mol Cancer Ther 2009;8(1):203–13]

BRCA1, FANCD2 and Chk1 are potential molecular targets for the modulation of a radiation-induced DNA damage response in bystander cells

Radiotherapy is an important treatment option for many human cancers. Current research is investigating the use of molecular targeted drugs in order to improve responses to radiotherapy in various cancers. The cellular response to irradiation is driven by both direct DNA damage in the targeted cell and intercellular signalling leading to a broad range of bystander effects. This study aims to elucidate radiation-induced DNA damage response signalling in bystander cells and to identify potential molecular targets to modulate the radiation induced bystander response in a therapeutic setting. Stalled replication forks in T98G bystander cells were visualised via bromodeoxyuridine (BrdU) nuclear foci detection at sites of single stranded DNA. γH2AX co-localised with these BrdU foci. BRCA1 and FANCD2 foci formed in T98G bystander cells. Using ATR mutant F02-98 hTERT and ATM deficient GM05849 fibroblasts it could be shown that ATR but not ATM was required for the recruitment of FANCD2 to sites of replication associated DNA damage in bystander cells whereas BRCA1 bystander foci were ATM-dependent. Phospho-Chk1 foci formation was observed in T98G bystander cells. Clonogenic survival assays showed moderate radiosensitisation of directly irradiated cells by the Chk1 inhibitor UCN-01 but increased radioresistance of bystander cells. This study identifies BRCA1, FANCD2 and Chk1 as potential targets for the modulation of radiation response in bystander cells. It adds to our understanding of the key molecular events propagating out-of-field effects of radiation and provides a rationale for the development of novel molecular targeted drugs for radiotherapy optimisation.

Radiation microbeams as spatial and temporal probes of subcellular and tissue response.

Understanding the effects of ionizing radiations are key to determining their optimal use in therapy and assessing risks from exposure. The development of microbeams where radiations can be delivered in a highly temporal and spatially constrained manner has been a major advance. Several different types of radiation microbeams have been developed using X-rays, charged particles and electrons. For charged particles, beams can be targeted with sub-micron accuracy into biological samples and the lowest possible dose of a single particle track can be delivered with high reproducibility. Microbeams have provided powerful tools for understanding the kinetics of DNA damage and formation under conditions of physiological relevance and have significant advantages over other approaches for producing localized DNA damage, such as variable wavelength laser beam approaches. Recent studies have extended their use to probing for radiosensitive sites outside the cell nucleus, and testing for mechanisms underpinning bystander responses where irradiated and non-irradiated cells communicate with each other. Ongoing developments include the ability to locally target regions of 3D tissue models and ultimately to target localized regions in vivo. With future advances in radiation delivery and imaging microbeams will continue to be applied in a range of biological studies.

Protein-Protein Interactions Occur Between p53 Phosphoforms and ATM and 53BP1 at Sites of Exogenous DNA Damage

Abstract We have previously shown that the Ser15-phosphorylated p53 phosphoform, p53Ser15, can localize at sites of ionizing radiation-induced DNA damage. In this study, we hypothesized that the non-specific DNA binding domain (NSDBD) of the p53 carboxy-terminus (C-terminus) mediates chromatin anchoring at sites of DNA damage to interact with two key mediators of the DNA damage response (DDR): ATM and 53BP1. Exogenous YFP-p53 fusion constructs expressing C-terminus deletion mutants of p53 were transfected into p53-null H1299 cells and tracked by microscopy and biochemistry to determine relative chromatin-binding pre- and postirradiation. We observed that exogenous YFP-p53WT and YFP-p53Δ367–393 associated with ATMSer1981 and 53BP1 in the nuclear, chromatin-bound fractions after DNA damage. Of interest, YFP-p53Δ1–299 fusion proteins, which lack transcriptional trans-activation and the Ser15-residue, bound to ATMSer1981 but not to 53BP1. In support of these data, we used subnuclear UV-microbeam and immunoprecipitation analyses of irradiated normal human fibroblasts (HDFs) that confirmed an interaction between endogenous p53 and ATM or 53BP1. Based on these observations, we propose a model whereby a pre-existing pool of p53 responds immediately to radiation-induced DNA damage using the C-terminus to spatially facilitate protein-protein interactions and the DDR at sites of DNA damage.

Abstract 2227: Effects of cathepsin S in differentiated and stem-like glioblastoma cells

Background: Glioblastoma (GBM) is the most common and aggressive brain tumour subtype with a poor prognosis of ∼15 months. A GBM stem-like population within the tumour is hypothesised to promote resistance to current treatment and invasion through normal tissue. Cathepsin S (Cat S) is a lysosomal cysteine protease that remains catalytically active at neutral pH and has been shown to play a role in radiation resistance and invasion of cancer cells. It is also an independent predictor of GBM patient survival. Materials and methods: GBM cell lines (U87MG and LN229) with lentiviral shRNA knockdown of Cat S are grown in serum-free medium with growth factors that allow de-differentiation of the cells into a stem-like state. A transwell system is used for quantifying monolayer invasion. Under stem conditions, spheroid structures are formed and their invasion in 3D is analysed with GFP expression. Protein expression determined via Western blot. Radiation response determined via clonogenic survival assay. Proliferation measured using MTT assay. Cat S-like activity measured using fluorogenic peptide substrate cleavage. Results: Radiation induces Cat S protein expression and protease activity in both GBM cell lines at 24 hours. Knockdown of Cat S induces radiation sensitivity in both cell lines when differentiated. In stem-like conditions, this is observed for U87MG but not for LN229 cells. Similarly, Cat S knockdown has no effect on proliferation of cell lines, except LN229 stem-like cells where it is increased. Finally, Cat S knockdown reduces the invasive capacity of both differentiated cell lines and U87MG stem-like cells 3D invasion through both collagen and matrigel, but has no effect on LN229 stem-like cells. Cat S-like activity is reduced in all but stem LN229, despite complete knockdown. Further investigation into differential effects in stem-like cells will examine possible role of autophagy, which is linked to radiation resistance and Cat S inhibition. Conclusion Cat S is a potential drug target in GBM as it reduces invasion and radio-resistance of glioma cell lines. The differential effect seen in stem-like cells suggests that the genetic background of tumours may be an important factor in determining utility of Cat S therapy in targeting the stem-like GBM population thought to be responsible for recurrence. Citation Format: Robyn A. Foster, Shahnaz T. Al Rashid, Roberta E. Burden, Christopher J. Scott, Kevin M. Prise, Thomas J. Flannery. Effects of cathepsin S in differentiated and stem-like glioblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2227. doi:10.1158/1538-7445.AM2015-2227