Farid Jalali

Gold Nanoparticles as Radiation Sensitizers in Cancer Therapy

Abstract Among other nanoparticle systems, gold nanoparticles have been explored as radiosensitizers. While most of the research in this area has focused on either gold nanoparticles with diameters of less than 2 nm or particles with micrometer dimensions, it has been shown that nanoparticles 50 nm in diameter have the highest cellular uptake. We present the results of in vitro studies that focus on the radiosensitization properties of nanoparticles in the size range from 14–74 nm. Radiosensitization was dependent on the number of gold nanoparticles internalized within the cells. Gold nanoparticles 50-nm in diameter showed the highest radiosensitization enhancement factor (REF) (1.43 at 220 kVp) compared to gold nanoparticles of 14 and 74 nm (1.20 and 1.26, respectively). Using 50-nm gold nanoparticles, the REF for lower- (105 kVp) and higher- (6 MVp) energy photons was 1.66 and 1.17, respectively. DNA double-strand breaks were quantified using radiation-induced foci of γ-H2AX and 53BP1, and a modest increase in the number of foci per nucleus was observed in irradiated cell populations with internalized gold nanoparticles. The outcome of this research will enable the optimization of gold nanoparticle-based sensitizers for use in therapy.

Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR

The promyelocytic leukemia (PML) nuclear body (NB) is a dynamic subnuclear compartment that is implicated in tumor suppression, as well as in the transcription, replication, and repair of DNA. PML NB number can change during the cell cycle, increasing in S phase and in response to cellular stress, including DNA damage. Although topological changes in chromatin after DNA damage may affect the integrity of PML NBs, the molecular or structural basis for an increase in PML NB number has not been elucidated. We demonstrate that after DNA double-strand break induction, the increase in PML NB number is based on a biophysical process, as well as ongoing cell cycle progression and DNA repair. PML NBs increase in number by a supramolecular fission mechanism similar to that observed in S-phase cells, and which is delayed or inhibited by the loss of function of NBS1, ATM, Chk2, and ATR kinase. Therefore, an increase in PML NB number is an intrinsic element of the cellular response to DNA damage.

Defective DNA strand break repair after DNA damage in prostate cancer cells: implications for genetic instability and prostate cancer progression.

Together with cell cycle checkpoint control, DNA repair plays a pivotal role in protecting the genome from endogenous and exogenous DNA damage. Although increased genetic instability has been associated with prostate cancer progression, the relative role of DNA double-strand break repair in malignant versus normal prostate epithelial cells is not known. In this study, we determined the RNA and protein expression of a series of DNA double-strand break repair genes in both normal (PrEC-epithelial and PrSC-stromal) and malignant (LNCaP, DU-145, and PC-3) prostate cultures. Expression of genes downstream of ATM after ionizing radiation-induced DNA damage reflected the p53 status of the cell lines. In the malignant prostate cell lines, mRNA and protein levels of the Rad51, Xrcc3, Rad52, and Rad54 genes involved in homologous recombination were elevated ∼2- to 5-fold in comparison to normal PrEC cells. The XRCC1, DNA polymerase-β and -δ proteins were also elevated. There were no consistent differences in gene expression relating to the nonhomologous end-joining pathway. Despite increased expression of DNA repair genes, malignant prostate cancer cells had defective repair of DNA breaks, alkali-labile sites, and oxidative base damage. Furthermore, after ionizing radiation and mitomycin C treatment, chromosomal aberration assays confirmed that malignant prostate cells had defective DNA repair. This discordance between expression and function of DNA repair genes in malignant prostate cancer cells supports the hypothesis that prostate tumor progression may reflect aberrant DNA repair. Our findings support the development of novel treatment strategies designed to reinstate normal DNA repair in prostate cancer cells.

A novel poly(ADP-ribose) polymerase inhibitor, ABT-888, radiosensitizes malignant human cell lines under hypoxia

The chemo- and radioresponse of tumor cells can be determined by genetic factors (e.g., those that modify cell cycle arrest, DNA damage repair or cell death) and microenvironmental factors, such as hypoxia. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that rapidly recognizes and binds to DNA breaks to facilitate DNA strand break repair. Pre-clinical data suggest that PARP inhibitors (PARPi) may potentiate the effects of radiotherapy and chemotherapy. However, it is unclear as to whether PARPi are effective against hypoxic cells. We therefore tested the role for a novel PARPi, ABT-888, as a radiosensitizing agent under hypoxic conditions. Using human prostate (DU-145, 22RV1) and non-small cell lung (H1299) cancer cell lines, we observed that ABT-888 inhibited both recombinant PARP activity and intracellular PARP activity (86% to 92% decrease in all 3 cells lines following 2.5 microM treatment). ABT-888 was toxic to both oxic and hypoxic cells. When ABT-888 was combined with ionizing radiation (IR), clonogenic radiation survival was decreased by 40-50% under oxic conditions. Under acute hypoxia, ABT-888 radiosensitized malignant cells to a level similar to oxic radiosensitivity. To our knowledge, this is the first study to demonstrate that inhibition of PARP activity can sensitize hypoxic cancer cells and the combination of IR-PARPi has the potential to improve the therapeutic ratio of radiotherapy.

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]

Late Residual γ-H2AX Foci In Murine Skin are Dose Responsive and Predict Radiosensitivity In Vivo

Abstract Accurate biodosimetry is needed to estimate radiation doses received in vivo from accidental or unwarranted radiation exposures. We investigated the use of DNA repair foci (e.g. γ-H2AX) at late times after irradiation in vivo as a biodosimeter of initial ionizing radiation dose. Two radiosensitive strains (SCID and BALB/c) and two radioresistant strains (C57BL/6 and C3H/HeJ) were used to quantify γ-H2AX foci in a skin tissue microarray after doses of 1 to 10 Gy at early and late times after irradiation (1 and 7 days). Using a 3D quantitative immunofluorescence microscopy analysis, we observed a dose response for γ-H2AX foci for all strains at 30 min, 24 h and 7 days after irradiation. The numbers of residual foci were significantly different between each of the four strains and reflected the relative radiosensitivity in vivo. In comparing γ-H2AX focus and micronucleus formation after irradiation, we also observed association between the number of micronuclei and number of foci after 1 and 7 days between radiosensitive and radioresistant strains. We conclude that 3D image analysis of γ-H2AX in skin can be used to detect relative radiosensitivity based on late residual γ-H2AX foci. This technique may be a useful biodosimeter to determine dose at times up to 1 week after accidental or catastrophic radiation exposure in vivo.

Microscopic imaging of DNA repair foci in irradiated normal tissues

Purpose: It is now feasible to detect DNA double strand breaks (DSB) in tissues by measuring the induction and resolution of DNA repair foci, such as γ-H2AX, using immunofluorescent microscopy and digital image analysis. This review will highlight principal tools and approaches to tissue microscopy and analysis. It will also discuss the practical considerations of using microscopy in vitro and in vivo in measuring intranuclear foci following irradiation. Conclusions: Computer-based image analysis algorithms allow an objective and quantitative analysis of foci and protein-protein interactions using 3D confocal images. Finally, we review the literature in which DNA repair foci have been investigated as a biodosimeter or a biomarker of DNA repair in normal tissues.

WTp53 induction does not override MTp53 chemoresistance and radioresistance due to gain-of-function in lung cancer cells.

New molecular cancer treatment strategies aim to reconstitute wild-type p53 (WTp53) function in mutant p53 (MTp53)–expressing tumors as a means of resensitizing cells to chemotherapy or radiotherapy. The success of this approach may depend on whether MTp53 proteins are acting in a dominant-negative or independent gain-of-function mode. Herein, we describe an isogenic, temperature-sensitive p53 model (p53A138V) in p53-null human H1299 lung cancer cells in which WTp53 can be selectively coexpressed with a temperature-sensitive MTp53 allele (A138V) during initial DNA damage and subsequent DNA repair. Cells expressing MTp53 alone or coexpressing induced WTp53 and MTp53 were tested for p53 transcription, G1 and G2 cell cycle checkpoints, apoptosis, and long-term clonogenic survival following DNA damage. Transient transfection of WTp53 into H1299 cells, or shift-down of H1299-p53A138V stable transfectants to 32°C to induce WTp53, led to increased p21WAF1 expression and G1 and G2 arrests following DNA damage but did not increase BAX expression or apoptosis. In contrast, both transient and stable expression of the p53A138V mutant in p53-null H1299 cells (e.g. testing gain-of-function) at 37°C blocked p21WAF1 induction following DNA damage. Cell death was secondary to mitotic catastrophe and/or tumor cell senescence. Overexpression of WTp53 did not resensitize resistant MTp53-expressing cells to ionizing radiation, cisplatinum, or mitomycin C. Our results suggest that human MTp53 proteins can lead to resistant phenotypes independent of WTp53-mediated transcription and checkpoint control. This should be considered when using p53 as a prognostic factor and therapeutic target. [Mol Cancer Ther 2008;7(4):980–92]

The therapeutic ratio is preserved for radiotherapy or cisplatin treatment in BRCA2-mutated prostate cancers

Prostate cancers in patients with a mutation in BRCA2 have earlier disease onset and an aggressive course, often necessitating the use of systemic therapy. However, these tumours are DNA repair-defective and could respond favourably to Parp inhibitors or DNA-damaging agents, depending on the therapeutic ratio (ratio of tumour response to normal tissue toxicity). We describe 3 patients treated with precision radiotherapy or cisplatin who responded favourably to both agents, yet did not suffer undue toxicity. We review the concept of treating such patients with agents that are selectively toxic to repair-deficient tumours.