Abstract DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PKCS (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by γH2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation–induced and etoposide-induced DSB. NU7441 appreciably increased G2-M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use. (Cancer Res 2006; 66(10): 5354-62)
Abstract The aim of this study is to identify the underlying molecular genetic mechanisms of radiation induced breast carcinogenesis. Studies have shown that women exposed to ionising radiation at a young age are more at risk of developing breast cancer than older women exposed to the same level of radiation. Higher levels of estrogen are present in young women and estrogen has a known transforming effect on breast epithelial cells. One hypothesis suggests that radiation and estrogen synergise to drive breast epithelial cell transformation. We have developed an in vitro model of radiation-induced breast epithelial cell transformation in order to investigate genetic alterations associated with breast cell transformation. The immortalised, non-transformed breast epithelial cell line MCF-10A was exposed to fractionated doses of X-rays in the presence or absence of exogenous estrogen. We have shown that radiation treated cells display evidence of transformation, including loss of contact inhibition, increased cell invasion, disrupted acini formation and tumor formation in immunocompromised Rag2−/− γ−/− mice. Analysis of radiation treated cells by SNP array karyotyping identified focal gene deletion (EP300 and OCT-1) and amplification (c-MYC) which may be linked to radiation-induced breast cell transformation. Gene deletion and amplification was confirmed by Fluorescent In Situ Hybridization analysis and alterations in expression have been confirmed by western analysis. EP300 is a transcriptional co-activator and a putative tumor suppressor. Somatic mutations in EP300 have been found in solid tumours and the gene is located in a region of chromosome 22 affected by loss of heterozygosity in numerous cancers. Oct-1 encodes an octamer binding transcription factor that has been associated with regulating DNA damage response through interactions with BRCA1 and GADD45. c-MYC is a known proto-oncogene that encodes a transcription factor involved in cell proliferation, cell cycle regulation and apoptosis. Analysis of our in vitro model of radiation-induced breast epithelial cell transformation has identified genetic alterations that have established roles in mediating cellular response to DNA damage and which might be key events in the development of radiogenic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2011-5093
<div>Abstract<p>Poly(ADP-ribose) polymerase (PARP)-1 (EC 2.4.2.30) is a nuclear enzyme that promotes the base excision repair of DNA breaks. Inhibition of PARP-1 enhances the efficacy of DNA alkylating agents, topoisomerase I poisons, and ionizing radiation. Our aim was to identify a PARP inhibitor for clinical trial from a panel of 42 potent PARP inhibitors (<i>K</i><sub>i</sub>, 1.4–15.1 nmol/L) based on the quinazolinone, benzimidazole, tricyclic benzimidazole, tricyclic indole, and tricyclic indole-1-one core structures. We evaluated chemosensitization of temozolomide and topotecan using LoVo and SW620 human colorectal cells; <i>in vitro</i> radiosensitization was measured using LoVo cells, and the enhancement of antitumor activity of temozolomide was evaluated in mice bearing SW620 xenografts. Excellent chemopotentiation and radiopotentiation were observed <i>in vitro</i>, with 17 of the compounds causing a greater temozolomide and topotecan sensitization than the benchmark inhibitor AG14361 and 10 compounds were more potent radiosensitizers than AG14361. In tumor-bearing mice, none of the compounds were toxic when given alone, and the antitumor activity of the PARP inhibitor-temozolomide combinations was unrelated to toxicity. Compounds that were more potent chemosensitizers <i>in vivo</i> than AG14361 were also more potent <i>in vitro</i>, validating <i>in vitro</i> assays as a prescreen. These studies have identified a compound, AG14447, as a PARP inhibitor with outstanding <i>in vivo</i> chemosensitization potency at tolerable doses, which is at least 10 times more potent than the initial lead, AG14361. The phosphate salt of AG14447 (AG014699), which has improved aqueous solubility, has been selected for clinical trial. [Mol Cancer Ther 2007;6(3):945–56]</p></div>
4999 Enzyme-mediated repair of DNA double-strand breaks (DSBs) is a major mechanism of resistance to both ionizing radiation (IR) and drugs that cause DSBs as intermediates in repair processes. The major DNA DSB repair pathway in eukaryotes is nonhomologous end joining. An important component in this repair pathway is the DNA-dependent protein kinase (DNA-PK), whose catalytic subunit is a member of the phosphatidylinositol 3-kinase (PI 3-K)-related protein kinase (PIKK) family of enzymes. DNA-PK deficient cells are hypersensitive to IR and some DNA-damaging anticancer drugs, and inhibition of DNA-PK therefore represents a potential strategy for radio- and chemo-sensitization. NU7441 is a competitive and highly selective inhibitor of DNA-PK (IC 50 = 14 nM). The cellular specificity of NU7441 for DNA-PK was studied in V3 and V3-YAC cells, deficient and proficient in DNA-PK respectively. V3 cells were inherently more sensitive to IR and etoposide than V3-YAC cells and NU7441 increased the cytotoxicity of IR and etoposide in V3-YAC cells but not in V3 cells, confirming that DNA-PKcs is the cellular target of NU7441. Exposure of human colon cancer cell lines; SW620 and LoVo, to 1 μM NU7441 alone for 16 hr did not affect cell survival but did significantly enhance the cytotoxicity of IR, doxorubicin and etoposide. Survival of SW620 cells exposed to etoposide (1 μM), in combination with 1 μM NU7441 gave a dose modification ratio of 5. Similarly, NU7441 caused a 2.3 fold enhancement of doxorubicin (100 nM) and 7.3 fold of IR (5 Gy) cytotoxicity in SW620 cells. In LoVo cells the dose modification ratio of etoposide(1 μM), doxorubicin(100 nM) and IR (5 Gy) was 2.1, 10 and 5.5 fold respectively. NU7441 alone had no effect on cell cycle distribution, but accumulation in G2/M induced by exposure to IR (2 Gy) or the topoisomerase II poisons, doxorubicin (10 nM) or etoposide (0.1 μM) was increased 1.2 to 2 fold by NU7441 in SW620 cells. In vivo anti tumor efficacy studies indicate that inhibition of DNA-PK can enhance the effect of etoposide against SW620 tumour xenografts. Tumor growth delay following treatment with etoposide alone (10 mg/kg i.p. daily x5) to a RTV4 was 8 days and for etoposide plus NU7441 (10 mg/kg i.p daily x 5) was 11 days, representing a 60% enhancement of etoposide efficacy. NU7441 alone did not cause any measurable toxicity (max body weight loss = 4%) and only caused a marginal enhancement of etoposide toxicity ( max body weight loss = 12%). These experiments demonstrate that the cellular effects of NU7441 are specific for DNA-PKcs. Furthermore, at non cytotoxic concentrations in vitro and non toxic doses in vivo this compound is a potent chemo- and radiosensitizer in models of human colon cancer.
Ewing sarcoma and osteosarcoma represent the two most common primary bone tumours in childhood and adolescence, with bone metastases being the most adverse prognostic factor. In prostate cancer, osseous metastasis poses a major clinical challenge. We developed a preclinical orthotopic model of Ewing sarcoma, reflecting the biology of the tumour-bone interactions in human disease and allowing in vivo monitoring of disease progression, and compared this with models of osteosarcoma and prostate carcinoma. Human tumour cell lines were transplanted into non-obese diabetic/severe combined immunodeficient (NSG) and Rag2−/−/γc−/− mice by intrafemoral injection. For Ewing sarcoma, minimal cell numbers (1000–5000) injected in small volumes were able to induce orthotopic tumour growth. Tumour progression was studied using positron emission tomography, computed tomography, magnetic resonance imaging and bioluminescent imaging. Tumours and their interactions with bones were examined by histology. Each tumour induced bone destruction and outgrowth of extramedullary tumour masses, together with characteristic changes in bone that were well visualised by computed tomography, which correlated with post-mortem histology. Ewing sarcoma and, to a lesser extent, osteosarcoma cells induced prominent reactive new bone formation. Osteosarcoma cells produced osteoid and mineralised "malignant" bone within the tumour mass itself. Injection of prostate carcinoma cells led to osteoclast-driven osteolytic lesions. Bioluminescent imaging of Ewing sarcoma xenografts allowed easy and rapid monitoring of tumour growth and detection of tumour dissemination to lungs, liver and bone. Magnetic resonance imaging proved useful for monitoring soft tissue tumour growth and volume. Positron emission tomography proved to be of limited use in this model. Overall, we have developed an orthotopic in vivo model for Ewing sarcoma and other primary and secondary human bone malignancies, which resemble the human disease. We have shown the utility of small animal bioimaging for tracking disease progression, making this model a useful assay for preclinical drug testing.
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