Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells.</p

Non-homologous DNA end joining in normal and cancer cells and its dependence on break structures

DNA double-strand breaks (DSBs) are a serious threat to the cell, for if not or miss-repaired, they can lead to chromosomal aberration, mutation and cancer. DSBs in human cells are repaired via non-homologous DNA end joining (NHEJ) and homologous recombination repair pathways. In the former process, the structure of DNA termini plays an important role, as does the genetic constitution of the cells, through being different in normal and pathological cells. In order to investigate the dependence of NHEJ on DSB structure in normal and cancer cells, we used linearized plasmids with various, complementary or non-complementary, single-stranded or blunt DNA termini, as well as whole-cell extract isolated from normal human lymphocytes, chronic myeloid leukemia K562 cells and lung cancer A549 cells. We observed a pronounced variability in the efficacy of NHEJ reaction depending on the type of ends. Plasmids with complementary and blunt termini were more efficiently repaired than the substrate with 3' protruding single-strand ends. The hierarchy of the effectiveness of NHEJ was on average, from the most effective to the least, A549/ normal lymphocytes/ K562. Our results suggest that the genetic constitution of the cells together with the substrate terminal structure may contribute to the efficacy of the NHEJ reaction. This should be taken into account on considering its applicability in cancer chemo- or radiotherapy by pharmacologically modulating NHEJ cellular responses

A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

BACKGROUND: The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst approximately 8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs. CONCLUSIONS/SIGNIFICANCE: In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress

Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta.

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polbetaDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polbetaDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polbetaDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H(2)O(2) treatment, polbetaDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H(2)O(2), these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polbetaDN induced increase in residual gammaH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual gammaH2AX foci, indicating an escape from gammaH2AX-mediated DSB repair. In addition, we show that in the polbetaDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner