Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

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Human breast cancer cell lines, as well as human breast cancer biopsies, possess specific high-affinity epidermal growth factor receptors (EGF-r). However, reports on the presence of EGF-r in human breast cancer biopsies are still controversial, especially concerning the relationship between EGF-r and other biological variables whose prognostic relevance is well known, such as the estrogen (ER) and progesterone (PgR) receptors. In the present study, the EGF-r content was estimated in a series of 136 unselected breast cancer primaries along with cytoplasmic (ERc) and nuclear (ERn) ER and cytoplasmic PgR. EGF-binding activity consisted of a single class of high-affinity binding sites (K_d = 0.55 nM) and ranged from 0 to 275 fmol/mg protein. We observed a strong inverse association between EGF-r and ERc (p < 0.001); in fact, about two thirds of the tumors were ERc-positive/EGF-r-negative or ERc-negative/EGF-r-positive. The same type of association was found between EGF-r and either ERn or PgR.Kendall’s rank correlation test confirmed that the EGF-r concentrations were correlated with the levels of ERc (τ = -0.291, p < 0.0001), ERn (τ = -0.27, p < 0.0005) and PgR (τ = -0.162, p < 0.01). The EGF-r content was significantly higher (p < 0.0001) in the ERc-negative tumors (72.6 ± 54.4 fmol/mg protein) as compared to the ERc-positive ones (33.0 ± 37.4 fmol/mg protein). Similarly, the subset of PgR-positive tumors was characterized by lower EGF-r mean concentrations when compared to PgR-negative cases (35.4 ± 54.4 vs. 63.8 ± 54.4 fmol/mg protein). These results confirm the previously described inverse relationship between EGF-r and steroid receptors. Moreover, they suggest the existence of an interaction between steroid hormones and EGF and support the need for further studies to better understand their respective roles in modulating breast cancer growth.

In the lizard Lacerta s. sicula sex steroids induce the synthesis of avidin in the oviduct in vivo (Botte, Segal & Koide, 1974). We report here evidence that these effects could be mediated, in vivo and in vitro, by RNA.Adult female Lacerta s. sicula were maintained in terraria at 28°C , and under a natural photoperiod. The lizards were ovariectomized and used 15 days after the operation. For the RNA preparation, three groups of ovariectomized females, each comprising 40 animals, were treated as follows: Group 1, four doses of 50 µg oestradiol dipropionate in 0-05 ml almond oil (every 3 days), administered intramuscularly; Group 2, treated with oestradiol as in Group 1, but only three times; on the fourth treatment given 0-5 mg progesterone in 0 5 ml almond oil; Group 3, injected with almond oil only.Three days after the last injection the oviducts and livers were removed and RNA was extracted by the method of Segal, Davidson & Wada (1965). When digested with ribonuclease (RNAase), it was found that more than 90% of the extracts were RNA. The avidin content of these preparations was negligible. These preparations were done twice.RNA from 5-6 oviducts or 2-3 livers of experimental animals was dissolved in 50 µ physiological saline and instilled into the oviducts of ovariectomized female lizards. For each test at least four different lizards were used. Forty-eight hours after the instillation, the animals were killed and the tubai sections of the oviducts were removed and assayed singly for avidin content.For in-vitro studies the tubai regions of the oviducts of lizards pretreated with oestradiol (Group 1) were used. Three minced tubai segments, 0-3 ml of the various RNA solutions (about 500 µg) and 500 000 i.u. penicillin were placed in 2 ml biotin-free Medium 199. The incubations were carried out at 28°C, for 4 h, in an atmosphere of 95% air: 5% C02. In parallel flasks, either physiological saline or RNA pretreated with RNAase was added instead of RNA solutions. After incubation, tissues and media were homogenized and assayed for avidin content.

Ionising radiation causes cell death through the induction of DNA damage, particularly double-stranded DNA (dsDNA) breaks. Evidence suggests that adenoviruses inhibit proteins involved in the DNA damage response (DDR) to prevent recognition of double-stranded viral DNA genomes as cellular dsDNA breaks. We hypothesise that combining adenovirus treatment with radiotherapy has the potential for enhancing tumour-specific cytotoxicity through inhibition of the DDR and augmentation of virus production. We show that EnAd, an Ad3/Ad11p chimeric oncolytic adenovirus currently being trialled in colorectal and other cancers, targets the DDR pathway at a number of junctures. Infection is associated with a decrease in irradiation-induced 53BP1 and Rad51 foci formation, and in total DNA ligase IV levels. We also demonstrate a radiation-associated increase in EnAd production in vitro and in a pilot in vivo experiment. Given the current limitations of in vitro techniques in assessing for synergy between these treatments, we adapted the plaque assay to allow monitoring of viral plaque size and growth and utilised the xCELLigence cell adhesion assay to measure cytotoxicity. Our study provides further evidence on the interaction between adenovirus and radiation in vitro and in vivo and suggests these have at least an additive, and possibly a synergistic, impact on cytotoxicity.