The high-fat diet (HFD)-fed mouse is a model of obesity, impaired glucose tolerance, and insulin resistance. The main objective of this study was to elucidate the molecular mechanisms underlying the antidiabetogenic and weight-lowering effects of 17β-estradiol (E₂) in this mouse model. C57BL/6 female mice (8 wk old) were fed on a HFD for 10 mo. E₂, given daily (50 μg/kg sc) during the last month of feeding, decreased body weight and markedly improved glucose tolerance and insulin sensitivity. Plasma levels of insulin, leptin, resistin, and adiponectin were decreased. We demonstrated that E₂treatment decreased the expression of genes encoding resistin and leptin in white adipose tissue (WAT), whereas adiponectin expression was unchanged. Furthermore, in WAT we demonstrated decreased expression levels of sterol regulatory element-binding protein 1c (SREBP1c) and its lipogenic target genes, such as fatty acid synthase and stearoyl-CoA desaturase 1 (SCD1). In the liver, the expression levels of transcription factors such as liver X receptor α and SREBP1c were not changed by E₂treatment, but the expression of the key lipogenic gene SCD1 was reduced. This was accompanied by decreased hepatic triglyceride content. Importantly, E₂decreased the hepatic expression of glucose-6-phosphatase (G-6-Pase). We conclude that E₂treatment exerts antidiabetic and antiobesity effects in HFD mice and suggest that this is related to decreased expression of lipogenic genes in WAT and liver and suppression of hepatic expression of G-6-Pase. Decreased plasma levels of resistin probably also play an important role in this context.

Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal γH2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Krüppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC TICs are possible factors contributing to their therapy resistance, which may be exploited for DNA damage-sensitizing purposes.

Cells react differently to clustered and dispersed DNA double strand breaks (DSB). Little is known about the initial reaction to simultaneous induction of DSBs with different complexities. Here, we used live cell microscopy to analyse the behaviour of 53BP1-GFP (green fluorescence protein) foci formation at DSBs induced in U2OS cells by alpha particles, X-rays or mixed beams over a 75 min period post irradiation. X-ray-induced foci rapidly increased and declined over the observation interval. After an initial increase, mixed beam-induced foci remained at a constant level over the observation interval, similarly as alpha-induced foci. The average areas of radiation-induced foci were similar for mixed beams and X-rays, being significantly smaller than those induced by alpha particles. Pixel intensities were highest for mixed beam-induced foci and showed the lowest level of variability over time as compared to foci induced by alphas and X-rays alone. Finally, mixed beam-exposed foci showed the lowest level of mobility as compared to alpha and X-ray exposure. The results suggest paralysation of chromatin around foci containing clustered DNA damage.

The clonogenic cell survival assay is a basic method to study the cytotoxic effect of radiation and chemical toxins. In large experimental setups, counting of colonies by eye is tiresome and prone to bias. Moreover, it is often interesting to quantify the size of individual colonies. Such analyses are largely facilitated by computerised image analysis systems. Although a number of such systems exist, they all focus on enumerating colonies and not on analysing the colony size. We have developed a new software package for both counting colonies and plotting their size distributions. The software called count and Plot HIstograms of Colony Size (countPHICS) consists of two parts: (1) a macro written for ImageJ which analyses computerised images of cell culture dishes or 6-well plates, counts colonies, estimates their size and saves the results in a text file; (2) a program written with QT Creator which reads the text file, plots histograms of colony size distribution and fits the best function. The full program is freely available at: http://www.fuw.edu.pl/~bbrzozow/FizMed/countPHICS.html. In conclusion, our new publically available software will facilitate colony counting and provide additional information on the colony growth rate, which is relevant especially for radiosensitisation studies.