Įvadas. Radioterapija yra vienas iš pagrindinių vėžio gydymo metodų, taikomų daugiau kaip pusei onkologinių pacientų. Manoma, kad pagrindinė onkologinės ligos pasikartojimo ir nesėkmingo gydymo priežastis yra vėžinių ląstelių atsparumas radioterapijai. Viena iš pagrindinių atsparumo radioterapijai priežasčių yra vėžinių ląstelių gebėjimas išvengti apoptozės. Manoma, kad ląstelės ciklas taip pat yra vienas iš procesų, turinčių reikšmės radioatsparumui. Šio tyrimo tikslas – įvertinti skirtingų krūties vėžio ląstelių linijų atsparumą radioterapijai, lyginant jų gyvybingumą, ląstelių pasiskirstymą tarp ciklo fazių ir apoptozės intensyvumą. Metodika. MCF-7 ir MDA-MB-231 ląstelių gyvybingumas buvo ištirtas, naudojant kolonijų formavimo testą. Ląstelės ciklo analizei buvo dažomos propidžio jodidu ir analizuojamos, naudojant tėkmės citometrą Muse Cell Analyzer. Apoptozės intensyvumui nustatyti naudotas aneksinas V ir Guava PCA tėkmės citometras. Rezultatai. Šiame tyrime nustatėme, kad MDA-MB-231 ląstelių gyvybingumas po radioterapijos poveikio buvo didesnis, nei MCF-7 ląstelių. Ciklo analizė parodė, kad po radioterapijos poveikio MCF-7 ląstelių ciklo sustabdymas vyko G0/G1 fazėje, o MDA-MB-231 ląstelių – G2/M fazėje. Po radioterapijos poveikio MCF-7 ląstelėse apoptozė prasidėjo anksčiau ir buvo intensyvesnė, o MDA-MB-231 ląstelės reagavo vėliau ir turėjo uždelstą apoptozinį atsaką. Išvados. MDA-MB-231 ląstelės buvo atsparesnės radioterapijai, negu MCF-7 ląstelės.

IntroductionPatients with Philadelphia-negative (Ph-) myeloproliferative neoplasms: primary myelofibrosis, essential thrombocythemia and polycythemia vera; often develop thrombotic events. It was suggested that the genetic variants that are responsible for blood coagulation and elevated homocysteine level play causal role in the occurrence of thrombosis. Our aim was to evaluate the single nucleotide polymorphisms in PROS1, EPCR, PROC, MTHFR, MS genes and their associations with the risk of developing thrombosis as well as clinical characteristics in patients with myeloproliferative disorders.Material and methodsThe screening of PROS1 g.66847T>C, EPCR c.4678G>C, EPCR c.6936A>G, PROC c.565C>T, MTHFR c.677C>T, MTHFR c.1298A>C, MS c.2756A>G polymorphisms was performed at Lithuanian University of Health Sciences, Kaunas, Lithuania. The PCR-RFLP method was applied.ResultsAfter genotyping 88 patients with Ph- myeloproliferative disorders, the association was found between venous thrombosis and MTHFR c.1298A>C (p=0.019). Regression analysis revealed that carriers of PROS1 66847TC, EPCR 4678GC, 6936AG or GG, PROC 565CT or TT, MTHFR 677CT, MS 2756AG genotypes were associated with a lower risk of developing venous thrombosis. EPCR 6936AG genotype could be considered as a protective factor against arterial thrombosis. Genotypes PROC 565CT and 565TT were associated with a lower risk of decreased levels of MPV, 565TT carriers were less likely to develop arterial or venous thrombosis, compared with those carrying the CC or CT genotype.ConclusionsIt can be concluded that more research into these polymorphisms needs to be performed, since there are many conflicting results published regarding the complexity of the possible interactions between these gene variants and predisposition to thrombotic events.

The purpose of this study was to determine characteristics potentially related to NBS1 mutations and polymorphisms in young (≤50 years of age) breast cancer patients. Blood from 80 breast cancer patients was collected. NBS1 mutations c.657_661del, p.R215W, p.I171V, and polymorphisms c.8360G>C, c.30537G>C were genotyped by the PCR-RFLP method. Two-sided Chi-square test was used for univariate analysis and logistic regression analysis was used to evaluate the odds ratio. No carriers of the c.657_661del, p.R215W and p.I171V mutations were found. NBS1 c.8360G>C logistic regression analysis showed that GC and CC genotypes compared with GG genotype had decreased risk of low grade tumour, 2.885-fold (OR = 2.885, 95% CI 0.173–0.735, P = 0.005) and 2.186-fold (OR = 2.186, 95% CI 0.188–0.888, P = 0.024), respectively. 8360 CC genotype (OR = 3.034, 95% CI 0.156–0.778, P = 0.010) significantly increased the chances of HER2 amplification compared to GG genotype. NBS1 8360 GC genotype had a higher risk for breast cancer progression (OR = 1.673, 95% CI 0.233–0.915, P = 0.027). The homozygote 8360 CC carriers had approximately a six times higher risk for the disease progress (OR = 5.946, 95% CI 0.098–0.585, P = 0.002). The prevalence of triple negative breast cancer type was significantly higher in individuals with NBS1 8360 CC genotype (OR = 2.186, 95% CI 0.188–0.888, P = 0.024). Regarding c.30537G>C polymorphism, none of the genotypes had a significant influence on pathological characteristics. NBS1 gene c.8360G>C polymorphism might be associated with breast cancer aggressiveness in young breast cancer patients.

Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO4 as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.

Constitutively activated JAK/STAT signaling pathway is a common feature of the BCR/ABL-negative classic myeloproliferative neoplasms (MPN). JAK2 small-molecule inhibitors have been proven to be clinically efficacious; however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. There is a need for exploring new therapeutic strategies for MPN. Additional signaling systems, such as PI3K/Akt/mTOR and Hedgehog, are a potential treatment target. The aim of this study was to characterise and compare the effects of specific JAK/STAT, PI3K/Akt/mTOR, and Hedgehog signaling inhibitors in haematological cell cultures. JAK2 p.V617F mutated SET-2 and JAK2 wild-type UT-7 human cell lines were employed in our study. The effect of specific signaling pathway inhibitors was studied as time- and dose-response experiments. Viability was measured by trypan blue exclusion and alamarBlue assays. IC50 values were used to compare the effectiveness of inhibitors in decreasing cell viability. Independent sample t-test was used for statistical comparisons between experimental groups. p < 0.05 was considered significant. Our results indicate that all specific inhibitors progressively reduced the number of viable cells as the concentration and exposure duration increased. Inhibitors impaired the proliferation of JAK2 mutated cells at significantly lower doses compared to wild-type JAK2 cell line. These in vitro data indicate that JAK/STAT and alternative PI3K/Akt/mTOR and Hedgehog inhibitors have a potential anti-proliferative efficacy. Future studies, involving direct screening of PI3K/Akt/ mTOR, JAK/STAT, and Hedgehog signaling molecules activity, at gene and protein level in cell-based MPN model, are required.