Ramunas Janavicius

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample

BackgroundPresently available flow cytometric methods of bromodeoxyuridine (BrdUrd) labelling do not provide information on the cell cycle time (TC) and the growth fraction (GF). In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling.MethodsThe proposed method is based on two assumptions: (1) the number of labelled cells traversing the cell cycle per unit time is constant and (2) the total number of labelled cells is constant throughout the cycle, provided that cells produced after division are excluded. The total numbers of labelled divided G1 cells, labelled divided S cells, labelled undivided S cells, and labelled undivided G2 cells were obtained for DNA histograms of BrdUrd-positive cells in a collected sample. These cell numbers were used to write equations to determine the durations of cell cycle phases, TC and GF. To illustrate the application of the proposed formulae, cell cycle kinetic parameters were analysed in solid SL2 tumours growing in DBA/2 mice and in human T-leukaemia Jurkat cells in culture.ResultsThe suitability of the proposed method for estimating durations of the cell cycle phases, TC and GF was demonstrated. TC in SL2 tumours was found to be relatively constant at 4 and 10 days after tumour implantation (20.3 ± 1.1 h and 21.6 ± 0.9 h, respectively). GF in tumours at day 10 was lower than GF at day 4 (54.2 ± 7.7% vs. 79.2 ± 5.9%, p = 0.0003). Approximate values of TC and GF of cultured Jurkat cells were 23.9 h and 79.3%, respectively.ConclusionThe proposed method is relatively simple and permits estimation of the cell cycle parameters, including TC and GF, from a single tumour sample after labelling with BrdUrd. We have shown that this method may be useful in preclinical studies, allowing estimation of changes in GF during growth of murine tumours. Experiments with human Jurkat cells suggest that the proposed method might also prove suitable for measurement of cell kinetics in human tumours. Development of suitable software enabling more objective interpretation of the DNA profile in this method would be desirable.

Haplotype analysis and ancient origin of the BRCA1 c.4035delA Baltic founder mutation

Uncertainty exists about the origin of BRCA1 c.4035delA mutation which is prevalent in Baltic countries, with the highest frequency being in Lithuania (53% of all BRCA1 mutations), although formal founder mutation analysis by haplotype has not yet been undertaken. In this study we genotyped 78 unrelated BRCA1 c.4035delA mutation carriers families from Lithuania, Latvia, Poland and Russia. The results from the haplotype analyses were used to estimate the age of the mutation. Using maximum likelihood methods we estimated that the mutation arose approximately 1550 years (62 generations of 25 years) ago (ca. 5th century) somewhere in the present territory of Lithuania, in the area inhabited by ancient Baltic tribes at that time. Our results show that this mutation gradually entered the gene pool in the neighboring countries.

Novel germline MSH2 mutation in lynch syndrome patient surviving multiple cancers

Lynch syndrome (LS) individuals are predisposed to a variety of cancers, most commonly colorectal, uterine, urinary tract, ovarian, small bowel, stomach and biliary tract cancers. The risk of extracolonic manifestations appears to be highest in MSH2 mutations carriers.We present a carrier case with a novel MSH2 gene mutation that clearly demonstrates the broad extent of LS phenotypic expression and highlights several important clinical aspects. Current evidence suggests that colorectal tumors from LS patients tend to have better prognoses than their sporadic counterparts, however survival benefits for other cancers encountered in LS are unclear.In this article we describe a family with a novel protein truncating mutation of c.2388delT in the MSH2 gene, particularly focusing on one individual carrier affected with multiple primary cancers who is surviving 25 years on. Our report of multiple primary tumors occurring in the 12-25 years interval might suggest these patients do not succumb to other extracolonic cancers, provided they are regularly followed-up.

BRCA1 testing in Lithuania.

Background The germline mutations in BRCA1/2 genes are the most significant and well characterized genetic risk factors for breast and/or ovarian cancer. Detection of mutations in these genes is an effective method of cancer prevention and early detection. Different ethnic and geographical regions may have different BRCA1 and BRCA2 mutation spectrum and prevalence due to founder effect. The population of Lithuania has over several centuries undergone limited mixing with surrounding populations and is mostly of indigenous Baltic origin, which is different from Slavs. The aim of our study was to asses full BRCA1/2 mutational profile in Lithuanian population.