Błażej Rubiś

Bystander senescence in human peritoneal mesothelium and fibroblasts is related to thrombospondin-1-dependent activation of transforming growth factor-β1.

Senescence bystander effect refers to a phenomenon in which senescent cells elicit the development of senescence phenotype in their nearby young counterparts. In this paper we examined the mechanism of senescence bystander effect triggered by senescent human peritoneal mesothelial cells (HPMCs) in proliferating HPMCs and peritoneal fibroblasts (HPFBs). The results showed that conditioned medium (CM) derived from senescent HPMCs elicited a senescence response (growth inhibition coupled with increased expression of senescence-associated β-galactosidase and accumulation of histone γ-H2A.X) in either early-passage HPMCs or HPFBs. Samples of CM from senescent HPMCs contained increased amounts of numerous soluble mediators of which only transforming growth factor-β1 (TGF-β1) was able to induce senescence phenotype in the both types of peritoneal cells, likely through an induction of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (MAPK). At the same time, senescent HPMCs released increased amounts of thrombospondin-1 (TSP-1), a major activator of TGF-β1. Significantly, TSP-1 itself was unable to induce senescence phenotype in HPMCs or in HPFBs. The experiments employing anti-TSP-1 antibodies and specific TSP-1 blocking peptide revealed that neutralization of TSP-1 in CM prevented TGF-β1-dependent development of senescence phenotype. Collectively, our findings indicate that senescent HPMCs exhibit senescence-promoting activity toward neighboring young cells (HPMCs and HPFBs), and this effect is, at least partly, related to TSP-1-dependent activation and further ROS- and p38 MAPK-related activity of TGF-β1.

Study of ABCB1 polymorphism frequency in breast cancer patients from Poland

BACKGROUND The accumulation of mutagenic substances in the human body may result in DNA metabolism disruption followed by carcinogenesis. As a consequence of mutations in the genes coding for transmembrane protein pumps, the intracellular concentration of xenobiotics may significantly increase. This, in turn, may provoke altered risk for cancer development. The gene known to be the most relevant in the transport of numerous compounds is ABCB1 (also known as MDR1). Numerous mutations and polymorphisms that affect the encoded proteins (PgP) function were identified in this gene. The aim of the study was to define the frequency of 2677G>A,T and 3435C>T polymorphisms in a population of Polish breast cancer patients and to estimate their contribution to cancer development. METHODS The polymorphism frequency analysis (209 patients vs. 202 control subjects) was performed either by allele-specific amplification (2677G>A,T) or by restriction fragment length polymorphism (RFLP) using the SAU3AI restriction enzyme (3435C>T) followed by verification with hybridization probe assays in a Real-Time system and sequencing. RESULTS In the control group the frequency of individual 2677 genotypes was: wild homozygous GG = 34%, heterozygous G/T or G/A = 52.5% and variant homozygous AA or TT = 13.5%, while the genotype frequency in the group of studied patients was 43.5, 44.5 and 12%, respectively. In the control group, the frequency of individual 3435 genotypes was: CC = 25.4%, CT = 50.2%, TT = 24.4%, while the genotype frequency in the group of studied patients was 23, 46 and 31%, respectively. CONCLUSION Thus, no significant differences in the studied polymorphism frequencies were observed. It is then suggested that the studied polymorphisms, although probably good candidates in other tissue cancer types, might not be good predictive factors in breast cancer risk or development in Caucasians.

Universal Real-Time PCR-Based Assay for Lentiviral Titration

Lentiviral vectors are efficient vehicles for stable gene transfer in both dividing and non-dividing cells. This feature among others makes lentiviral vectors a powerful tool in molecular research. However, the use of lentiviruses in research studies and clinical trials requires a precise and validated titration method. In this study, we describe a qPCR-based approach for estimation of lentiviral vector titer (pLV-THM-GFP). The use of WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) and albumin genes as templates for an SYBR green-based real-time qPCR method allows for a rapid, sensitive, reproducible, and accurate assessment of lentiviral copy number at an integrated lentiviral DNA level. Furthermore, this optimization enables measurement of lentiviral concentration even in very poor quality and small quantity material. Consequently, this approach provides researchers with a tool to perform low-cost assessment with highly repeatable results.

Oleanolic acid derivative methyl 3,11-dioxoolean-12-en-28-olate targets multidrug resistance related to ABCB1

Multidrug resistance (MDR) in leukemia patients is a great incentive to the development of new drugs. In a search for potential multidrug resistance modulators we tested a group of oleanolic acid (OA) analogues modified at C-3, C-11, C-12 and C-28 using an experimental model consisting of three human acute lymphoblastic leukemia cell lines (CCRF-CEM and the multidrug resistant sublines CCRF-VCR1000 and CCRF-ADR5000). The most effective compound, methyl 3,11-dioxoolean-12-en-28-olate (DIOXOL) was more potent in cell viability inhibition than its precursor - OA, and showed similar or even higher activity in the drug resistant than in the wild-type cells. Resistance factor (RF) values obtained for CCRF-VCR1000 and CCRF-ADR-5000 cells using MTT assay were 0.7 and 0.8 (24 h of treatment) and after 72 h of treatment 0.9 and 1.1, respectively. Moreover, 5 μM DIOXOL significantly reduced the expression of the ABCB1 gene in MDR cells by around 30%, and also decreased the level of P-gp protein. Compared to untreated control cells, DIOXOL treatment resulted in a significant P-gp decrease (30% in CCRF-ADR5000 and 50% in CCRF-VCR1000), that was detected by western blot and confirmed by flow cytometry analysis. Moreover, DIOXOL (at 10 μM) significantly inhibited P-gp transport function by more than twofold comparing to control, untreated cells that was demonstrated using rhodamine 123-based functional test. The compound exhibited synergistic activity with ABCB1 substrate - adriamycin in CCRF-VCR1000 cells, indicating partial but significant MDR reversing ability.

Proapoptotic activity and ABCC1-related multidrug resistance reduction ability of semisynthetic oleanolic acid derivatives DIOXOL and HIMOXOL in human acute promyelocytic leukemia cells.

One of the main problems of present-day oncology is the ability of neoplastic cells to develop different mechanisms of resistance to chemotherapeutic agent. A natural compound oleanolic acid (OA) was found to be active against many types of neoplastic cells. This paper examines the influence of eight semisynthetic oleanolic acid derivatives on drug-sensitive human acute promyelocytic leukemia cell line HL-60 and its multidrug resistant subline ABCC1 overexpressing HL-60/AR. Viability inhibition, proapoptotic activity, as well as influence on the ABCC1 gene expression level, ability to inhibit the transport function of multidrug resistance associated protein 1 (ABCC1) and to alter its level by the tested compounds, were evaluated. The most potent compounds were DIOXOL (methyl 3,11-dioxoolean-12-en-28-oate) and HIMOXOL (methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate). DIOXOL was most efficient in inducing apoptosis of HL-60 cells. It activated both intrinsic and extrinsic pathways of apoptotic cell death. Proapoptotic properties of DIOXOL were probably related to the significant decrease of p65 NFκB level and inhibition of its translocation to the nucleus. In turn, HIMOXOL was the most potent compound against resistant HL-60/AR cells. It inhibited ABCC1 transport function (short time response) and decreased the level of ABCC1 protein (long time response) as a result of reduction of ABCC1 expression.

Telomere shortening in Down syndrome patients--when does it start?

Down syndrome (DS) is one of the most common aneuploidy. In general population, its prevalence is 1:600-1:800 live births. It is caused by a trisomy of chromosome 21. DS is phenotypically manifested by premature aging, upward slant to the eyes, epicanthus, flattened face, and poor muscle tone. In addition to physical changes, this syndrome is characterized by early onset of diseases specific to old age, such as Alzheimers disease, vision and hearing problems, and precocious menopause. Since DS symptoms include premature aging, the shortening of telomeres might be one of the markers of cellular aging. Consequently, the aim of the study was to determine the length of the telomeres in leukocytes from the blood of juvenile patients with DS (n=68) compared to an age-matched control group (n=56) and also to determine the diagnostic or predictive value for this parameter. We show that, for the first time, in juveniles, the average relative telomere length in studied subjects is significantly longer than in the control group (50.46 vs. 40.56, respectively arbitrary units [AU]; p=0.0026). The results provide interesting basis for further research to determine the causes and consequences of telomere maintaining and the dynamics of this process in patients with DS.

Serum from patients with chronic obstructive pulmonary disease induces senescence-related phenotype in bronchial epithelial cells

Chronic obstructive pulmonary disease (COPD) is a risk factor for the development of lung cancer (LC). The mechanism of interplay between both diseases remains poorly recognized. This report examines whether COPD may cause a senescence response in human bronchial epithelial cells (HBECs), leading to the progression of LC in a senescence-dependent manner. The results show that HBECs exposed to serum from COPD patients manifest increased expression of markers of cellular senescence, including senescence-associated β-galactosidase (SA-β-Gal), histone γ-H2A.X, and p21, as compared to the serum of healthy donors. This effect coincides with an increased generation of reactive oxygen species by these cells. The clinical analysis demonstrated that COPD may cause the senescence, independently on smoking status and disease severity. The concentrations of CXCL5, CXCL8/IL-8 and VEGF were higher in conditioned medium (CM) harvested from HBECs after exposure to COPD serum as compared to controls. In addition, CM treated with serum from COPD patients stimulated adhesion of A549 cancer cells to HBECs, as well as accelerating cancer cell proliferation and migration in vitro. Collectively, these findings indicate that COPD may induce senescence-like changes in HBECs and thus enhance some processes associated with the progression of lung cancer.

Strain-dependent mutational effects for Pepino mosaic virus in a natural host

BackgroundPepino mosaic virus (PepMV) is an emerging plant pathogen that infects tomatoes worldwide. Understanding the factors that influence its evolutionary success is essential for developing new control strategies that may be more robust against the evolution of new viral strains. One of these evolutionary factors is the distribution of mutational fitness effect (DMFE), that is, the fraction of mutations that are lethal, deleterious, neutral, and beneficial on a given viral strain and host species. The goal of this study was to characterize the DMFE of introduced nonsynonymous mutations on a mild isolate of PepMV from the Chilean 2 strain (PepMV-P22). Additionally, we also explored whether the fitness effect of a given mutation depends on the gene where it appears or on epistatic interactions with the genetic background. To address this latter possibility, a subset of mutations were also introduced in a mild isolate of the European strain (PepMV-P11) and the fitness of the resulting clones measured.ResultsA collection of 25 PepMV clones each containing a single nucleotide nonsynonymous substitution was created by site-directed mutagenesis and the fitness of each mutant was determined. PepMV-P22 genome showed a high degree of robustness against point mutations, with 80% of mutations being either neutral or even beneficial and only 20% being deleterious or lethal. We found that the effect of mutations strongly depended on the gene in which they were introduced. Mutations with the largest average beneficial effects were those affecting the RdRp gene, in contrast to mutations affecting TGB1 and CP genes, for which the average effects were deleterious. Moreover, significant epistatic interactions were observed between nonsynonymous mutations and the genetic background, meaning that the effect of a given nucleotide substitution on a particular genomic context cannot be predicted by knowing its effect in a different one.ConclusionsOur results indicated that PepMV genome has a surprisingly high robustness against mutations. We also found that fitness consequences of a given mutation differ between the two strains analyzed. This discovery suggests that the strength of selection, and thus the rates of evolution, vary among PepMV strains.

Contribution of ABCB1 gene polymorphisms to breast cancer cells response to chemotherapy

Każdego roku odnotowuje się 11 000 nowych przypadków zachorowań na raka piersi, a prawie połowa chorych umiera. Tak wysoka umieralność może być spowodowana zbyt późnym rozpoznaniem choroby, brakiem specyficznych i skutecznych leków, a także wystąpieniem zjawiska oporności wielolekowej (multiple drug resistance – MDR) w komórkach nowotworowych. Podstawowym mechanizmem wywołującym to zjawisko jest nadekspresja i zwiększona aktywność transporterów błonowych, z których większość należy do nadrodziny transporterów ABC (ATP binding casette). Spośród tych białek najlepiej poznanym jest glikoproteina P, produkt ekspresji genu ABCB1, odpowiedzialna m.in. za usuwanie ksenobiotyków i leków poza obszar komórki. Szerokie spektrum substratowe glikoproteiny P obejmuje: ksenobiotyki oraz leki cytotoksyczne, inhibitory białek, immunosupresanty, steroidy, statyny, blokery kanału wapniowego, beta-blokery, leki antyhistaminowe, przeciwdepresyjne oraz przeciwwymiotne. W sekwencji genu ABCB1 opisano szereg polimorfizmów, spośród których największe znaczenie kliniczne mają trzy – zamiana cytozyny na tyminę w pozycji 1236 egzonu 12 (C1236T, rs1128503), zamiana w egzonie 21 G2677A/T (rs2032582) oraz w egzonie 26 C3435T (rs1045642). Występowanie tych polimorfizmów może wpływać na zmianę ekspresji genu ABCB1 (C3435T), strukturę glikoproteiny P (G2677T/A) czy zdolność wiązania się białka z substratem (C1236T). Jak się podejrzewa, obecność tych polimorfizmów może w efekcie modyfikować odpowiedź komórek nowotworowych na chemioterapię. Niniejsza praca opisuje modulujący wpływ polimorfizmów genu ABCB1 na odpowiedź komórek raka piersi na chemioterapię. Słowa kluczowe: rak piersi, glikoproteina P, chemioterapia.