Activating Mutations in β-Catenin in Colon Cancer Cells Alter Their Interaction with Macrophages; the Role of Snail

Background

Tumor cells become addicted to both activated oncogenes and to proliferative and pro-survival signals provided by the abnormal tumor microenvironment. Although numerous soluble factors have been identified that shape the crosstalk between tumor cells and stroma, it has not been established how oncogenic mutations in the tumor cells alter their interaction with normal cells in the tumor microenvironment.

Principal Findings

We showed that the isogenic HCT116 and Hke-3 cells, which differ only by the presence of the mutant kRas allele, both stimulate macrophages to produce IL1β. In turn, macrophages enhanced Wnt signaling, proliferation and survival in both HCT116 and Hke-3 cells, demonstrating that signaling by oncogenic kRas in tumor cells does not impact their interaction with macrophages. HCT116 cells are heterozygous for β-catenin (HCT116^WT/MT), harboring one wild type (WT) and one mutant (MT) allele, but isogenic lines that carry only the WT (HCT116^WT) or MT β-catenin allele (HCT116^MT) have been generated. We showed that macrophages promoted Wnt signaling in cells that carry the MT β-catenin allele, but not in HCT116^WT cells. Consistent with this observation, macrophages and IL1β failed to stabilize Snail in HCT116^WT cells, and to protect these cells from TRAIL-induced apoptosis. Finally, we demonstrated that HCT116 cells expressing dominant negative TCF4 (dnTCF4) or HCT116 cells with silenced Snail failed to stimulate IL1β production in macrophages, demonstrating that tumor cells activate macrophages via a Wnt-dependent factor.

Significance

Our data demonstrate that oncogenic β-catenin mutations in tumor cells, and subsequent activation of Wnt signaling, not only trigger cell-intrinsic alterations, but also have a significant impact on the crosstalk of tumor cells with the tumor associated macrophages.     

Abscisic acid promotes root system development in birch tissue culture: a comparison to aspen culture and conventional rooting‐related growth regulators

The research aim was to assess the effects of the plant hormone abscisic acid (ABA) and the growth regulator paclobutrazol (PBZ) on root system development during the in vitro culture of different birch and aspen genotypes. The studied genotypes involved two aspen (Populus tremula and Populus tremuloides × P. tremula) and two silver birch (Betula pendula) trees, with one of the birches characterized by its inability to root in vitro. For experiments, apical shoot segments were cultured on nutrient medium enriched with either ABA or PBZ. Additionally, the analysis of the endogenous hormones in shoots developed on hormone‐free medium was conducted by high‐performance liquid chromatography. The endogenous concentration of auxin indole‐3‐acetic acid was much higher in the aspens than that in the birches, while the highest concentration of ABA was found in the root‐forming birch. The culturing of this birch genotype on medium enriched with ABA resulted in an increased root length and a higher number of lateral roots without any negative effect on either shoot growth or adventitious root (AR) formation, although these two processes were largely inhibited by ABA in the aspens. Meanwhile, PBZ promoted AR formation in both aspen and birch cultures but impaired secondary root formation and shoot growth in birches. These results suggest the use of ABA for the in vitro rooting of birches and PBZ for the rooting of aspens.     

Human papillomavirus DNA typing in the cervical specimens among women of Split and Dalmatian County

Human papillomavirus (HPV) infection is the main cause of cervical cancer, the second most common cancer in women worldwide. More than 200 types of HPV have been described, and within this range more than 40 types attack epithelium of genital tract. The types that were most commonly related to the development of cervical cancer are called high-risk types (HR HPV). There are very few studies about HPV prevalence in Croatia and there is an absolute lack of data for Split and Dalmatian County. Therefore, during a 6 month period, we routinely screened 570 women for HPV DNA at the Educational Public Health Institute of Split and Dalmatian County. HR HPV was detected in cervical exfoliated cells, by using Hybrid Capture 2 HPV DNA test. Out of the total number of tested women, 200 (35%) of them were positive to HR HPV Polymerase chain reaction (PCR) based assays were employed for HR HPV genotyping in positive specimens. The following frequency was observed: HPV 16 in 10%, HPV 18 in 6.1%, HPV 31 in 2.6%, HPV 33 in 1.9%, HPV 52 in 1.4%, HPV 59 in 0.7%, HPV 45 in 0.4% specimens, while 11.9% of tested specimens currently remained untyped. It is necessary to expand this study to a larger number of women, in order to better evaluate genital HPV types distribution among women in this region.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Oncogenic Ras promotes butyrate-induced apoptosis through inhibition of gelsolin expression

Activation of Ras promotes oncogenesis by altering a multiple of cellular processes, such as cell cycle progression, differentiation, and apoptosis. Oncogenic Ras can either promote or inhibit apoptosis, depending on the cell type and the nature of the apoptotic stimuli. The response of normal and transformed colonic epithelial cells to the short chain fatty acid butyrate, a physiological regulator of epithelial cell maturation, is also divergent: normal epithelial cells proliferate, and transformed cells undergo apoptosis in response to butyrate. To investigate the role of k-ras mutations in butyrate-induced apoptosis, we utilized HCT116 cells, which harbor an oncogenic k-ras mutation and two isogenic clones with targeted inactivation of the mutant k-ras allele, Hkh2, and Hke-3. We demonstrated that the targeted deletion of the mutant k-ras allele is sufficient to protect epithelial cells from butyrate-induced apoptosis. Consistent with this, we showed that apigenin, a dietary flavonoid that has been shown to inhibit Ras signaling and to reverse transformation of cancer cell lines, prevented butyrate-induced apoptosis in HCT116 cells. To investigate the mechanism whereby activated k-ras sensitizes colonic cells to butyrate, we performed a genome-wide analysis of Ras target genes in the isogenic cell lines HCT116, Hkh2, and Hke-3. The gene exhibiting the greatest down-regulation by the activating k-ras mutation was gelsolin, an actin-binding protein whose expression is frequently reduced or absent in colorectal cancer cell lines and primary tumors. We demonstrated that silencing of gelsolin expression by small interfering RNA sensitized cells to butyrate-induced apoptosis through amplification of the activation of caspase-9 and caspase-7. These data therefore demonstrate that gelsolin protects cells from butyrate-induced apoptosis and suggest that Ras promotes apoptosis, at least in part, through its ability to down-regulate the expression of gelsolin.     

Phytoseiid mites in protected crops: the effect of humidity and food availability on egg hatch and adult life span of Iphiseius degenerans, Neoseiulus cucumeris, N. californicus and Phytoseiulus persimilis (Acari: Phytoseiidae)

The effect of relative humidity on egg hatch success for Iphiseius degenerans, Neoseiulus californicus and N. cucumeris was described by a binomial model with a parallel slope. The shape of the response differed for Phytoseiulus persimilis and a model with separate parameters gave a significantly better fit. Fitted response curves showed that I. degenerans, N. cucumeris, N. californicus and P. persimilis were ranked by decreasing tolerance to low humidity, with egg mortalities of < 0.5, 3, 12 and 16% respectively at 75-80% RH at 20 degrees C. Egg stage duration for I. degenerans and N. cucumeris was unaffected over the range 60-82% RH. For N. californicus and P. persimilis egg duration was significantly longer at 60 and 70% than for either 82 or 90% RH. No effect of relative humidity was found on the mean life span of adult females when food was available continuously to the mites. N. californicus lived significantly longer (58 days after the first egg was laid) than the other species. No significant difference was observed in mean life span between adult females of I. degenerans and N. cucumeris (25 and 28 days respectively). The mean life span of adult female P. persimilis (19 days) was significantly shorter than the other species. In the absence of both food and water, the survival of adult female mites was reduced to 2-4 days. Survival time was at least doubled when free water was available in the absence of food. Mean survival of adult female mites with water but without food was 10 days for N. cucumeris, 18 days for N. californicus, 6 days for P. persimilis and 4 days for I. degenerans. Survival of adult female N. cucumeris and N. californicus was increased significantly, to 20 and 22 days respectively, when fungal hyphae were present along with water but in the absence of other food.     

Make love to forget: two cases of transient global amnesia triggered by sexual intercourse

Transient global amnesia (TGA) is characterized by a sudden onset and by a typical resolution within several hours. Several precipitating events have been proposed: physical exertion, emotional experiences, etc. The aim of this paper was to present two cases of TGA triggered by sexual intercourse and to suggest a possible mechanism for the development of TGA. In both patients, clinical examination revealed elevated blood pressure. Laboratory examinations and brain CTs were normal. EEG demonstrated diffuse dysrhythmia and slow spike-waves, respectively. SPECT revealed hypoperfusion in the left frontal and right medial temporal regions. Various explanations of the mechanism of TGA are discussed. Based on the observed hypoperfusion in the medial temporal regions, a new hypothesis is advanced, suggesting the possibility that TGA occurs due to a pathologically changed or less adaptable anterior chorioid artery, initially constricted by hypotension following a blood shift from the center towards periphery.