Genetic Diversity of EBV-Encoded LMP1 in the Swiss HIV Cohort Study and Implication for NF-Κb Activation

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1

Secretion of bioactive granulocyte-macrophage colony-stimulating factor by human colorectal carcinoma cells

Secretion of several cytokines by colorectal carcinoma cells has been substantiated. These do not include granulocyte-macrophage colony-stimulating factor (GM-CSF) thus far. We show that the supernatant of two human colorectal carcinoma cell lines, LS1034 and SW480, stimulates proliferation of GM-CSF-dependent M07e cells. The activity was constitutively secreted by LS1034 cells and could be induced by serum-free culture conditions in SW480 cells. Addition of a neutralizing anti-GM-CSF antibody completely inhibited this activity. Preabsorption with anti-GM-CSF antibody removed all M07e growth-stimulating activity from LS1034 and SW480 supernatant. Western blot analysis revealed the presence of GM-CSF in LS1034 supernatant. Our results indicate that human colorectal carcinoma cells secrete indeed biologically active GM-CSF

Blockade of the insulin-like growth-factor-I receptor inhibits growth of human colorectal cancer cells: evidence of a functional IGF-II-mediated autocrine loop

Insulin-like growth factors (IGFs) are potent proliferation stimulators for numerous tumor cells and often function as autocrine growth factors. We have previously shown that exogenous IGF-I and IGF-II enhance proliferation of colorectal carcinoma cells. The biological signal of both factors is transmitted through the IGF-I receptor (IGF-I-R). This receptor was expressed in 12/12 colorectal carcinoma cell lines tested. alpha IR3, a neutralizing monoclonal antibody (MAb) directed against the human IGF-I-R, inhibited proliferation in 7/12 lines (Caco-2, HT-29, LS411N, LS513, LS1034, WiDr and SW620), as reflected by a reduction of MTT conversion (19 to 42%), a decrease in cell number (39 to 72%) and an increase in doubling time (up to 2-fold). In addition, in 4 cell lines (Caco-2, LS513, LS1034, WiDr) alpha IR3 suppressed colony formation in methylcellulose (40 to 84%). Excess of exogenous IGF completely neutralized alpha IR3-mediated inhibitory effects. Northern blot analysis revealed abundant expression of 2 IGF-II transcripts of 5.0 and 4.3 kb in LS1034 cells. In addition, we observed that growth inhibition by alpha IR3 was correlated with a more differentiated phenotype. Our results suggest that growth of many colorectal carcinoma cell lines is regulated by autocrine IGF-II-mediated stimulation of the IGF-I-R

Interleukin 4 down-regulates expression of c-kit and autocrine stem cell factor in human colorectal carcinoma cells

Stem cell factor (SCF) is a cytokine which plays an important role in the development of precursor cells. We have investigated the expression of SCF and its receptor, the c-kit proto-oncogene, in human colorectal carcinoma cell lines. Using reverse transcription-PCR, we confirmed the expression of c-kit in two lines (151 74T and 151 034) and of SCF in 9 of 1 1 cell lines tested. In a Northern blot, a single transcript of 6.6 kb was detected for SCF mRNA. In addition, two lines (LS1 74T and HT29) synthesized SCF protein, as detected by Western blot analysis. SCF stimulated proliferation and colony formation of 151 74T in a dose-dependent manner up to 160%. A half-maximal effect was obtained with about 5.5 ng/ml of SCF under both growth conditions. 151 74T cells expressed the Mr 1 45,000 c-kit protein on the cell surface and a neutralizing anti-c-kit mAb inhibited colony formation of 151 74T by 40%. Interleukin 4 (11-4) completely inhibited SCF-induced proliferation of 151 74T cells. Interestingly, 11-4 induced an almost complete down-regulation of both c-kit and SCF expression in [Si 74T. Our findings suggest that in LS1 74T cells, an SCF-mediated autocrine loop is functional and that 11-4 down-regulates the expression of both the receptor and the ligand of this circuit

Growth inhibition of human colorectal-carcinoma cells by interleukin-4 and expression of functional interleukin-4 receptors

The growth-inhibitory effect of interleukin-4 (IL-4) was investigated in a panel of 7 human colorectal-carcinoma cell lines. In 5 cell lines (HT29, WiDr, LS411N, LS513, LS1034) a dose-dependent reduction of proliferation was documented. At 100 U/ml, IL-4 inhibited thymidine incorporation between 45 and 75% and MTT conversion (26 to 41%). The ability of LS513 and WiDr cells to form colonies after IL-4 treatment was reduced by 85 and 62% respectively. LS513 was the most sensitive cell line, with IL-4 inducing half-maximal inhibition at 5 to 6 U/ml. The inhibitory effect of IL-4 was completely neutralized by anti-IL-4 antibodies. Northern-blot analysis revealed the presence of IL-4-receptor (IL-4R) mRNA in all cell lines. The membrane expression of the 130-kDa IL-4R was assessed by FACS, utilizing an anti-IL-4R monoclonal antibody and was confirmed by biotinylated IL-4 binding. Our results attribute an important role for IL-4 as a negative regulator of colorectal-carcinoma cell growth, thus indicating a possible avenue for intervention in this disease

Why do red and dark-coloured cars lure aquatic insects? The attraction of water insects to car paintwork explained by reflection–polarization signals

We reveal here the visual ecological reasons for the phenomenon that aquatic insects often land on red, black and dark-coloured cars. Monitoring the numbers of aquatic beetles and bugs attracted to shiny black, white, red and yellow horizontal plastic sheets, we found that red and black reflectors are equally highly attractive to water insects, while yellow and white reflectors are unattractive. The reflection–polarization patterns of black, white, red and yellow cars were measured in the red, green and blue parts of the spectrum. In the blue and green, the degree of linear polarization p of light reflected from red and black cars is high and the direction of polarization of light reflected from red and black car roofs, bonnets and boots is nearly horizontal. Thus, the horizontal surfaces of red and black cars are highly attractive to red-blind polarotactic water insects. The p of light reflected from the horizontal surfaces of yellow and white cars is low and its direction of polarization is usually not horizontal. Consequently, yellow and white cars are unattractive to polarotactic water insects. The visual deception of aquatic insects by cars can be explained solely by the reflection–polarizational characteristics of the car paintwork