Isabel Soto-Cruz

A role for galectin-3 in CD13-mediated homotypic aggregation of monocytes

Abstract We recently reported that anti-CD13 mAbs induce homotypic aggregation of monocytic cells. This phenomenon is signal transduction dependent and does not require CD13 aminopeptidase activity. Since CD13 is heavily glycosylated and a member of the galectin family (galectin-4) has been shown to associate with CD13 in the intestinal epithelium, we hypothesized that CD13-mediated aggregation might proceed through a carbohydrate-dependent mechanism involving galectin-3, the most highly expressed galectin on monocytes. We report here that lactose and anti-galectin-3 antibodies completely abrogate homotypic aggregation induced by anti-CD13 antibodies. Furthermore, galectin-3 co-immunoprecipitates with CD13 from resting U-937 cells and this association decreases during the aggregation process, a phenomenon that may have functional implications. Together, the results presented here point to a key role for galectin-3 in CD13-mediated homotypic aggregation of monocytic cells.

Interferon gamma induces actin polymerization, Rac1 activation and down regulates phagocytosis in human monocytic cells

IFNγ is a potent activator and IL-10 a powerful inhibitor of macrophage functions. However, neither all cellular functions are enhanced by IFNγ nor IL-10 inhibits all cellular responses. Thus, FcγRs-mediated phagocytosis in monocyte-derived macrophages (MDM) increases after IL-10 treatment, and decreases after treatment with IFNγ, although both IL-10 and IFNγ up regulate FcγRI expression. In this work we investigated the effect of IFNγ and IL-10 on phagocytic signaling by FcγRs in MDM. Treatment with IFNγ diminished phagocytosis of IgG-opsonized SRBC (IgG-SRBC) while treatment with IL-10 increased it. These opposite effects cannot be attributed to changes in FcγR expression induced by each cytokine. Early biochemical responses mediated by FcγRs were distinctly affected by cytokine treatment. Syk phosphorylation and the rise in [Ca(2+)](i) were higher after IL-10 treatment, whereas IFNγ treatment also increased Syk phosphorylation but had no effect on the rise in [Ca(2+)](i). IFNγ treatment led to increased basal levels of F-actin and this effect correlated with the decrease in phagocytosis of both IgG-SRBC and non-opsonized Escherichia coli. IL-10 did not alter F-actin basal levels, and enhanced the phagocytosis of E. coli and IgG-SRBC. The level of F-actin reached after IFNγ treatment was not further increased after stimulation with IgG-SRBC or CCL5, whereas MDM treated with IL-10 showed a slightly higher response than control cells to CCL5. IFNγ increased Rac1-GTP levels. Inhibition of PI3K with LY294002 prevented IFNγ-mediated actin polymerization. Our data suggest that IFNγ induces a higher basal level of F-actin and activation of Rac1, affecting the response to stimuli that induce cytoskeleton rearrangement such as phagocytic or chemotactic stimuli.

Expression of MICA, MICB and NKG2D in human leukemic myelomonocytic and cervical cancer cells

BackgroundCancer cells are known to secrete the stress molecules MICA and MICB that activate cytotoxicity by lymphocytes and NK cells through their NKG2D receptor as a mechanism of immunological defense. This work was undertaken to evaluate if cancer cells can also express this receptor as a possible mechanisms of depletion of MIC molecules and thus interfere with their immune recognition.MethodsMyelomonocytic leukemic (TPH-1 and U-937) and cervical cancer (CALO and INBL) cell lines were evaluated by Western Blot, ELISA, flow cytometry and immunocytochemistry to evaluate their capacity to express and secrete MICA and MICB and to be induced to proliferate by these molecules as well as to express their receptor NKG2D. Statistical analysis was performed by two-way ANOVA for time course analysis and Students t-test for comparison between groups. Values were considered significantly different if p < 0.05.ResultsTHP-1 and U-937 produce and secrete the stress MICA and MICB as shown by Western Blot of lysed cells and by ELISA of their conditioned media. By Western Blot and flow cytometry we found that these cells also express the receptor NKG2D. When THP-1 and U-937 were cultured with recombinant MICA and MICB they exhibited a dose dependent induction for their proliferation. CALO and INBL also produce MICA and MICB and were induced to proliferate by these stress molecules. By Western Blot, flow cytometry and immunocytochemistry we also found that these cells express NKG2D.ConclusionsOur novel results that tumor cells can simultaneously secrete MIC molecules and express their receptor, and to be induced for proliferation by these stress molecules, and that tumor epithelial cells can also express the NKG2D receptor that was thought to be exclusive of NK and cytotoxic lymphocytes is discussed as a possible mechanism of immunological escape and of tumor growth induction.

Evidence that cervical cancer cells secrete IL-2, which becomes an autocrine growth factor.

We present evidence that cervical cancer cells express a functional IL-2 receptor (IL-2R). In fact, by RT-PCR we obtained that the IL-2R is present in CALO, and INBL cells, and that it consisted of the alphaIL-2R, betaIL-2R, and gammaIL-2R chains. We also found that IL-2 is a growth factor for these cell lines, and unexpectedly that CALO and INBL themselves being cancer cells produce, and secrete IL-2. Antibodies against the alpha and beta subunits of the IL-2R inhibited cell proliferation thus hinting to a cell growth dependency on this factor. Our results thus provide evidence that the IL-2R on cervical cancer cells is part of an autocrine mechanism for its growth to the extent that, like lymphocytes, they produce and become partially dependent on this growth factor. We think that in view of our results caution should be taken when IL-2 is being considered for cancer therapy; in particular when the patients cancer cells present the IL-2R, because as indicated by our results, the use of this factor could promote tumor growth. Finally, the possible implications of the expression of both IL-2, and IL-2R on cervical cancer cells on the immune escape mechanism of tumor cells are discussed.

Analysis of a p53 mutation associated with cancer susceptibility for biochemistry and genetic laboratory courses

We have devised and implemented a module for an upper division undergraduate laboratory based on the amplification and analysis of a p53 polymorphism associated with cancer susceptibility. First, students collected a drop of peripheral blood cells using a sterile sting and then used FTA cards to extract the genomic DNA. The p53 region is then PCR amplified, and the PCR products are digested with the BstUI enzyme to detect the 72 codon polymorphism. Polyacrylamide gel electrophoresis is used to resolve the PCR products, and the results are statistically analyzed in the context of human population genetics. Blood samples in FTA cards were also collected from 50 women to detect the mutation in a wide range of ages and assess its relationship to familial cancer susceptibility. This module enables students to use materials and methods that are routinely used by scientific researchers to analyze polymorphisms. Therefore, it can be used for laboratory exercises in traditional biochemistry curricula as well as in the growing field of genomic science and education.

Analysis of Proteins Binding to the ITAM Motif of the β-Subunit of the High-Affinity Receptor for IgE (FcεRI)

Aggregation of the multichain (α β γ2) high-affinity IgE receptor (Fcε RI) initiates a signaling cascade that results in the release of allergic mediators. The cytoplasmic tails of the Fcε RI-β and -γ subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the γ ITAM mediates activation of Syk kinase and is sufficient for triggering the responses induced by Fcε RI crosslinking. Phosphorylation of the β ITAM is insufficient to mediate cell activation. The rat β ITAM contains three tyrosines (Tyr218, Tyr224, and Tyr228) with an intermediate noncanonical tyrosine. Synthetic peptides based on the ITAM of the Fcε RI-β subunit were used to investigate the role of each phosphotyrosine in the binding of signaling proteins to this motif. Among the proteins that bind to phosphorylated β ITAM are Syk, Grb2, Shc, SHIP, and SHP-1, and binding does not depend on previous cell activation. Nonphosphorylated peptides do not bind these proteins. Syk binding to β -peptides is dependent on the number and position of phosphotyrosines in the ITAM. Phosphorylation of Tyr218 seems to be most important for Syk binding. Recruitment of Syk and other signaling proteins to the β -subunit might be important for its amplifier role.

IL-2 Enhances Cervical Cancer Cells Proliferation and JAK3/STAT5 Phosphorylation at Low Doses, While at High Doses IL-2 Has Opposite Effects

The IL-2R signaling is critical for normal lymphocyte proliferation. However, the role of the IL-2 signaling in cervical cancer is not yet fully understood. We show that in IL-2R-expressing cervical cancer cells, JAK1 molecules are not phosphorylated. At low doses of IL-2, the constitutive phosphorylation of JAK3 and STAT5 increases in the tumor cells and decreases in lymphocytes, whereas the opposite occurs at high doses of IL-2. Using AG-490, the activation of JAK3 and the proliferation of cervical cancer cells were inhibited. We describe differences in the response of molecules downstream the IL-2R in lymphocytes and tumor cells.

The Tyrphostin B42 Inhibits Cell Proliferation and HER-2 Autophosphorylation in Cervical Carcinoma Cell Lines

The HER family receptors have an important role controlling cell growth and differentiation. Although the activity of the HER-2 receptor is strictly controlled in normal cells, its overexpression plays a pivotal role in transformation and tumorigenesis. Constitutive phosphorylation of HER-2 protein has been implicated in conferring uncontrolled growth to mammary cancer cells, and to a lesser extent, with adenocarcinoma of uterus, cervix, fallopian tube, and endometrium. This study addresses the role of HER-2 in cervical carcinoma. Firstly, we demonstrate the presence of HER-2 protein expression by flow cytometry in two new cervical carcinoma cell lines CALO and INBL. Secondly, we use the specific tyrosine kinase inhibitors, Tyrphostins to examine HER-2 regulation by the crystal violet assay. Thirdly, we use western blot analysis to assess the state of HER-2 phosphorylation. The most efficient agent, Tyrphostin B42, known as an inhibitor of epithelial growth factor receptor, arrested cervical carcinoma cell lines growth in vitro at micromolar concentrations within 72 h of application. Tyrphostin B42 inhibited the HER2 signal-regulated kinase pathway, as observed by the reduction in the phosphorylated forms of HER2. The loss of phosphorylated forms of HER2 at early time points after Tyrphostin B42 application was associated with suppression of cell growth. Thus, the inhibition of the proliferation of our cervical carcinoma cell lines by Tyrphostin B42 is associated with inhibition of HER2 protein kinase signal.

Pleiotropic Effects of IL-2 on Cancer: Its Role in Cervical Cancer

IL-2 receptor (IL-2R) signalling is critical for normal lymphocyte proliferation, but its role in cervical cancer is not fully understood. The receptor is composed of three chains: IL-2α, IL-2β, and IL-2γ. Intracellular signalling is initiated by ligand-induced heterodimerization of the IL-2β and IL-2γ chains, resulting in the activation of multiple intracellular kinases. Recently, IL-2R was shown to be expressed on nonhaematopoietic cells, especially on several types of tumour cells. However, the function of this receptor on malignant cells has not been clearly defined. The expression of IL-2R and the production of IL-2 in cervical cancer cells have been documented as well as expression of molecules of the JAK-STAT pathway. In the current review we have highlighted the differences in the responses of molecules downstream from the IL-2R in normal lymphocytes and tumour cells that could explain the presence of tumour cells in an environment in which cytotoxic lymphocytes also exist and compete and also the effect of different concentrations of IL-2 that could activate effector cells of the immune system cells, which favour the elimination of tumour cells, or concentrations that may promote a regulatory microenvironment in which tumour cells can easily grow.

Detection of an ABCA1 variant associated with type 2 diabetes mellitus susceptibility for biochemistry and genetic laboratory courses

We selected diabetes mellitus for this laboratory exercise to provide students with an explicit model for scientific research concerning the association between the R230C polymorphism and susceptibility to type 2 diabetes mellitus, which is highly prevalent in the Mexican population. We used a collaborative project‐based learning to engage students to direct their own learning process. Students worked in small groups with the same learning goal to research, organize data, and present seminars to experimentally genotype the C230 variant and correctly interpret their results. At the conclusion of this laboratory exercise, the students were able to demonstrate a clear understanding of the relevant biological molecular principles to genotype the C230 variant, showed technical competency to carry out the experimental protocols with proficiency, and interpret their results using statistical analyses. The students discussed their understanding of the genetic technologies and the broader social and ethical implications of the research. A randomly selected team was trained to work as a “sentinel” to monitor their classmates and ensure the proper application of techniques. Moreover, the evaluation of this exercise is shared between the students and the instructors; the students evaluate their own work and the performance of their classmates. At the end of the course, the students complete a questionnaire to anonymously provide feedback and information regarding their perception of the learning outcomes. Overall, the student feedback was positive, indicating that the exercise was useful and that it would help to prepare the students for professional practice.