Francisco Gómez-Esquer

Nup88 mRNA overexpression is associated with high aggressiveness of breast cancer

The nuclear pore complex protein Nup88 is overexpressed in tumor cells. Immunohistochemical studies have shown that this overexpression is linked to higher aggressiveness of colorectal carcinoma and to enhanced metastatic potential of melanoma cells. However, the antibodies so far developed against Nup88 have the drawback of recognizing a number of other, up to now unspecified antigens besides Nup88. For this reason, we devised the present study on Nup88 expression at the mRNA level. RNA was extracted from fresh tumor tissue corresponding to 122 breast cancer patients. Nup88 mRNA expression was measured by means of differential RT‐PCR, standardizing against a constitutive internal control gene (β‐actin). The results were dichotomized into “high” and “low” expression levels, using the median value as cut‐off. High Nup88 mRNA expression levels correlated significantly with ductal and tubular histology (p = 0.012), histologic and nuclear grade 3 of tumors (p < 0.001), absence of hormone receptor expression (p < 0.001), expression of the c‐erb‐B2 oncogene (p < 0.001), expression of mutant p53 protein (p < 0.001), high proliferation (defined by Ki67 labeling index >20%, p < 0.001), DNA aneuploidy (p < 0.001) as well as the most important ominous clinical prognostic factor, axillary node invasion (p < 0.001). We also found an inverse correlation (p < 0.001) with expression of the H‐MAM (mammaglobin) gene, a marker of low biologic and clinical aggressiveness of breast cancer. All of these factors, without exception, define a highly aggressive tumor phenotype. These findings appear to be specific to Nup88 and not to nuclear pore proteins in general. Indeed, analysis of Nup107 (which is a limiting component of the nuclear pore complex) under the same conditions in the same tumors did not yield comparable results.

Positive correlation between the expression of X-chromosome RBM genes (RBMX, RBM3, RBM10) and the proapoptotic Bax gene in human breast cancer

In a recent report, it has been postulated that the ubiquitous RBM proteins might constitute a novel family of apoptosis modulators. We measured the expression of the X‐chromosome RBM genes (RBMX, RBM3, and RBM10) in 122 breast cancers by means of differential RT‐PCR. Using the same method, we also studied the expression of the apoptosis‐related genes Bcl‐2 and Bax. Markers of hormone dependence (estrogen and progesterone receptors), proliferation (Ki67 and DNA‐ploidy), angiogenesis (VEGF and CD105), as well as oncogene (c‐erb‐B2), and tumor suppressor gene (p53) expression were also analyzed. The expression of all X‐chromosome RBM genes was significantly associated with the expression of the proapoptotic Bax gene (RBMX, P = 0.039; RBM3, P < 0.001; RBM10 large variant, P < 0.001; RBM10 small variant, P < 0.001). Furthermore, the expression of both RBM10 variants was significantly associated with the expression of the VEGF gene (large variant, P = 0.004; small variant, P = 0.003). We also found an association of borderline significance (P = 0.05) between the expression of RBM3, the large variant of RBM10 and wild‐type p53. Expression of the small RBM10 variant, finally, was associated with high proliferation of the tumors (Ki67 ≥ 20%; P = 0.037). The expression of both RBM10 variants seems to be interdependent to a significant degree (r = 0.26, P = 0.006). From these results, it seems that the X‐chromosome, through its RBM genes, plays a formerly unknown role in the regulation of programmed cell death (apoptosis) in breast cancer. J. Cell. Biochem. 97: 1275–1282, 2006.

Protein Kinase Cθ Is a Specific Target for Inhibition of the HIV Type 1 Replication in CD4+ T Lymphocytes

Integration of HIV-1 genome in CD4+ T cells produces latent reservoirs with long half-life that impedes the eradication of the infection. Control of viral replication is essential to reduce the size of latent reservoirs, mainly during primary infection when HIV-1 infects CD4+ T cells massively. The addition of immunosuppressive agents to highly active antiretroviral therapy during primary infection would suppress HIV-1 replication by limiting T cell activation, but these agents show potential risk for causing lymphoproliferative disorders. Selective inhibition of PKCθ, crucial for T cell function, would limit T cell activation and HIV-1 replication without causing general immunosuppression due to PKCθ being mostly expressed in T cells. Accordingly, the effect of rottlerin, a dose-dependent PKCθ inhibitor, on HIV-1 replication was analyzed in T cells. Rottlerin was able to reduce HIV-1 replication more than 20-fold in MT-2 (IC50 = 5.2 μm) and Jurkat (IC50 = 2.2 μm) cells and more than 4-fold in peripheral blood lymphocytes (IC50 = 4.4 μm). Selective inhibition of PKCθ, but not PKCδ or -ζ, was observed at <6.0 μm, decreasing the phosphorylation at residue Thr538 on the kinase catalytic domain activation loop and avoiding PKCθ translocation to the lipid rafts. Consequently, the main effector at the end of PKCθ pathway, NF-κB, was repressed. Rottlerin also caused a significant inhibition of HIV-1 integration. Recently, several specific PKCθ inhibitors have been designed for the treatment of autoimmune diseases. Using these inhibitors in combination with highly active antiretroviral therapy during primary infection could be helpful to avoid massive viral infection and replication from infected CD4+ T cells, reducing the reservoir size at early stages of the infection.

The Presence of HIV-1 Tat Protein Second Exon Delays Fas Protein-mediated Apoptosis in CD4+ T Lymphocytes: A POTENTIAL MECHANISM FOR PERSISTENT VIRAL PRODUCTION*

Background: HIV-infected T cells are quite resistant to apoptosis. Results: Intracellular expression of HIV-1 Tat in T cells stabilized the mitochondrial membrane and reduced caspase activation mainly through NF-κB activation. Conclusion: Intracellular Tat induced resistance to FasL-mediated apoptosis in T cells mainly through the second exon. Significance: Tat-mediated protection against apoptosis may be a mechanism for HIV-1 persistence. HIV-1 replication is efficiently controlled by the regulator protein Tat (101 amino acids) and codified by two exons, although the first exon (1–72 amino acids) is sufficient for this process. Tat can be released to the extracellular medium, acting as a soluble pro-apoptotic factor in neighboring cells. However, HIV-1-infected CD4+ T lymphocytes show a higher resistance to apoptosis. We observed that the intracellular expression of Tat delayed FasL-mediated apoptosis in both peripheral blood lymphocytes and Jurkat cells, as it is an essential pathway to control T cell homeostasis during immune activation. Jurkat-Tat cells showed impairment in the activation of caspase-8, deficient release of mitochondrial cytochrome c, and delayed activation of both caspase-9 and -3. This protection was due to a profound deregulation of proteins that stabilized the mitochondrial membrane integrity, such as heat shock proteins, prohibitin, or nucleophosmin, as well as to the up-regulation of NF-κB-dependent anti-apoptotic proteins, such as BCL2, c-FLIPS, XIAP, and C-IAP2. These effects were observed in Jurkat expressing full-length Tat (Jurkat-Tat101) but not in Jurkat expressing the first exon of Tat (Jurkat-Tat72), proving that the second exon, and particularly the NF-κB-related motif ESKKKVE, was necessary for Tat-mediated protection against FasL apoptosis. Accordingly, the protection exerted by Tat was independent of its function as a regulator of both viral transcription and elongation. Moreover, these data proved that HIV-1 could have developed strategies to delay FasL-mediated apoptosis in infected CD4+ T lymphocytes through the expression of Tat, thus favoring the persistent replication of HIV-1 in infected T cells.

Changes in the cellular microRNA profile by the intracellular expression of HIV-1 Tat regulator: A potential mechanism for resistance to apoptosis and impaired proliferation in HIV-1 infected CD4+ T cells

HIV-1 induces changes in the miRNA expression profile of infected CD4+ T cells that could improve viral replication. HIV-1 regulator Tat modifies the cellular gene expression and has been appointed as an RNA silencing suppressor. Tat is a 101-residue protein codified by two exons that regulates the elongation of viral transcripts. The first exon of Tat (amino acids 1–72) forms the transcriptionally active protein Tat72, but the presence of the second exon (amino acids 73–101) results in a more competent regulatory protein (Tat101) with additional functions. Intracellular, full-length Tat101 induces functional and morphological changes in CD4+ T cells that contribute to HIV-1 pathogenesis such as delay in T-cell proliferation and protection against FasL-mediated apoptosis. But the precise mechanism by which Tat produces these changes remains unknown. We analyzed how the stable expression of intracellular Tat101 and Tat72 modified the miRNA expression profile in Jurkat cells and if this correlated with changes in apoptotic pathways and cell cycle observed in Tat-expressing cells. Specifically, the enhanced expression of hsa-miR-21 and hsa-miR-222 in Jurkat-Tat101 cells was associated with the reduced expression of target mRNAs encoding proteins related to apoptosis and cell cycle such as PTEN, PDCD4 and CDKN1B. We developed Jurkat cells with stable expression of hsa-miR-21 or hsa-miR-222 and observed a similar pattern to Jurkat-Tat101 in resistance to FasL-mediated apoptosis, cell cycle arrest in G2/M and altered cell morphology. Consequently, upregulation of hsa-miR-21 and hsa-miR-222 by Tat may contribute to protect against apoptosis and to anergy observed in HIV-infected CD4+ T cells.

Gallus gallus orthologous to human alpha-dystroglycanopathies candidate genes: Gene expression and characterization during chicken embryogenesis.

Alpha-dystroglycanopathies are a heterogenic group of human rare diseases that have in common defects of α-dystroglycan O-glycosylation. These congenital disorders share common features as muscular dystrophy, malformations on central nervous system and more rarely altered ocular development, as well as mutations on a set of candidate genes involved on those syndromes. Severity of the syndromes is variable, appearing Walker-Warburg as the most severe where mutations at protein O-mannosyl transferases POMT1 and POMT2 genes are frequently described. When studying the lack of MmPomt1 in mouse embryonic development, as a murine model of Walker-Warburg syndrome, MmPomt1 null phenotype was lethal because Reitcherts membrane fails during embryonic development. Here, we report gene expression from Gallus gallus orthologous genes to human candidates on alpha-dystroglycanopathies POMT1, POMT2, POMGnT1, FKTN, FKRP and LARGE, making special emphasis in expression and localization of GgPomt1. Results obtained by quantitative RT-PCR, western-blot and immunochemistry revealed close gene expression patterns among human and chicken at key tissues affected during development when suffering an alpha-dystroglycanopathy, leading us to stand chicken as a useful animal model for molecular characterization of glycosyltransferases involved in the O-glycosylation of α-Dystroglycan and its role in embryonic development.