RESUMO
Endemic Burkitt's lymphoma (BL) is considered to preferentially develop in equatorial Africa because of chronic co-infection with Epstein-Barr virus (EBV) and the malaria pathogen Plasmodium falciparum. The interaction and contribution of both pathogens in the oncogenic process are poorly understood. Earlier, we showed that immune activation with a synthetic Toll-like receptor 9 (TLR9) ligand suppresses the initiation of EBV lytic replication in primary human B cells. In this study we investigate the mechanism involved in the suppression of EBV lytic gene expression in BL cell lines. We show that this suppression is dependent on functional TLR9 and MyD88 signaling but independent of downstream signaling elements, including phosphatidylinositol-3 kinase, mitogen-activated protein kinases and nuclear factor-kappaB. We identified TLR9 triggering resulting in histone modifications to negatively affect the activation of the promoter of EBV's master regulatory lytic gene BZLF1. Finally, we show that P. falciparum hemozoin, a natural TLR9 ligand, suppresses induction of EBV lytic gene expression in a dose-dependent manner. Thus, we provide evidence for a possible interaction between P. falciparum and EBV at the B-cell level and the mechanism involved in suppressing lytic and thereby reinforcing latent EBV that has unique oncogenic potential.
Assuntos
Linfoma de Burkitt/patologia , Herpesvirus Humano 4/genética , Histonas/metabolismo , Receptor Toll-Like 9/metabolismo , Transativadores/genética , Transcrição Gênica , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Sequência de Bases , Linfoma de Burkitt/virologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ilhas de CpG/genética , Hemeproteínas/metabolismo , Hemeproteínas/farmacologia , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/fisiologia , Humanos , Ligantes , Fator 88 de Diferenciação Mieloide/genética , Plasmodium falciparum/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Ativação Viral/efeitos dos fármacosRESUMO
Cellular resistance to etoposide has been correlated both with reduced levels and an aberrant cytoplasmic accumulation of the drug's target, topoisomerase IIalpha (topo IIalpha). It is not known, however, whether a cytoplasmic pool of topo IIalpha is sufficient to confer drug resistance on cultured mammalian cells. In our study, we have transfected mouse fibroblasts and human 293 cells with truncated forms of human topo IIalpha fused to GFP and have examined the transformants for the subcellular localization of topo IIalpha and their resistance to etoposide. Transient transfection resulted in high-level expression of all GFP-topo IIalpha fusions tested, whereas in stably transfected cells the levels varied significantly. Transfectants expressing a central or a carboxy-terminal topo IIalpha domain (aa 428-1504, 639-1028 or 1028-1504) accumulated high levels of the fusion proteins, while only very low amounts of GFP-topo IIalpha proteins were observed in cell lines expressing constructs that retain the amino-terminus of the enzyme (aa 1-1214, aa 1-939, aa 1-611). Our results suggest that the topo IIalpha amino-terminus affects the stability of truncated forms of the enzyme in mammalian cells, perhaps due to targeted degradation. Assays that screen for cell vitality and DNA synthesis reveal no significant changes in etoposide sensitivity in transfected cells expressing high levels of cytoplasmic or nuclear localized topo II fusion proteins. Retroviral expression of a cytoplasmically anchored domain of human topo IIalpha also failed to confer drug resistance. These results suggest that a cytoplasmic pool of topo IIalpha is not sufficient to render cultured mammalian cells drug resistant.
Assuntos
DNA Topoisomerases Tipo II , DNA Topoisomerases Tipo II/biossíntese , Etoposídeo/farmacologia , Isoenzimas/biossíntese , Células 3T3/efeitos dos fármacos , Células 3T3/enzimologia , Animais , Antígenos de Neoplasias , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA , Resistência a Medicamentos , Proteínas de Fluorescência Verde , Humanos , Isoenzimas/genética , Rim/citologia , Rim/efeitos dos fármacos , Rim/enzimologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos , Microscopia de Fluorescência , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
The human surface antigen CD24 is over-expressed in small-cell lung cancer. Here we describe the isolation, sequencing and functional characterization of the 5'-flanking region of the human CD24 gene. A sequence (accession number: Y14692) of 3.4 kb regulates the activity of a luciferase reporter gene in CD24-expressing small-cell-lung-cancer cell lines up to 1.6-fold more than the control SV40 promoter and enhancer. In contrast, little or no luciferase activity was detected in 4 non-small-cell-lung-cancer cell lines. A deletion fragment of 269 bp had maximal activity in small-cell-lung-cancer cell lines (15- to 20-fold higher than control), while activity remained 2- to 10-fold below control in non-small-cell-lung-cancer cell lines. We conclude that the CD24 promoter has a strong and cell-type-specific activity, and propose its exploitation to drive the expression of therapeutic genes in small-cell lung cancer.