Fabio Aristizábal

Isolation of the LEMMI9 gene and promoter analysis during a compatible plant-nematode interaction

Plant-endoparasitic root-knot nematodes feed on specialized giant cells that they induce in the vascular cylinder of susceptible plants. Although it has been established that a number of plant genes change their expression pattern during giant cell differentiation, virtually no data are available about the mechanisms involved in that change. One possibility is differential promoter recognition by the transcription factor(s) responsible for the expression of specific genes. We have isolated and characterized a genomic clone from tomato containing the promoter region of LEMMI9, one of the few plant genes that have been reported to be highly expressed in galls (predominantly in giant cells). The analysis of transgenic potato plants carrying a LEMMI9 promoter-beta glucuronidase (GUS) fusion has demonstrated that the tomato promoter was activated in Meloidogyne incognita-induced galls in a heterologous system. We have located putative regulatory sequences in the promoter and have found that nuclear proteins from the galls formed specific DNA-protein complexes with the proximal region of the LEMMI9 promoter. The nuclear protein-binding sequence mapped to a region of 111 bp immediately upstream from the TATA box. This region contains a 12-bp repeat possibly involved in the formation of DNA-protein complexes, which might be related to the LEMMI9 transcriptional activation in the giant cells.

Cytotoxicity of withanolides isolated from Acnistus arborescens.

A new withanolide identified by spectroscopic analysis as 12beta-acetoxy-4-deoxy-5,6-deoxy-Delta(5)-withanolide D and Withanolide D, were isolated from the leaves of Acnistus arborescens. Cytotoxic activity of these two compounds against human tumor cell lines HT-29, MCF-7, MKN-45, HEp-2, HeLa, U-937 and two human normal fibroblast cultures, Fib04 and Fib05, were assessed. Withanolide D presented in vitro cytotoxic activity against tumor cell lines at the low micromolar range (LC(50):1.0 to 1.69 microM) and showed a slightly lower activity against Fib04, suggesting moderated selectivity among tumoral and normal cells. No cytotoxic effect was observed for 12beta-acetoxy-4-deoxy-5,6-deoxy-Delta(5)-withanolide D.

Efecto citotóxico de algunos compuestos naturales aislados de plantas Laureaceae y derivados sintéticos

Introduccion. El efecto contra la proliferacion celular de once neolignanos, dos lignanos y un diterpeno, aislados de tres plantas de la familia Lauraceae, y cuatro benzofuranos y dos biciclooctanos sinteticos, fue evaluado in vitro sobre cinco lineas celulares derivadas de tumores solidos de alta incidencia en Colombia. Objetivo. Evaluar el efecto citotoxico de veinte compuestos sobre las lineas tumorales HeLa, A-549, Hep-2, PC-3 y MCF-7. Materiales y metodos. Los 14 compuestos de origen natural fueron aislados de tres plantas nativas colombianas (Pleurothyrium cinereum, Ocotea macrophylla y Nectandra amazonum) por tecnicas cromatograficas y se establecieron sus estructuras por metodos espectroscopicos, y los seis derivados sinteticos fueron preparados mediante reaccion de oxiarilacion y metilacion con diazometano. El efecto contra la proliferacion y la recuperacion celular se hicieron mediante tratamiento in vitro de las lineas tumorales con los compuestos , evaluando la viabilidad celular por tincion con resazurina. Resultados. Entre los compuestos evaluados, solamente ocofilal A, cinerina D, acido kaurenoico, dos benzofuranos y la (-)-cinerina A sintetica presentaron actividad contra la proliferacion celular en diferentes niveles. Los biciclooctanos, asi como el acido kaurenoico, fueron activos contra todas las lineas celulares, mientras que los benzofuranoides mostraron actividad selectiva contra HeLa. Ademas, la (-)-cinerina A exhibio un efecto letal total contra todas las lineas celulares, mientras que el acido kaurenoico presento efecto letal total contra PC-3, Hep-2 y A549. Conclusion. Los compuestos evaluados que exhibieron actividad contra la proliferacion celular mostraron resultados interesantes, lo cual sugiere su potencial uso como cabezas de serie o moleculas plantilla en el desarrollo de agentes anticancerigenos.

Cytotoxic effect of some natural compounds isolated from Lauraceae plants and synthetic derivatives

INTRODUCTION The antiproliferative effect of eleven neolignans, two lignans and one diterpene isolated from three Lauraceae plants, four benzofurans and two bicyclooctanes synthetic derivatives was evaluated in vitro on a set of five human cancer cells from solid tumors with a high incidence in Colombia. OBJECTIVE To evaluate the cytotoxic effect of twenty compounds on the tumor cell lines HeLa, A-549, Hep-2, PC-3, and MCF-7. MATERIALS AND METHODS. Fourteen natural compounds were isolated by chromatographic techniques from three native Colombian plants (Pleurothyrium cinereum, Ocotea macrophylla and Nectandra amazonum), whose structures were established by spectroscopic methods; six synthetic derivatives were prepared by oxyarylation and diazomethane methylation. Antiproliferative effect and cell recovery were performed by means of in vitro treatment of tumor cell lines with test compounds, evaluating cell viability by resazurin staining. RESULTS Among test compounds, only neolignans ocophyllal A, cinerin D, kaurenoic acid, two benzofuran-derivatives, and synthetic (-)-cinerin A were found to have antiproliferative effect at different levels. Bicyclooctanoids as well as kaurenoic acid exhibited activity against all human cancer cells while benzofuranoids showed selective activity against HeLa. Furthermore, compounds (-)-cinerin A and kaurenoic acid exhibited total lethal effect against all-five cell lines and PC-3, Hep-2, and A549 cell lines, respectively. CONCLUSION Test compounds exhibiting antiproliferative activity showed interesting results, which would promote their use as lead compounds on further studies for anticancer agents development.

Activation of geminivirus V-sense promoters in roots is restricted to nematode feeding sites

Obligate sedentary endoparasitic nematodes, such as the root-knot and cyst nematodes, elicit the differentiation of specialized nematode nurse or feeding cells [nematode feeding sites (NFS), giant cells and syncytia, respectively]. During NFS differentiation, marked changes in cell cycle progression occur, partly similar to those induced by some geminiviruses. In this work, we describe the activation of V-sense promoters from the Maize streak virus (MSV) and Wheat dwarf virus (WDV) in NFS formed by root-knot and cyst nematodes. Both promoters were transiently active in microinjection experiments. In tobacco and Arabidopsis transgenic lines carrying promoter-beta-glucuronidase fusions, the MSV V-sense promoter was activated in the vascular tissues of aerial plant parts, primarily leaf and cotyledon phloem tissue and some floral structures. Interestingly, in roots, promoter activation was restricted to syncytia and giant cells tested with four different nematode populations, but undetectable in the rest of the root system. As the activity of the promoter in transgenic rootstocks should be restricted to NFS only, the MSV promoter may have utility in engineering grafted crops for nematode control. Therefore, this study represents a step in the provision of some of the much needed additional data on promoters with restricted activation in NFS useful in biotechnological nematode control strategies.

Oncogene amplification as tumor marker in a group of Colombian lung cancer patients

Introduction: In spite of recent treatment advances, lung cancer continues to be the first world cancer related death cause; its mortality associated occupied the fifth place in Colombia in 2004. Complete surgical resection is the therapeutic option with the greatest cure probability, however it results frequently ineffective given the current incapacity in Colombia to an early detection of the disease. This study reports the characterization of a group of 30 lung cancer patients regarding the gene dose (gene copy number) found at the loci corresponding to genes EGFR (erb B1), PIK3CA and C-myc in tumor samples, and compares the results with the dose found in adjacent lung from the same patients. Methods: The gene dose of EGFR (erbB1), PIK3CA, and C-myc were measured by real time PCR in matched tumor and normal lung tissue samples. Results are expressed as the multiplicity of each gene dose with respect to a single copy reference gene. In this case the gene HHB (human hemoglobin). Antiquity of the cases ranged from 5 to 10 years. Results: An increased gene dose for EGFR and PIK3CA was a feature clearly associated to the tumor phenotype of the sample (found in 96 and 100% of the tumors respectively). Quantitative measure of this feature demonstrated for both genes a high sensitivity and specificity for tumor/normal discrimination as confirmed by the ROC analysis. On the other hand, the Spearman test showed a great correlation between EGFR and PIK3CA doses (ρ=0.75). C-myc was the gene whose dose was less consistently correlated to the tumor phenotype, however most of the patients with amplified C-myc presented distant spread of tumor cells (metastasis) at diagnosis. Conclusion: Quantitative measurement of EGFR, PIK3CA, and C-myc gene dose by real time PCR provides a method for tumor phenotype recognition in DNA samples from lung tissue. These markers can be considered at the construction of a marker panel for lung cancer detection on alternative, non-invasive clinical samples. However clinical value will depend on the use of additional molecular markers, some of which could be of epigenetic character.

DNA methylation pattern in high-grade cervical intraepithelial neoplasia and cancer revealed by genome-wide methylation analysis of cervical DNA

Cancer of the uterine cervix is caused by a subset of oncogenic human Papillomavirus (HPV)-types with mucosal tropism. Besides the known effects of the viral oncoproteins on cellular functions there is evidence suggesting that cervical carcinogenesis involves epigenetic changes in the host DNA. In this study, we have examined the global promoter methylation profile associated with progression to cervical cancer at wide genome scale. The methylation pattern of nearly 14,000 genes was analyzed in cervical swabs at different stages of cervical carcinogenesis: low-grade cervical intraepithelial neoplasia (CIN I and II), high-grade CIN (CIN III) and invasive cancer, as well as healthy individuals. Unsupervised analysis (Hierarchical Clustering) identified two groups: A) healthy, CIN I and CIN II; and B) CIN III/cancer. Supervised T-Test analysis showed 1069 promoter regions hypermethylated and 85 hypomethylated in CIN III/cancer compared to CIN I/CIN II and healthy samples (p<0.0001). Overall, the differentially methylated genes act in transcription, cell cycle, apoptosis and cell adhesion pathways. Of the hypermethylated genes, 132 (12.3%) were down-regulated in a matched cervical cancer group. In turn, only 4 (4.7%) of the hypomethylated genes were overexpressed in that group. These data suggest that, although significant changes in methylation of a large number of cellular promoters take place during progression to cervical cancer, correlation between methylation and altered gene expression occurs only in a reduced number of genes. Nevertheless, these genes are relevant to cell transformation. Our results support the prognostic value of methylation profiling of a larger number of genes than previously thought. Introduction Cervical cancer (CC) of the uterus is the third most frequent cancer in women worldwide with over half a million new cases estimated in the year 2012 [1]. Over 85% of the cases occur in developing countries. In Colombia, the estimated incidence of CC is 21.5 cases per 100.000 inhabitants [1]. The cervical carcinogenesis begins with non-invasive dysplastic lesions, which progress to cervical intraepithelial neoplasia (CIN) grades I to III and, in some cases, the lesions persist and progress to invasive carcinoma [2]. A few high-risk human papillomaviruses (HPVs), most notably HPV16 and HPV18, are in the etiology of the majority of CCs, as well as other anogenital tumors and an increasing number of head and neck cancers [3]. Two proteins encoded by these viruses, E6 and E7 have transforming capabilities by inactivating RB1 and promoting degradation of TP53 thereby interfering with signaling pathways and cell cycle control mechanisms [4]. However, the natural history of the disease shows that only a minority of women infected with high-risk HPVs cannot clear infection and develop CC [5]. The molecular mechanisms that determine clearance or persistence of the HPV infection are mainly unknown. Thus, to date there are no reliable molecular markers that may help predicting whether low-grade lesions will progress to cancer or rather regress and be cleared. Cancer cells develop their malignant phenotype through a series of concurrent genetic and epigenetic changes that cooperatively influence gene transcription and contribute to overall malignant transformation [6]. The epigenetic machinery plays an important role in the control of the transcriptional activity through DNA modifications, most importantly CpG cytosine-5 methylation [6]. The human genome can be envisioned as immense regions of DNA sequence containing Correspondence to: Angel Cid Arregui, Applied Tumor Immunity, German Cancer Research Center (DKFZ), Heidelberg, Germany, E-mail: a.cid@dkfz-heidelberg.de D Adriana García, CIO, Pontificia Universidad Javeriana, Bogota, Colombia, E-mail: garciad@javeriana.edu.co