Expression of Wnt5A and Wnt10B in non-immortalized breast cancer cells.

Wnt signaling is usually divided into two pathways: the 'canonical', acting through beta-catenin, and the 'non-canonical' acting through the Ca2+ and planar cell polarity pathway. Both pathways have been implicated in different types of cancer. Most results obtained with established cell lines have been contradictory. Here, we have investigated the expression of Wnt10B (canonical) and Wnt5A (non-canonical) in a panel of finite life-span and established normal and breast cancer cells using quantitative RT-PCR. It was found that there were both significant overexpression of Wnt5A and underexpression of Wnt10B in the metastasis-derived finite life-span breast cancer cells when they were compared to the finite life-span normal and established normal and breast tumor cells. Since expression profiles of primary breast cancer cultures are closer to the original tumor than the established cell lines, future research in this area should take into consideration these differences.

Transcription profiles of non-immortalized breast cancer cell lines.

BACKGROUND: Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. METHODS: Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs) were used in addition to commercially-available normal breast epithelial cells (HMECs), established breast cancer cell lines (T-est) and established normal breast cells (N-est). The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. RESULTS: According to Significance Analysis of Microarray (SAM) and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p < 0.01). Some of these genes have already been directly linked with breast cancer, metastasis and malignant progression, whilst others encode receptors linked to signal transduction pathways or are otherwise related to cell proliferation. Fifty genes showed at least a 2.5-fold difference between MSSMs and T-est cells according to AtlasImage, 2-fold according to SAM. Most of these classified as genes related to metabolism and cell communication. CONCLUSION: The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research.

Kinetic analysis of thapsigargin-induced thymocyte apoptosis.

Thapsigargin addition to thymocytes increased cytosolic Ca2+ by a factor of 8.5 with a time for half maximal effect (t1/2) of 2.5 min. Calcium signaling increased mitocondrial and endoplasmic reticulum nitric oxide synthase (NOS) activities by five and six times, with t1/2 of 16 and 48 min, respectively, followed by increases of 140% in intracellular [H2O2], 73% in hydroperoxide content, and 250% in thiobarbituric reactive substance content, with t1/2 of 13, 27, and 30 min, respectively. Mitochondrial dysfunction followed, and was characterized by decreased respiratory control, membrane depolarization, and decrease cytochrome c content release, processes with t1/2 of 101, 129, and 133 min, respectively. Increased UDP-GT gene expression, observed by mRNA synthesis, and the enzymatic activity of this protein had t1/2 of 52 and 187 min, respectively. These events were followed by caspase-3 activation (t1/2 = 210 min) and DNA laddering (t1/2 = 260 min) at the completion of the cell death program. Preincubation of thymocytes with NOS inhibitors (NG-methyl-L-arginine and L-Nomega-nitro-L-arginine methylester) halted the whole process through inhibition of mitochondrial and endoplasmic reticulum NOS activities and of DNA laddering.

Transcription profile of a human breast cancer cell line expressing MMTV-like sequences.

BACKGROUND: It has been postulated that inflammation caused by certain viruses might result in cancer. Recently, it was shown that childhood lymphoblastic leukemia, breast and ovarian cancers express an interferon-related signature, providing support for this notion. We have previously shown that 38% of the sporadic breast cancers contain MMTV-like env gene sequences. To find out if the presence and expression of MMTV-like sequences correlated with an inflammatory phenotype, we have compared the expression profile of two sublines of MCF-7 cells, one containing the MMTV-like sequences (env+), the other one lacking them (env-). RESULTS: The results indicated that there were 47 differentially expressed genes between the two sublines. Among 27 upregulated genes in the env+ cells there were 7 interferon-related genes, 5 TNF-connected genes and 2 TGFbeta-related genes. CONCLUSION: These results suggest that the env+ cells were most likely responding to an infectious agent, and support the hypothesis that a viral infection may play a role in breast cancer pathogenesis.

Strains of JC virus in Amerind-speakers of North America (Salish) and South America (Guaraní), Na-Dene-speakers of New Mexico (Navajo), and modern Japanese suggest links through an ancestral Asian population.

Previously we showed that strains of human polyoma virus JC among the Navajo in New Mexico, speakers of an Athapaskan language in the Na-Dene language phylum, and among the Salish people in Montana, speakers of a language of the Salishan group in the Amerind family, were mainly of a northeast Asian genotype found in Japan (type 2A). We now report partial VP1-gene, regulatory region, and complete genome sequences of JC virus (JCV) from the Guaraní Indians of Argentina. The Tupí-Guaraní language represents the Equatorial branch of the Amerind language family proposed by Greenberg ([1987] Language in the Americas, Stanford: Stanford University Press). The partial VP1 gene sequences of the Guaraní revealed several variants of strains found in northeast Asia (Japan), as did the Salish. In contrast, the strains in the Navajo largely conformed to the prototype type 2A sequence (MY). Phylogenetic reconstruction with both the neighbor-joining and maximum parsimony methods utilized three complete Guaraní JCV genome sequences, three genomes from the Salish people, and 27 other complete JCV genomes, including three from the Navajo and three from Japan. Both trees showed that all type 2A JCV strains from the North and South Americans are closely related phylogenetically to strains in present-day Japan. However, variant sites in the coding regions, the T-antigen intron, and the regulatory region link the type 2A strains in Amerind groups (Guaraní and Salish), but differentiate them from those in a Na-Dene-speaking (Navajo) population. The data suggest separation from a population ancestral to modern Japanese, followed by a second division within the ancestral group that led to Amerind- and Na-Dene-speaking groups. The data cannot, however, localize the latter split to the Asian mainland (two migrations) or to North America (one migration).

Heterogeneidad genomica de virus de la hepatitis B, genotipo A, circulantes en el area metropolitana de Buenos Aires, Argentina / Genomic heterogenity of virus B Hepatitis, genotype A circulating in Buenos Aires, metropolitan area, Argentine

Se purificaron partículas de Dane, a partir de sueros de pacientes virémicos del area metropolitana de Buenos Aires (Argentina) y se extrajo HBV DNA. El HBV DNA fué clonado en el vector pUC18, amplificado en Escherichia coli DH5 alpha F. Los plasmidos fueron aislados y el contenido de los insertos de HBV DNA se analizó por tratamiento con diversas enzimas de restricción para estabelecer las características genómicas clonadas. Se seleccionaron tres plasmidos y sus estructuras fueron determinadas, identificandolos como: pHB4, pHB7 y pHB20. Los insertos presentes en cada uno fueron secuenciados y el resultado incorporado a la base de datos Gen Bank, con los siguientes números de código: PHB4P3= U33188; PHB4P5= U33189; PHB7P3=U33190; PHB7P5=U33191 y PHB20=U33187. Todos los insertos pertenencen a genotipos A, pHB4 y pHB20 tienen una estrecha homología, que comparten con el L 13994, de circulación americana. pHB7 presenta una marcada diferencia con los clones pHB4 y pHB20 y una discreta relación con el agente m57663, aislando originalmente en Filipinas. pHB4 muestra una mutación en el nucleótico T3182(Leu en la región preS1, que cambia Pro. por Leu. Esta mutación est ausente en 125 secuencias estudiadas, que mantienen por lo menos un 65 por ciento de homología con los clones aislados en el area metropolitana de Buenos Aires. Esta selección se efectuó en el banco National Center for Biotechnology information (NCBI) utilizando el algoritmo Blast. El an lisis de estos clones no reveló la presencia de mutantes del tipo e o de escape HBV DNA, con este tipo de variantes han sido descriptas recientemente en esta area geografica por López y col. (Ref.7). Si bien los genotipos aislados pertencen al tipo A, es de importancia tener en cuenta la manifesta divergencia de por lo menos uno de los clones estudiados. (AU)

Heterogeneidad genomica de virus de la hepatitis B, genotipo A, circulantes en el area metropolitana de Buenos Aires, Argentina / Genomic heterogenity of virus B Hepatitis, genotype A circulating in Buenos Aires, metropolitan area, Argentine

Se purificaron partículas de Dane, a partir de sueros de pacientes virémicos del area metropolitana de Buenos Aires (Argentina) y se extrajo HBV DNA. El HBV DNA fué clonado en el vector pUC18, amplificado en Escherichia coli DH5 alpha F'. Los plasmidos fueron aislados y el contenido de los insertos de HBV DNA se analizó por tratamiento con diversas enzimas de restricción para estabelecer las características genómicas clonadas. Se seleccionaron tres plasmidos y sus estructuras fueron determinadas, identificandolos como: pHB4, pHB7 y pHB20. Los insertos presentes en cada uno fueron secuenciados y el resultado incorporado a la base de datos Gen Bank, con los siguientes números de código: PHB4P3= U33188; PHB4P5= U33189; PHB7P3=U33190; PHB7P5=U33191 y PHB20=U33187. Todos los insertos pertenencen a genotipos A, pHB4 y pHB20 tienen una estrecha homología, que comparten con el L 13994, de circulación americana. pHB7 presenta una marcada diferencia con los clones pHB4 y pHB20 y una discreta relación con el agente m57663, aislando originalmente en Filipinas. pHB4 muestra una mutación en el nucleótico T3182(Leu en la región preS1, que cambia Pro. por Leu. Esta mutación est ausente en 125 secuencias estudiadas, que mantienen por lo menos un 65 por ciento de homología con los clones aislados en el area metropolitana de Buenos Aires. Esta selección se efectuó en el banco National Center for Biotechnology information (NCBI) utilizando el algoritmo Blast. El an lisis de estos clones no reveló la presencia de mutantes del tipo e o de escape HBV DNA, con este tipo de variantes han sido descriptas recientemente en esta area geografica por López y col. (Ref.7). Si bien los genotipos aislados pertencen al tipo A, es de importancia tener en cuenta la manifesta divergencia de por lo menos uno de los clones estudiados.

Detección de una nueva variación genómica de Adenovirus 7 asociada a infecciones severas del tracto respiratorio inferior / Detection of a new genomic variation of Adenovirus 7 associated to severe infections of the lower respiratory tract

We report a new genomic variation of Adenovirus 7, associated to severe infections of the lower respiratory tract isolated during September 1990, from children under 3 years of age and living in Buenos Aires city. The restriction analysis with the BamHI, BglI, BglII and SmaI restriction endonucleases demonstrated that the new variation is highly related to the recently described Adenovirus 7h.

Detección de una nueva variación genómica de Adenovirus 7 asociada a infecciones severas del tracto respiratorio inferior / Detection of a new genomic variation of Adenovirus 7 associated to severe infections of the lower respiratory tract

We report a new genomic variation of Adenovirus 7, associated to severe infections of the lower respiratory tract isolated during September 1990, from children under 3 years of age and living in Buenos Aires city. The restriction analysis with the BamHI, BglI, BglII and SmaI restriction endonucleases demonstrated that the new variation is highly related to the recently described Adenovirus 7h.(Au)