mRNA expression of MAGE-A3 gene in leukemia cells.

Leukemia-associated antigens such as proteins encoded by MAGE genes might provide tools for immunotherapy of leukemia. Positive and negative results of MAGE-A gene expression in hematological malignancies have been reported. This led us to study MAGE-A gene expression in human leukemias using RT-PCR. Among 115 leukemias from various subtypes, 14/34 (41.17%) AML were positive for one of the three genes analyzed (MAGE-A1 1/32; MAGE-A3 10/32; MAGE-B2 3/12). Expression was also detected in 23/76 (30.26%) B-cell ALL patients (MAGE-A1 2/53; MAGE-A3 20/53; MAGE-B2 1/32). One of these patients expressed both MAGE-A1 (weak signal) and -A3 (strong signal) genes. Other patient with CML were positive for MAGE-B2 (1/5, 20%). MAGE-A3 expression data were corroborated by real time RT-PCR through determination of MAGE-A3 transcript levels. We concluded that the MAGE-A3 gene is expressed at the mRNA level in a proportion of human leukemias.

Epidemiología molecular de cánceres de alta incidencia en México / Molecular epidemiology of high incidence cancers in Mexico

En este trabajo presentamos la situación epidemiológica molecular actual en México con respecto a la presencia de secuencias de ADN de virus de papiloma humano (VPH) en pacientes afectadas por cáncer cérvicouterino (CaCu) y en mujeres asintomáticas clínicamente normales. Encontramos, por PCR, que en un 82-85 por ciento de las neoplasias cervicales y en el 31 por ciento de las mujeres normales están presentes dichas secuencias. En cuanto a leucemias, otro cáncer de muy alta incidencia en México, investigamos la frecuencia de los rearreglos brc-abl y e2a-pbxl en pacientes con Leucemia Linfoblástica Aguda (LLA) y Leucemia Granulocítica Crónica (LGC) mediante la tecnología de RT-PCR. Encontramos que un 66 por ciento de los niños con pre B-LLA presentan el rearreglo e2a-pbxl, un 46 por ciento de los adultos con LLA CALLA(+) presentan el rearreglo bcr-abl y el 100 por ciento de los pacientes con LGC tuvieron el rearreglo bor-abl. Esta tecnología y los resultados presentados permiten un mejor diagnóstico, pronóstico y terapia de las mencionadas neoplasias

Induced mitotic death of HeLa cells by abnormal expression of c-H-ras.

When HeLa cells were selected for stable expression of a neo gene, linked either to mutated or wt c-H-ras genes, morphological examination of selected clones from several experiments revealed formation of giant multinucleated cells. These morphological alterations culminate in cell death, as a consequence of mitotic catastrophe (or mitotic death). Although clones expressing the mutated gene produced significantly larger numbers of these giant cells, those transfected with the normal allele were also found to produce significantly more giant multinucleated cells than non-transfected HeLa cells. Northern blot analysis of mRNA revealed overexpression of the normal H-ras gene in these clones. Chromatin structure analysis of these clones showed gross alterations, including the presence of micronuclei and heteroploid nuclei. Interestingly, odd numbers of nuclei were found in colonies of these giant cells. In addition, alterations in cell cycle parameters were observed, including the appearance of a subpopulation of cells with an abnormal content of DNA, probably representing dying cells. Our data support the notion that abnormal expression of H-ras contributes to mitotic catastrophe and death of a subpopulation of HeLa cells.

Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.

Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin.