Identification of molecular mechanisms for cellular drug resistance by combining drug activity and gene expression profiles.

Acquired drug resistance is a major problem in cancer treatment. To explore the genes involved in chemosensitivity and resistance, 10 human tumour cell lines, including parental cells and resistant subtypes selected for resistance against doxorubicin, melphalan, teniposide and vincristine, were profiled for mRNA expression of 7400 genes using cDNA microarray technology. The drug activity of 66 cancer agents was evaluated on the cell lines, and correlations between drug activity and gene expression were calculated and ranked. Hierarchical clustering of drugs based on their drug-gene correlations yielded clusters of drugs with similar mechanism of action. Genes correlated with drug sensitivity and resistance were imported into the PathwayAssist software to identify putative molecular pathways involved. A substantial number of both proapoptotic and antiapoptotic genes such as signal transducer and activator of transcription 1, mitogen-activated protein kinase 1 and focal adhesion kinase were found to be associated to drug resistance, whereas genes linked to cell cycle control and proliferation, such as cell division cycle 25A and signal transducer of activator of transcription 5A, were associated to general drug sensitivity. The results indicate that combined information from drug activity and gene expression in a resistance-based cell line panel may provide new knowledge of the genes involved in anticancer drug resistance and become a useful tool in drug development.

Gene survey of the pathogenic protozoan Trypanosoma cruzi.

We have performed a survey of the active genes in the important human pathogen Trypanosoma cruzi by analyzing 5013 expressed sequence tags (ESTs) generated from a normalized epimastigote cDNA library. Clustering of all sequences resulted in 771 clusters, comprising 54% of the ESTs. In total, the ESTs corresponded to 3054 transcripts that might represent one-fourth of the total gene repertoire in T. cruzi. About 33% of the T. cruzi transcripts showed similarity to sequences in the public databases, and a large number of hitherto undiscovered genes predicted to be involved in transcription, cell cycle control, cell division, signal transduction, secretion, and metabolism were identified. More than 140 full-length gene sequences were derived from the ESTs. Comparisons with all open reading frames in yeast and in Caenorhabditis elegans showed that only 12% of the T. cruzi transcripts were shared among diverse eukaryotic organisms. Comparison with other kinetoplastid sequences identified 237 orthologous genes that are shared between these evolutionarily divergent organisms. The generated data are a useful resource for further studies of the biology of the parasite and for development of new means to combat Chagas' disease.

Trypanosoma rangeli and Trypanosoma cruzi: molecular characterization of genes encoding putative calcium-binding proteins, highly conserved in trypanosomatids.

Genes encoding a 29-kDa flagellar calcium-binding protein (F29) in Trypanosoma cruzi, strongly homologous to EF-hand calcium-binding protein-encoding genes previously reported in this parasite, were isolated by immunoscreening. F29 is encoded by a number of very similar genes, highly conserved among different T. cruzi isolates. The genes are located on a pair of homologous chromosomes, arranged in one or two clusters of tandem repeats. PCR amplification of Trypanosoma rangeli genomic DNA, using primers derived from the T. cruzi F29 sequence made it possible to isolate the homologous gene in T. rangeli, encoding a 23-kDa protein called TrCaBP. Gene sequence comparisons showed homology to EF-hand calcium-binding proteins from T. cruzi (82.8%), Trypanosoma brucei brucei (60.2%), and Entamoeba histolytica (28.4%). Northern blot analysis revealed that the TrCaBP gene is expressed in T. rangeli as a polyadenylated transcript. The TrCaBP-encoding genes are present in at least 20 copies per cell, organized in tandem arrays, on large T. rangeli chromosomes in some isolates and on two smaller ones in others. This gene, however, seems to be absent from Leishmania.

Karyotype variability in Trypanosoma rangeli.

The molecular karyotypes of several different protozoan parasites show high intra-species variation, including different kinetoplastids such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp. In this study, the molecular karyotype of Trypanosoma rangeli was examined. To evaluate potential intra-species molecular karyotype variations, 16 different samples were studied by pulsed field gel electrophoresis (PFGE) followed by ethidium bromide staining and hybridizations with 6 different probes. The result showed that different T. rangeli populations are highly polymorphic regarding the molecular karyotype, and thus suggests that PFGE analysis can be used for classification of different T. rangeli isolates. In addition, the molecular karyotype of T. rangeli was compared to molecular karyotypes of other kinetoplastids, and was shown to be distinctly different from that of T. cruzi, but shows some similarities with the karyotype described for T. brucei. Among the probes used one was identified as highly polymorphic, and thus informative for studies of different T. rangeli populations, and another was useful for differentiation between T. rangeli and T. cruzi.

Chromosome specific markers reveal conserved linkage groups in spite of extensive chromosomal size variation in Trypanosoma cruzi.

The karyotypes of three cloned stocks, CL Brener (CL), CA I/72 (CA) and Sylvio X10/7 (X10), of Trypanosoma cruzi were studied by pulsed-field gel electrophoresis followed by ethidium bromide staining and hybridization with 35 different probes, 30 of which identified single chromosomes. The chromosome-specific probes identified between 26 and 31 chromosomal bands in the three cloned stocks, corresponding to 20 unique chromosomes in CL and 19 in CA and X10. Considering the DNA content of the parasite, it was predicted that the markers recognise at least half of all T. cruzi chromosomes. A majority of identified chromosomes showed large differences in size among different strains, in some cases by up to 50%. Interestingly, CL had in general larger chromosomes than the two other studied cloned stocks. Several of the markers showed linkage and nine different linkage groups were identified, each comprising 2-4 markers. The linkage between the markers was maintained in 8 of the 9 linkage groups when a panel comprising 26 different T. cruzi strains representing major T. cruzi populations was tested. One linkage group was found to be maintained in some strains but not in others. This result shows that chromosomal rearrangements occur in the T. cruzi genome, albeit with a low frequency. Repetitive DNA, both non-coding and in one case coding, was more abundant in the cloned stock CL Brener than in CA and X10. The information presented will make it possible to select chromosomes for the construction of physical chromosomal maps required for the T. cruzi genome project.

Genes for cysteine proteinases from Trypanosoma rangeli.

PCR amplification of genomic DNA from the American trypanosome, Trypanosoma rangeli, using as primers oligonucleotides derived from the gene of cruzipain, the major cysteine proteinase (CP) from Trypanosoma cruzi, allowed the production of a probe which was used to obtain three clones encoding a CP with 70% overall identity with cruzipain. The genes are organized in tandem, with a monomere size of approximately 2 kbp, located on two chromosomes which, in some parasite isolates, have a high molecular mass (higher than 5.7 Mbp), and in others are much smaller (about 500 kbp). The low expression of this CP at the protein level correlates well with the low level of specific mRNA found in Northern blots.

A gene family encoding heterogeneous histone H1 proteins in Trypanosoma cruzi.

A gene family encoding a set of histone H1 proteins in Trypanosoma cruzi is described. The sequence of 3 genomic and 4 cDNA clones revealed the presence of several motifs characteristic of histone H1, although heterogeneity at the polypeptide level was evident. The clones encode histone H1 proteins of an unusually small size (74-97 amino acids), which lack the globular domain found in histone H1 of higher eukaryotes. All histone H1 mRNAs from T. cruzi are polyadenylated, although no typical polyadenylation signal was found. Furthermore, the genes encoding the histone H1 proteins in T. cruzi are found in a tandem array containing 15-20 gene copies per haploid genome. This tandem array is located on a large chromosome of 2.2 Mb.

Ugandan HIV-1 V3 loop sequences closely related to the U.S./European consensus.

The third variable (V3) loop of the human immunodeficiency virus type 1 (HIV-1) envelope protein is an important determinant for virus neutralization and cell tropism. V3 loop sequences from uncultured lymphocytes obtained in 1990 from 22 Ugandan HIV-1-infected patients could, with the exception of two patients' sequences, be divided into two groups (A and B) on the basis the V3 loop size and sequence. The V3 loop consensus sequences from both groups showed a high degree of homology to a U.S./European consensus, a characteristic also reflected by the results of peptide serology. In the case of group B the difference in sequence was only five amino acids. In contrast, the V3-flanking regions for both groups showed greater homology to an earlier (1986/1987) Ugandan consensus. The discovery of these two new Ugandan V3 loop genotypes, which are closely related to the U.S./European consensus, has implications for the understanding of the evolution of HIV-1 and for the future design of a vaccine for use in Africa.