Specific block of androgen receptor activity by antisense oligonucleotides.

Claims about molecular mechanisms underlying the resistance to anti-hormones of prostate cancer cells find support in biological experiments, which involve hormone-independent activation of the androgen receptor's (AR) transcriptional activity. In order to test this hypothesis, we attempted to shut down the expression of AR by the means of target-directed antisense oligonucleotides. A set of 49 oligonucleotides matching sequences of the AR mRNA either in the coding sequence or in the 3' and 5' untranslated regions were synthesized and examined in a cellular AR-dependent reporter system. Five antisense oligonucleotides were identified as highly potent inhibitors of AR-driven gene expression in a cellular reporter assay. These five were further profiled using point-mutated control sequences for the assessment of AR inhibition. In addition the expression of another AR-driven gene, the modulator of PSA expression (gene for inhibition of prostate specific antigen, an endogenous, AR-driven gene) was examined. Finally, we observed that the hormone-independent but AR-mediated transactivation by IGF-1 could also be specifically shut-down by these antisense oligonucleotides. The selection of highly target-restricted antisense oligonucleotides in the prostate cancer cell line LNCaP provided tools to study a central role of the androgen receptor in growth regulation of prostatic cancer cell lines and could be of utility in cancer situations in vivo.

Conservation and synteny of SSR loci and QTLs for vegetative propagation in four Eucalyptus species.

Conservation of microsatellite loci, heterozygous in Eucalyptus grandis, Eucalyptus urophylla, Eucalyptus tereticornis and Eucalyptus globulus, allowed us to propose homeologies among genetic linkage groups in these species, supported by at least three SSR loci in two different linkage groups. Marker-trait associations for sprouting and adventitious rooting ability were also compared in the four species. Putative quantitative trait loci (QTLs) influencing vegetative propagation traits were located on homeologous linkage groups. Our findings indicate high transferability of microsatellite markers between Eucalyptus species of the Symphyomyrtus subgenus and establish foundations for the use of synteny in the genetic analysis of this genus. Microsatellite markers should help integrate eucalypt genetic linkage maps from various sources. The availability of comparative linkage maps provides a basis of more-efficient use of genetic information for molecular breeding and evolutionary studies in Eucalyptus.

QTL mapping and introgression of yield-related traits from Oryza glumaepatula to cultivated rice ( Oryza sativa) using microsatellite markers.

Rice ( Oryza sativa) cultivar development currently faces the task of overcoming yield plateaus, which is difficult due to the narrow genetic base of breeding programs. Oryza glumaepatula is a diploid wild relative of cultivated rice, native to Central and South America, and is therefore a potential source of alleles of agronomic importance to rice breeding programs. We studied 11 agronomic traits in BC(2)F(2) families of the interspecific cross Oryza sativa x O. glumaepatula. Transgressive lines which are almost isogenic to the elite recurrent O. sativa parent were identified for most of these traits. Quantitative trait locus (QTL) analysis was performed by single-point and interval mapping using a molecular map based on 157 microsatellite and STS markers. Marker regions accounting for 14.5 to 72.9% of a phenotypic variation trait were identified in 9 of the 12 rice chromosomes. Positive QTL effects from O. glumaepatula were observed in chromosomal regions associated with tillering and panicle-number traits.

Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines.

In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression, referred to as RNA interference (RNAi). In invertebrates, RNAi can be triggered effectively by either long dsRNAs or 21- to 23-nt-long short interfering (si) duplex RNAs, acting as effectors of RNAi. siRNAs recently have been shown to act as potent inducers of RNAi in cultured mammalian cells. However, studies of RNAi activated by long dsRNA are impeded by its nonspecific effects, mediated by dsRNA-dependent protein kinase PKR and RNase L. Here, we report that the RNAi response can be induced effectively by long dsRNA in nondifferentiated mouse cells grown in culture. Transfection of dsRNA into embryonal carcinoma (EC) P19 and F9 cells results in a sequence-specific decrease in the level of proteins expressed from either exogenous or endogenous genes. dsRNA-mediated inhibition of the reporter gene also occurs in mouse embryonic stem cells. The RNAi effect is mediated by siRNAs, which are generated by cleavage of dsRNA by the RNaseIII-like enzyme, Dicer. We demonstrate that extracts prepared from EC cells catalyze processing of dsRNA into approximately 23-nt fragments and that Dicer localizes to the cytoplasm of EC and HeLa cells.

Retinoic acid switches differential expression of FGF8 isoforms in LNCaP cells.

Retinoic acid (RA) is described as an inhibitor of prostate cancer cell growth. We utilized reverse transcription-polymerase chain reaction (RT-PCR) to analyze expression of different isoforms of fibroblast growth factor 8 (FGF8) in response to RA. Results in the prostate cancer cell line LNCaP show that whereas overall expression levels of FGF8 appear to remain constant, RA addition induces an inversion of the ratio between FGF8a and -b mRNAs. Along with this observed "isoform switch," unexpected expression of retinoic acid receptor alpha was detected. Although preliminary, these data allow one to hypothesize on the existence of a possible link between the morphogenic hormone RA and the regulation of the potent mitogen FGF8.

Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway.

B-Myb, a highly conserved member of the Myb oncoprotein family, is a 110 kDa sequence-specific DNA binding protein expressed in virtually all proliferating cells. B-myb expression reaches its maximum at the G1/S phase boundary and during the S phase of the cell cycle. We have previously shown that B-Myb activity is cell cycle regulated and it is controlled by the antagonistic effects of cyclin D1 and A. Here we show that ectopic expression of cyclin A causes a pronounced reduction of B-Myb protein level. We provide evidence that in addition to triggering B-Myb activity an important effect of cyclin A is to facilitate multiple ubiquitination of B-Myb. The C-terminal domain of B-Myb is of key importance in mediating this effect of cyclin A. Contrary to full-length B-Myb, a C-terminal deletion mutant displays activity irrespective of cyclin A expression, does not undergo ubiquitination, and its half-life is not affected by cyclin A. Ectopic expression of either Cdc34 or the F-box protein p45Skp2, respectively the E2 and E3 components of a ubiquitination pathway that regulates the G1/S transition, accelerates degradation of B-Myb. We show that B-Myb physically and functionally interacts with components of the Cdc34-SCFp45Skp2 ubiquitin pathway and propose that B-Myb degradation may be required for controlling the correct alternation of events during progression through the cell division cycle. Oncogene (2000).

A new class of HIV-1 Tat antagonist acting through Tat-TAR inhibition.

The main transcriptional regulator of the human immunodeficiency virus, the Tat protein, recognizes and binds to a small structured RNA element at the 5' end of every viral mRNA, termed TAR. On the basis of published structural data of the molecular interactions between TAR and Tat-related peptides, we defined requirements for potential low-molecular weight inhibitors of TAR recognition by the Tat protein. In accordance with the resulting concept, a series of compounds was synthesized. In vitro evaluation of their potential to directly interfere with Tat-TAR interaction was used to define a new chemical class of potent Tat antagonistic substances. The most active compound competed with Tat-TAR complexation with a competition dose CD50 of 22 nM in vitro and blocked HIV expression in a cellular Tat transactivation system with an IC50 of 1.2 microM. The close relation between structural features of the interaction between TAR and a new type of inhibitory agent, "In-PRiNts" (for inhibitor of protein-ribonucleotide sequences), such as CGP 40336A and those of the Tat-TAR complex was confirmed by RNase A footprinting and by two-dimensional NMR. Structural implications for the complex between this class of compounds and TAR RNA will be presented.