Upregulation of Bfl-1/A1 in leukemia cells undergoing differentiation by all-trans retinoic acid treatment attenuates chemotherapeutic agent-induced apoptosis.

All-trans retinoic acid (ATRA) induces differentiation of NB4 and HL-60 leukemia cells, but not R4 and HL-60/Res cells. Three agents used in cancer therapy, doxorubicin (Dox), arsenic trioxide (As(2)O(3)) and paclitaxel, induce apoptosis, but not differentiation, in all of these cell lines. The induction of apoptosis by these agents is decreased in ATRA-pretreated NB4 and HL-60 cells, but not in ATRA-pretreated R4 and HL-60/Res cells. The level of Bcl-2 protein is decreased by ATRA treatment in NB4, HL-60 and HL-60/Res cells. The level of Mcl-1 protein is increased by ATRA treatment in NB4 and R4 cells, but not in HL-60 and HL-60/Res cells. Bfl-1/A1 mRNA is not expressed in these cell lines, however, its expression is markedly induced by ATRA treatment in NB4 and HL-60 cells, but not in R4 or HL-60/Res cells, which correlates with inhibition of apoptosis. Inhibiting Bfl-1/A1 mRNA upregulation in ATRA-pretreated NB4 cells using small interfering RNA (siRNA) partly recovers cell sensitivity to Dox-induced apoptosis. These data demonstrate that ATRA induction of Bfl-1/A1 in differentiated NB4 and HL-60 cells contributes to a loss of sensitivity to chemotherapy-induced apoptosis.

Gonadotropin-releasing hormone receptor-coupled gene network organization.

An early gene cDNA microarray was developed to study genes that are regulated immediately following gonadotropin-releasing hormone (GnRH) receptor activation. 956 selected candidate genes were printed in triplicate, a t statistic-based regulation algorithm was used for data analysis, and the response to GnRH in a time course from 1 to 6 h was determined. Measurements were highly reproducible within arrays, between arrays, and between experiments. Accuracy and algorithm reliability were established by real-time polymerase chain reaction assays of 60 genes. Gene changes ranging from 1.3- to 31-fold on the microarray were confirmed by real-time polymerase chain reaction. Many of the genes were found to be highly regulated. The regulated genes identified were all elevated at 1 h of treatment and returned nearly or completely to baseline levels of expression by 3 h of treatment. This broad, robust, and transient transcriptional response to constant GnRH exposure includes modulators of signal transduction (e.g. Rgs2 and IkappaB), cytoskeletal proteins (e.g. gamma-actin), and transcription factors (e.g. c-Fos, Egr1, and LRG21). The interplay of the activators, repressors, and feedback inhibitors identified embodies a combinatorial code to direct the activity of specific downstream secondary genes.

Medaka eyeless is the key factor linking retinal determination and eye growth.

The complete absence of eyes in the medaka fish mutation eyeless is the result of defective optic vesicle evagination. We show that the eyeless mutation is caused by an intronic insertion in the Rx3 homeobox gene resulting in a transcriptional repression of the locus that is rescued by injection of plasmid DNA containing the wild-type locus. Functional analysis reveals that Six3- and Pax6- dependent retina determination does not require Rx3. However, gain- and loss-of-function phenotypes show that Rx3 is indispensable to initiate optic vesicle evagination and to control vesicle proliferation, by that regulating organ size. Thus, Rx3 acts at a key position coupling the determination with subsequent morphogenesis and differentiation of the developing eye.

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes.

Many cell fate decisions in higher animals are based on intercellular communication governed by the Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of Hairless (Su(H)) mediated transcription of target genes. In Drosophila, the majority of Notch target genes known so far is located in the Enhancer of split complex (E(spl)-C), encoding small basic helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. Here we show that the E(spl)-C contains three additional Notch responsive, non-bHLH genes: m4 and ma are structurally related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide.

In vivo structure-function analysis of Drosophila Hairless.

Hairless plays an important role as the major antagonist in the Notch signalling pathway in Drosophila. It appears to be a direct inhibitor of the signal transducer Su(H). Hairless encodes a pioneer protein which was dissected in a structure-function analysis; a series of deletion constructs was tested for wild type and gain of function activity in the fly as well as for Su(H) binding. Thereby, the Hairless protein was subdivided into the absolutely essential Su(H)-binding domain, similarly important N- and C-terminal domains and a central antimorphic domain. Therefore, Hairless protein might have additional functions apart from Su(H) binding and may antagonize Notch mediated cell-cell communication in a more complex way than currently anticipated.