Novel halogenated sulfonamides inhibit the growth of multidrug resistant MCF-7/ADR cancer cells.

In this report, we describe the synthesis of halogenated benzenesulfonamide compounds and their ability to inhibit the growth of HeLa, MCF-7 and MCF-7/ADR tumor cells in vitro. The multidrug resistance (MDR) phenotype of certain cells does not affect their sensitivity to these compounds. These agents belong to a family of compounds previously shown to bind irreversibly to cysteine-239 of beta-tubulin. Consistent with this mechanism of action, the cytotoxicities of these compounds appear to correlate with their ability to undergo nucleophilic aromatic substitution.

Identification of a nuclear receptor for bile acids.

Bile acids are essential for the solubilization and transport of dietary lipids and are the major products of cholesterol catabolism. Results presented here show that bile acids are physiological ligands for the farnesoid X receptor (FXR), an orphan nuclear receptor. When bound to bile acids, FXR repressed transcription of the gene encoding cholesterol 7alpha-hydroxylase, which is the rate-limiting enzyme in bile acid synthesis, and activated the gene encoding intestinal bile acid-binding protein, which is a candidate bile acid transporter. These results demonstrate a mechanism by which bile acids transcriptionally regulate their biosynthesis and enterohepatic transport.

Selective, covalent modification of beta-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors.

Microtubules are linear polymers of alpha- and beta-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis. Here we describe a synthetic compound, 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the beta1, beta2, and beta4 isotypes of beta-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymerization. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy. Subsequently, these cells undergo apoptosis. Furthermore, T138067 exhibits cytotoxicity against tumor cell lines that exhibit substantial resistance to vinblastine, paclitaxel, doxorubicin, and actinomycin D. T138067 is also equally efficacious in inhibiting the growth of sensitive and multidrug-resistant human tumor xenografts in athymic nude mice. These observations suggest that T138067 may be clinically useful for the treatment of multidrug-resistant tumors.

Novel antineoplastic agents with efficacy against multidrug resistant tumor cells.

A novel series of pentafluorobenzenesulfonamides has been shown to inhibit the growth of a variety of human tumor cell lines. Among the cell types against which these agents were evaluated were the multidrug resistant (MDR) cell lines MCF-7/ADR and P388/ADR. The cytotoxic activity of members of this series of compounds was not affected by the multidrug resistant pump in MCF-7/ADR or P388/ADR cells.

Light modulates the spatial patterns of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in Arabidopsis thaliana.

Although the coordinated biosynthesis of isoprenoid compounds is thought to be essential to the normal processes of plant growth and development, the mechanisms that regulate the mevalonate pathway in plants are not well understood. As the first committed step in the pathway, the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonic acid by HMG CoA reductase and the regulation of the genes encoding this enzyme have been implicated in the network that controls isoprenoid biosynthesis in higher plants. Using histochemical staining for beta-glucuronidase, as well as conventional RNA hybridization analysis, the temporal and spatial regulation of HMG1, one of the genes encoding HMG CoA reductase in the crucifer Arabidopsis thaliana, has been characterized. Furthermore, the HMG1 promoter is shown to be differentially responsive to illumination in different organs, and promoter activation by light deprivation is confined primarily to immature leaves. In contrast, expression of the HMG1 gene in roots is confined to the elongation zone and is not responsive to illumination. Light-mediated regulation of HMG1 expression is shown to be an organ-autonomous response that depends on direct illumination, and environmental cues regarding light do not appear to be exchanged between different organs in Arabidopsis. These studies reveal several new features of HMG1 regulation, and indicate that the high levels of HMG CoA reductase expression detected in immature leaves may be primarily attributed to the dark-induced expression of HMG1, and that HMG1 is expressed at low levels throughout the plant in response to light. Thus, environmental cues interact with the developmental program to define the pattern of HMG1 gene expression in Arabidopsis.

Light suppresses 3-Hydroxy-3-methylglutaryl coenzyme A reductase gene expression in Arabidopsis thaliana.

3-Hydroxy-3-methylglutaryl (HMG) coenzyme A reductase mRNA accumulates preferentially in dark-grown Arabidopsis plants. As one step toward understanding the role that light plays in the regulation of the mevalonate pathway in plants, we characterized the suppression of HMG1 gene expression in response to illumination wavelength, duration, and fluence rate. The accumulation of HMG1 mRNA by dark treatment is suppressed by continuous exposure to white light and is dependent on the amount of light perceived during the period of illumination. By using promoter/reporter gene fusions we also demonstrate that this reaction is mediated by cis-acting elements that reside in the Arabidopsis HMG1 promoter and, therefore, is likely to be controlled at the transcriptional level. HMG1 expression is differentially responsive to continuous blue and red light but not to far-red light. In contrast, changes in HMG1 mRNA levels were not observed in response to brief light pulses of any spectrum, suggesting that continuous illumination is required for sustained and maximal suppression of HMG coenzyme A reductase expression. Taken together, these data indicate that light-mediated control of the HMG1 gene is mediated by a regulatory circuit that monitors aspects of both spectral quality and fluence and involves either multiple photoreceptors or a single photoreceptor that is differentially sensitive to both blue and red light.

Co-expression of native and introduced genes reveals cryptic regulation of HMG CoA reductase expression in Arabidopsis.

In eukaryotes, all isoprenoid compounds share a common precursor, mevalonic acid, whose synthesis is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. As one step towards a better understanding of the role that this enzyme plays in coordinating isoprenoid biosynthesis in plants, Arabidopsis thaliana HMG CoA reductase was ectopically expressed in transgenic Arabidopsis plants. By using this molecular genetic approach, several novel and fundamental observations have been made regarding isoprenoid biosynthesis in Arabidopsis. First, it was demonstrated that the overexpression of authentic Arabidopsis HMG CoA reductase is not sufficient to alter the bulk synthesis and accumulation of the abundant end products of the plant isoprenoid pathway. Second, active transcription of the transgene appears to co-activate and deregulate expression of the native gene, resulting in a striking elevation of HMG CoA reductase mRNA levels. Finally, although very high levels of HMG CoA reductase mRNA were expressed in these transgenic plants, only modest increases in enzyme activity could be detected. Taken together, these data suggest that HMG CoA reductase expression is regulated at multiple levels in plants as well as animals, and they provide a foundation for elucidating the molecular mechanisms for mevalonate regulation in A. thaliana.

Molecular and genetic analysis of signal transduction in higher plants.

Plant scientists have long recognized the complexity of responses to environmental and hormonal signals that provide the basis for plant growth and development. The systematic isolation and analysis of mutations that disrupt signal transduction and prevent the appropriate physiological response provides an important resource for studying these processes and, ultimately, for describing the molecular events that control growth and developmental responses in plants.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis thaliana is structurally distinct from the yeast and animal enzymes.

We have isolated the Arabidopsis thaliana gene (HMG1) encoding 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; (S)-mevalonate:NAD+ oxido-reductase (CoA-acylating), EC 1.1.1.88], the catalyst of the first committed step in isoprenoid biosynthesis. cDNA copies of the plant gene were identified by hybridization with a short, highly conserved segment of yeast HMG-CoA reductase as probe. DNA sequence analysis reveals that the COOH-terminal domain of the Arabidopsis HMG-CoA reductase (containing the catalytic site of the enzyme) is highly conserved with respect to the yeast, mammalian, and Drosophila enzymes, whereas the membrane-bound amino terminus of the Arabidopsis protein is truncated and lacks the complex membrane-spanning architecture of the yeast and animal reductases. Expression of the Arabidopsis gene from the yeast GAL1 promoter in a yeast mutant lacking HMG-CoA reductase activity suppresses the growth defect of the yeast mutant. Taken together, the sequence similarity to other cloned HMG-CoA reductase genes and the suppression of the yeast hmg- mutant provide strong evidence that the novel Arabidopsis gene we have cloned encodes a functional HMG-CoA reductase enzyme.

Functional cooperativity between transcription factors UBF1 and SL1 mediates human ribosomal RNA synthesis.

The human ribosomal RNA promoter contains two distinct control elements (UCE and core) both of which are recognized by the sequence-specific DNA binding protein UBF1, which has now been purified to apparent homogeneity. The purified factor activates RNA polymerase I (RNA pol I) transcription through direct interactions with either control element. A second RNA pol I transcription factor, designated SL1, participates in the promoter recognition process and is required to reconstitute transcription in vitro. Although SL1 alone has no sequence-specific DNA binding activity, deoxyribonuclease I footprinting experiments reveal that a cooperative interaction between UBF1 and SL1 leads to the formation of a new protein-DNA complex at the UCE and core elements. In vitro transcription experiments indicate that formation of the UBF1-SL1 complex is vital for transcriptional activation by UBF1. Thus, protein-protein interactions between UBF1 and SL1 are required for targeting of SL1 to cis-control sequences of the promoter.

Analysis of clustered point mutations in the human ribosomal RNA gene promoter by transient expression in vivo.

We have mapped the cis regulatory elements required in vivo for initiation at the human rRNA promoter by RNA polymerase I. Transient expression in COS-7 cells was used to evaluate the transcription phenotype of clustered base substitution mutations in the human rRNA promoter. The promoter consists of two major elements: a large upstream region, composed of several domains, that lies between nucleotides -234 and -107 relative to the transcription initiation site and affects transcription up to 100-fold and a core element that lies between nucleotides -45 and +20 and affects transcription up to 1000-fold. The upstream region is able to retain partial function when positioned within 100-160 nucleotides of the transcription initiation site, but it cannot stimulate transcription from distances of greater than or equal to 600 nucleotides. In addition, we demonstrate, using mouse-human hybrid rRNA promoters, that the sequences responsible for human species-specific transcription in vivo appear to reside in both the core and upstream elements, and sequences from the mouse rRNA promoter cannot be substituted for them.

Human rRNA transcription is modulated by the coordinate binding of two factors to an upstream control element.

The human rRNA promoter contains two distinct cis-control sequences, the core and upstream control element (UCE), that serve as the target for binding cellular trans-activating proteins involved in transcription initiation by RNA polymerase I. One of these factors, SL1, has been extensively purified and shown to be a species-specific factor required to reconstitute in vitro RNA synthesis. DNAase footprinting revealed that although SL1 alone does not bind specifically to rRNA promoter sequences, a second factor, UBF1, recruits SL1 to the template and directs binding to an extended region encompassing sequences in the UCE. Analysis of mutant and human-mouse hybrid promoters indicate that protein-DNA interactions at the UCE modulate the efficiency of rRNA synthesis. Transcription from the human rRNA promoter appears to require an unusual set of protein-DNA transactions in which recognition and binding to an upstream cis-control element is carried out by one factor (UBF1), whereas activation requires an additional factor, SL1, acting in conjunction with UBF1 to trigger transcription.

Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I.

A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.

Regulation of human ribosomal RNA transcription.

We have used a cell-free polymerase I transcription system derived from HeLa cells to study the regulation of human rRNA synthesis. Analysis of deletion mutants spanning the start site of transcription at nucleotide +1 indicates that the control region affecting initiation of human rRNA synthesis is contained within sequences from nucleotides -158 to +18. This promoter region can be subdivided into (i) a central segment of approximately 40 base pair that is required for transcription and (ii) flanking sequences that influence the efficiency of transcription in vitro. We have examined the in vitro transcriptional activity of the human extract under various conditions that are thought to modulate rRNA synthesis in vivo. Cell-free extracts prepared from HeLa cells infected with adenovirus 2 synthesize human rRNA at levels greatly decreased relative to uninfected cell extracts. By contrast, in vitro transcription of human rRNA is stimulated 2- to 3-fold by the addition of purified simian virus 40 large tumor antigen to the transcription reaction. Moreover, a mutant tumor antigen known to be defective for rRNA activation in vivo is incapable of stimulating rRNA synthesis in vitro. The ability to detect these different regulatory phenomena in vitro provides us with an experimental basis for investigating the molecular mechanisms that control rRNA synthesis.

In vitro transcription of human ribosomal RNA genes by RNA polymerase I.

We have studied the initiation of human ribosomal RNA synthesis in vitro using a cell-free polymerase I transcription system derived from HeLa cells and cloned human ribosomal DNA containing the site of initiation for ribosomal RNA synthesis. Mapping of the RNA products by run-off assays and high-resolution S1 nuclease analysis indicated that transcription in vitro initiates at a unique site and that the RNA has the same 5' terminus as in vivo precursor ribosomal RNA isolated from nuclei of HeLa cells. To provide additional evidence for the initiation site of ribosomal RNA transcription, dinucleoside monophosphates complementary to a sequence on the template have been used as primers for ribosomal RNA synthesis. When the concentrations of the four nucleoside triphosphates in the transcription reaction were reduced to 10 microM, [alpha-32 P]GTP was no longer incorporated into the run-off transcript. Under these conditions, we then tested a variety of dinucleotides for their ability to initiate promoter-specific RNA synthesis, and found that GpC exhibited maximum stimulation. We have also determined that the human cell-free system exhibits a significant degree of template specificity and is able to transcribe ribosomal DNA derived from human and rhesus monkey cells but not from mouse cells.