S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism.

The injurious consequences of ionizing radiation (IR) to normal human cells and the acquired radioresistance of cancer cells represent limitations to cancer radiotherapy. IR induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair or apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly and independent of DNA damage, IR activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting radiation-induced apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in IR-resistant cancer cells. S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism. We show that SPL modulates the kinetics of DNA repair, speed of recovery from G2 cell cycle arrest and the extent of apoptosis after IR. SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death. Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection.

Overexpression of fatty acid synthase in SKBR3 breast cancer cell line is mediated via a transcriptional mechanism.

Overexpression of fatty acid synthase (FAS) in certain breast, prostate and ovarian tumors has been correlated with aggressive cancer phenotype and poor prognosis. The objective of this study was to use a breast cancer-derived cell line, SKBR3, as a model to define the underlying mechanism for overexpression of FAS in cancer cells. Different stages of gene expression where overproduction of FAS could potentially be achieved were investigated. Whereas gross chromosomal rearrangement at the FAS locus, amplification of the FAS gene, increases in FAS message stability and longer half-life of the FAS protein were not detected, an increase in the rate of transcription of the FAS gene, and consequently a higher abundance of FAS-mRNA, was found to be primarily responsible for FAS overexpression in this cell line.

The PLB2 gene of Saccharomyces cerevisiae confers resistance to lysophosphatidylcholine and encodes a phospholipase B/lysophospholipase.

The PLB1 gene of Saccharomyces cerevisiae encodes a protein that demonstrates phospholipase B, lysophospholipase, and transacylase activities. Several genes with significant homology to PLB1 exist in the S. cerevisiae genome, raising the possibility that other proteins may contribute to the total phospholipase B/lysophospholipase/transacylase activities of the cell. We report the isolation of a previously uncharacterized gene that is highly homologous to PLB1 and that, when overexpressed, confers resistance to 1-palmitoyllysophosphatidylcholine. This gene, which is located adjacent to the PLB1 gene on the left arm of chromosome XIII and which we refer to as PLB2, encodes a phospholipase B/lysophospholipase. Unlike PLB1, this gene product does not contain significant transacylase activity. The PLB2 gene product shows lysophospholipase activity toward lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylethanolamine. Whereas deletion of either PLB1 or PLB2 resulted in the loss of 80% of cellular lysophospholipase activity, a plb1/plb2 double deletion mutant is completely devoid of lysophospholipase activity toward the preferred substrate lysophosphatidylcholine. Overexpression of PLB2 was associated with an increase in total cellular phospholipase B/lysophospholipase activity, as well as the appearance of significant lysophospholipase activity in the medium. Moreover, overexpression of PLB2 was associated with saturation at a higher cell density, and an increase in total cellular phospholipid content, but no change in phospholipid composition or fatty acid incorporation into cellular lipids. Deletion of PLB2 was not lethal and did not result in alteration of membrane phospholipid composition or content. PLB2 gene expression was found to be maximal during exponential growth conditions and was decreased in late phase, in a manner similar to other genes involved in phospholipid metabolism.

YAP1 confers resistance to the fatty acid synthase inhibitor cerulenin through the transporter Flr1p in Saccharomyces cerevisiae.

In this study, we utilized a genetic approach to identify genes which render yeast cells resistant to cerulenin (Cer), a potent and noncompetitive inhibitor of fatty acid synthase (FAS). Overexpression of the yeast transcription factor Yap1p was found to confer Cer resistance (CerR). This resistance was shown to be less pronounced in a strain deleted for YCF1, a multidrug resistance ABC transporter, supporting previous observations that implicated YCF1 in mediating CerR. However, isolation of YAP1 as a high-copy CerR gene in a ycf1delta strain suggested that YAP1-induced CerR was mediated by additional downstream effectors. Overexpression of neither glutathione reductase nor a predicted aryl alcohol dehydrogenase (the products of two YAP1-regulated genes involved in detoxification) conferred CerR. Overexpression of ATR1, another YAP1-regulated gene previously implicated in conferring resistance to a number of cytotoxic drugs, was also incapable of making cells resistant to Cer. In contrast, overexpression of Flr1p, a yeast transporter of the major facilitator superfamily which is also under the control of YAP1, was sufficient to confer CerR in an otherwise wild-type background. Moreover, CerR was markedly diminished in a strain deleted for FLR1. These findings implicate members of both of the transporter superfamilies involved in multiple drug resistance (MDR) in the acquisition of CerR in yeast. Furthermore, our studies indicate that yeast may be a useful model system in which to investigate the role of FAS in cancer biology and the effects of Cer on eukaryotic cell growth.

Regulatory elements in the first intron of the rat fatty acid synthase gene.

Sequence elements have been identified within the 1.2 kb-long first intron of the fatty acid synthase (FAS) gene that mediate both positive and negative effects on transcription. The negative regulatory element, when positioned downstream of either the FAS or simian virus 40 promoter, down-regulates the expression of a coupled reporter gene in an orientation-dependent manner. Sequences mediating this effect have been mapped, by deletion mutagenesis, to two regions approximately within nucleotides +405 to +768 and +924 to +1083. Both regions contain sequence elements that are strongly protected from DNase I digestion by nuclear extracts prepared from liver, but not by those prepared from spleen. The results of run-on assays performed with nuclei derived from tissues that express FAS at either high or low levels indicate that the different rates of transcription of the endogenous FAS gene result from differences in the extent of initiation, so it is unlikely that the negative effect is caused by transcriptional pausing in the first intron. The positive element maps to nt +292 to +297 and corresponds to an authentic binding site for upstream stimulatory factor (USF). This USF-binding element can up-regulate transcription from a heterologous promoter in a position- and orientation-independent manner. However, in the context of the entire FAS first intron, the effect of the USF-binding site is masked unless the effect of the negative elements is ablated by mutagenesis. These results suggest that the dominant negative element of the first intron may play a role in determining the tissue-specific expression of the FAS gene.

Transcriptional regulation of the rat fatty acid synthase gene: identification and functional analysis of positive and negative effectors of basal transcription.

The gene for fatty acid synthase (FAS), which contains both GC-rich sequences and a TATA box in its promoter region, is expressed in a tissue-specific manner in response to developmental, nutritional and hormonal signals. Here we report the identification of sequence elements in the 5'-flanking region responsible for modulation of basal promoter activity. Transient transfection of H4IIE hepatoma cells and 3T3-30A5 preadipocytes with plasmids containing the chloroamphenicol acetyltransferase gene driven by FAS promoter sequences of different lengths revealed that two regions between nucleotides -249 and -30 contain elements capable of enhancing transcription. One of these positive regulatory elements was localized to nucleotides -241/-236 using DNase I footprinting, electrophoretic mobility-shift assays and mutagenesis. The sequence element is a typical GC box and the nuclear protein binding to this region appears immunochemically indistinguishable from Sp1. The second positive regulatory element, an inverted CCAAT box, was localized to nucleotides -98/-92 by electrophoretic mobility-shift assays and mutagenesis. A putative negative regulatory element, initially identified by reporter gene transfection experiments, was localized between nucleotides -319 and -301 by DNase I footprinting, electrophoretic mobility-shift assays and deletion mutagenesis; this region consists of 78% G residues. In conclusion, initiation of FAS transcription from a single start site is enhanced by the presence of an adjacent TATA motif, an inverted CCAAT box and an upstream binding site for the transcription factor Sp1; further modulation of transcription is achieved through complex interactions between these promoter elements and an upstream negative regulatory element.

Identification of an inverted CCAAT box motif in the fatty-acid synthase gene as an essential element for modification of transcriptional regulation by cAMP.

The antagonistic effect of cAMP on the insulin-induced expression of fatty acid synthase (FAS) in liver could be mimicked in vitro using H4IIE hepatoma cells, both by measuring the response of the endogenous FAS gene and by assaying expression of transfected reporter genes containing promoter elements of the FAS gene. 5'-Deletion analysis and replacement mutagenesis revealed that an essential element required for cAMP antagonism of the insulin effect is an inverted CCAAT box located between nucleotides -99 and -92. DNase I foot-printing and gel shift analysis revealed that this region can bind a protein present in nuclei of liver and spleen, organs that express high and undetectable levels of FAS, respectively. This protein is not a CCAAT/enhancerbinding protein, C/EBP. Thus, the FAS gene appears unusual in that the sequence element required for transcriptional regulation by cAMP is neither a cAMP response element (CRE) nor a binding site for AP-1, AP-2, or C/EBP. These results suggest that essential to the regulation of FAS transcription by cAMP is the interaction of an inverted CCAAT box motif with a constitutively produced trans-acting factor that either itself undergoes modification in response to cAMP or associated with a protein that is produced or modified by cAMP exposure.

Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway.

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus.

Repression and catabolite repression of the lactose operon of Staphylococcus aureus.

The lacR gene encodes the repressor of the lactose operon of S. aureus. The nucleotide sequence of this gene and the promoter-operator region of the operon are reported. The lacR gene encodes a protein with a molecular weight of 28,534. This protein was found to share sequence homology with the DeoR protein, the repressor of the E. coli deoxyribonucleotide operon. Directly and invertedly repeated sequences were found associated with the promoter for the structural genes of the operon. These sequences were examined by site-directed mutagenesis and found to be important in repressor binding and in the binding of a catabolite repressor. Evidence is presented in support of a model for catabolite repression of the operon which involves a negative-acting transcriptional regulator which binds to the promoter region of the operon and prevents transcription.

Cloning and characterization of the repressor gene of the Staphylococcus aureus lactose operon.

The genes responsible for utilization of lactose in Staphylococcus aureus are organized as an inducible operon, with galactose 6-phosphate being the intracellular inducer. To clone the repressor gene of this operon, we constructed an integration vehicle carrying 1.9 kilobases (kb) of DNA sequences from a region upstream of the structural genes of the operon. Through integration and subsequent rescue of this plasmid, we were able to clone approximately 7 kb of staphylococcal chromosomal DNA. We have shown that the plasmid insert complemented lac constitutive mutants. This repressor activity was localized to a 1.8-kb DNA fragment and, through maxicell analysis, was shown to correlate with the presence of a polypeptide with an apparent molecular weight of 32,000. Furthermore, a region between the repressor gene and the other genes of the operon was identified which, when carried on multicopy plasmids, resulted in expression of the operon in the absence of any exogenous induction. This region may represent an operator-type element capable of titrating repressor molecules away from chromosomal operator, allowing transcription of the operon in the absence of induction.