Identification of OAT1/OAT3 as Contributors to Cisplatin Toxicity.

Cisplatin is among the most widely used anticancer drugs and known to cause a dose-limiting nephrotoxicity, which is partially dependent on the renal uptake carrier OCT2. We here report a previously unrecognized, OCT2-independent pathway of cisplatin-induced renal injury that is mediated by the organic anion transporters OAT1 and OAT3. Using transporter-deficient mouse models, we found that this mechanism regulates renal uptake of a mercapturic acid metabolite of cisplatin that acts as a precursor of a potent nephrotoxin. The function of these two transport systems can be simultaneously inhibited by the tyrosine kinase inhibitor nilotinib through noncompetitive mechanisms, without compromising the anticancer properties of cisplatin. Collectively, our findings reveal a novel pathway that explains the fundamental basis of cisplatin-induced nephrotoxicity, with potential implications for its therapeutic management.

hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.

hsp82 is one of the most highly conserved and abundantly synthesized heat shock proteins of eucaryotic cells. The yeast Saccharomyces cerevisiae contains two closely related genes in the HSP82 gene family. HSC82 was expressed constitutively at a very high level and was moderately induced by high temperatures. HSP82 was expressed constitutively at a much lower level and was more strongly induced by heat. Site-directed disruption mutations were produced in both genes. Cells homozygous for both mutations did not grow at any temperature. Cells carrying other combinations of the HSP82 and HSC82 mutations grew well at 25 degrees C, but their ability to grow at higher temperatures varied with gene copy number. Thus, HSP82 and HSC82 constitute an essential gene family in yeast cells. Although the two proteins had different patterns of expression, they appeared to have equivalent functions; growth at higher temperatures required higher concentrations of either protein. Biochemical analysis of hsp82 from vertebrate cells suggests that the protein binds to a variety of other cellular proteins, keeping them inactive until they have reached their proper intracellular location or have received the proper activation signal. We speculate that the reason cells require higher concentrations of hsp82 or hsc82 for growth at higher temperatures is to maintain proper levels of complex formation with these other proteins.

Sharp boundaries demarcate the chromatin structure of a yeast heat-shock gene.

In both induced and basally transcribed states, the chromatin structure of the yeast HSP82 heat-shock locus exhibits a remarkable degree of organization with respect to DNA sequence. The promoter region contains a constitutive DNase I hypersensitive site. The transcription unit is markedly sensitive to DNase I, and exhibits a sharp transition from a phased half- to a whole nucleosomal cleavage periodicity at the 3' end. Distant upstream and downstream regions are also organized into distinct arrays of phased nucleosomes. Each array is demarcated by DNase I hypersensitive sites that display internal protected regions, suggesting the presence of DNA binding proteins. In addition, since these sites are of mononucleosomal DNA length, they may acquire a nucleosomal structure under certain environmental conditions without disrupting flanking nucleosomal phasing frames. Thus, the HSP82 locus is organized into specific, phased, chromatin structures that appear to function in transcriptional initiation, RNA polymerase passage, transcriptional termination, and the establishment of chromatin-domain microenvironments.

Complete sequence of the heat shock-inducible HSP90 gene of Saccharomyces cerevisiae.

We have sequenced the yeast HSP90 gene, which encodes the (apparent) Mr = 90,000 heat shock-inducible protein of this organism. All sequences required for the heat shock-regulated expression of this gene reside on a segment of DNA containing no more than 273 nucleotides 5' to the transcription origin. Transcript mapping reveals that the mature hsp90 mRNA contains a 59-nucleotide 5' untranslated segment, a coding segment of 2130 nucleotides (sufficient to encode a protein of Mr = 81,419), and a 3' untranslated segment of 128 nucleotides. Although the sequences surrounding the coding region of the HSP90 gene are quite similar to those surrounding other sequenced yeast genes, we find as well a limited homology between sequences located 5' to this gene and the putative heat shock-regulatory sequences located 5' to the heat shock-inducible genes of Drosophila.

Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae.

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.

Identification and expression of a cloned yeast heat shock gene.

We have isolated the yeast HSP90 gene which encodes the Mr = 90,000 heat shock-inducible protein of this organism. When this gene is introduced into yeast on a multicopy plasmid vector, a dramatic increase is observed in the level of synthesis of the Mr = 90,000 heat shock-inducible protein. This protein overproduction is due to expression of the plasmid-borne HSP90 gene, which is under the same heat shock regulation as its chromosomal counterpart. The presence of an increased dosage of the HSP90 gene has no effect on the synthesis of the other major heat shock-inducible proteins and does not alter the heat shock-associated phenotype of thermal tolerance.

Alterations of transcription during heat shock of Saccharomyces cerevisiae.

The pattern of polyadenylated RNA of the yeast Saccharomyces cerevisiae changes dramatically upon heat shock. By in vitro translation, we have demonstrated that a 2.9-kilobase heat shock RNA encodes the Mr = 90,000 yeast heat shock protein. Heat shock-responsive genes have been isolated by differential plaque filter hybridization of a recombinant library of yeast DNA inserted in the vector lambda Charon 4. The putative yeast gene products of a number of these recombinants molecules has been determined by in vitro translation of hybrid-selected RNA. We have used one of these hybrid phages (lambda Yhsil) to demonstrate that the heat shock-induced alteration in the level of the 2.9-kilobase polyadenylated RNA which encodes the Mr = 90,000 yeast heat shock protein is regulated at the level of transcription.

alpha-Factor-mediatd modification of a 32P-labeled protein by MATa cells of Saccharomyces cerevisiae.

Addition of the polypeptide mating pheromone alpha-factor to haploid MATa cells of Saccharomyces cerevisiae results in the modification of a 32P-labeled protein (P17) with an apparent Mr of 17,000 to a form having an apparent Mr of 17,500 (P17). 32P associated with both P17 and P17 exhibits an unusually rapid rate of turnover. The conversion of P17 to P17 precedes the appearance of morphologically abnormal cells and, in contrast to other responses elicited by this pheromone, this change in apparent molecular weight does not require protein synthesis. Upon removal of alpha-factor, the P17/P17 ratio returns to pretreatment levels.

Alterations in translatable ribonucleic acid after heat shock of Saccharomyces cerevisiae.

Changes in populations of translatable messenger ribonucleic acids (mRNA's) after heat shock of Saccharomyces cerevisiae were examined and found to correlate very closely with transient alterations in patterns of in vivo protein synthesis. Initial changes included an increase in translatable species coding for polypeptides synthesized during heat shock; this increase was found to be dependent on transcription but did not require ongoing protein synthesis. A decrease was observed in the level of translatable mRNA's coding for polypeptides whose synthesis was repressed after heat shock. This decrease was much more rapid than can be explained solely by termination of transcription. Requirements for this rapid loss of RNA from the translatable pool included both transcription and an active rna1 gene product but not protein synthesis. After the initial changes in translatable RNA induced by heat shock, the patterns of both in vivo and in vitro translation products began to revert to the preshock levels. This recovery period, unlike the earlier changes, was dependent upon a requisite period of protein synthesis.

Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae.

When a mating type cells of Saccharomyces cerevisiae are exposed to the mating pheromone alpha-factor in liquid cultures, there is a time-dependent loss of alpha-factor activity from the culture fluid. This loss of biological activity can be directly correlated with the proteolysis of the pheromone by a mating type cells. The metabolism of alpha-factor by a mating type cells may be measured by using either in vitro 125I-labeled or in vivo 35S-labeled pheromone. Addition of chloroquine to growing cultures of a mating type cells at concentrations which cause no detectable alterations in cell growth produces a potentiation of alpha-factor mediated cell cycle arrest. This potentiation of alpha-factor activity is directly correlated with the inhibition of alpha-factor proteolysis. Thus, while proteolytic digestion of alpha-factor appears to be related to the mechanism whereby a mating type cells "detoxify" alpha-factor and recover from cell cycle arrest, proteolysis of the mating factor is not necessary for alpha-factor mediated cell cycle arrest.

Altered patterns of protein synthesis induced by heat shock of yeast.

The products of protein synthesis from exponential phase cultures of Saccharomyces cerevisiae grown at 23 °C or at 36 °C appear to be essentially identical. However, yeast cells respond to a shift in culture temperature from 23 °C to 36 °C with the rapid de novo synthesis of a polypeptide species of molecular weight 100,000. Within 60-90 min after the shift this polypeptide represents approximately 2.5% of the total cellular protein, a 5-10 fold increase over the preshift level. The level of this polypeptide then decreases with continued growth of the cells at 36 °C. Analyses by SDS-polyacrylamide gel electrophoresis of polypeptides obtained from cells pulse labeled with [(35)S]methionine demonstrate that following a temperature shift from 23 °C to 36 °C the synthetic rate of the 100,000 molecular weight polypeptide (as well as a number of other polypeptide species) increases to a level at least 10 fold higher than that observed prior to the shift. A concomittant decrease is observed in the synthesis of a large number of polypeptide species which were actively synthesized before the shift. Maximum changes in synthetic rates are observed 20-30 min after the shift and preshift synthetic patterns are regained within 60-90 min. Synthetic changes of the same magnitude and time course can be produced by short (20-30 min) exposures to 36 °C implicating a heat shock response. Several of the transiently induced polypeptides, including the 100,000 molecular weight species, show an affinity for DNA as determined by DNA-cellulose chromatography.

Reaction order of Saccharomyces cerevisiae alpha-factor-mediated cell cycle arrest and mating inhibition.

Alpha-factor-mediated cell cycle arrest and mating inhibition of a mating-type cells of Saccharomyces cerevisiae have been examined in liquid cultures. Cell cycle arrest may be monitored unambiguously by the appearance of morphologically abnormal cells after administration of alpha factor, whereas mating inhibition is determined by comparing the mating efficiency in the absence or presence of added alpha factor. For both cell cycle arrest and mating inhibition, a dose-dependent response may be observed at limiting concentrations of the pheromone. If cell cycle arrest and mating inhibition require a small number of alpha-factor molecules, one might expect that responsive/nonresponsive cells = K(alpha factor)(N) where N is the order of dependence of cell cycle arrest (or mating inhibition) on alpha-factor concentration. The value of N has been determined to be 0.98 +/- 0.18 (standard error of the mean) for cell cycle arrest and 1.08 +/- 0.32 for mating inhibition. These results support the notion that saturation of a single site by alpha factor is sufficient to cause cell cycle arrest or mating inhibition of a mating-type cells.