FU chemoradiotherapy continues to play an important role in the management of many cancer sites. During the last four decades, optimal dosing schedules have produced a therapeutic gain. The introduction of oral prodrug analogs will likely further improve the results of FU therapy in several organ systems, such as the rectum, head and neck, and esophagus.
Mevalonate kinase [ATP:(R)-mevalonate 5-phosphotransferase, EC 2.7.1.36] may be a regulatory site in the cholesterol biosynthetic pathway, and a mutation in the gene coding for this enzyme is thought to cause the genetic disease mevalonic aciduria. To characterize this enzyme, a rat liver cDNA library was screened with a monospecific antibody, and a 1.7-kilobase cDNA clone coding for mevalonate kinase was isolated. A mutation in the gene coding for mevalonate kinase is presumed to be the cause of the recently discovered genetic disease mevalonic aciduria (4, 5). The genetic disease is transmitted as an autosomal recessive trait, and there are six reported cases (6). Subjects with mevalonic aciduria have extremely high levels of mevalonate in their plasma and urine, and cells from these subjects have <10% of the normal levels of mevalonate kinase activity (4-6). As a first step in identifying the molecular defect causing mevalonic aciduria and to study the regulation of mevalonate kinase activity, we have isolated and characterized a cDNA clone coding for rat mevalonate kinase.* Data are also presented to show that long-term regulation of enzyme activity in rat liver is controlled by changes in the levels of mevalonate kinase mRNA. MATERIALS AND METHODSPreparation of the Antiserum. Monospecific antisera to rat mevalonate kinase was prepared in rabbits by using the purified enzyme (3), and the IgG fraction was collected by affinity chromatography (7).Isolation of a cDNA Clone Coding for Mevalonate Kinase. A Agtll cDNA library (8) derived from mRNA purified from the livers of rats treated with diets containing 5% cholestyramine and 0.1% lovastatin was kindly provided by Peter Edwards (UCLA). Positive clones were identified by immunoscreening (9). To obtain a full-length cDNA clone, the cDNA insert from one of the clones was isolated and radiolabeled by random priming (10) to a specific activity of 109 cpm per Ag ofDNA. The radiolabeled probe was used to screen -2 x 105 recombinants. Plaque hybridization was performed as described (8), after which the filters were washed at 680C with 2 x SSC (1x SSC = 0.15 M NaCI/0.015 M sodium citrate, pH 7) containing 0.1% SDS, followed by washes with 0.lx SSC containing 0.1% SDS. Fifteen cDNA clones were isolated. The cDNA inserts were subcloned into the pGEM-3Z vector (Promega) for restriction enzyme mapping or into M13 vectors for DNA sequencing.DNA Sequencing. The DNA sequence of the cDNA was determined by the dideoxynucleotide chain-termination method (11). Sequencing reactions using the Klenow fragment of DNA polymerase I (New England Biolabs) or the modified T7 DNA polymerase (Sequenase, United States Biochemicals) were performed following the manufacturer's protocol. The DNA and protein sequences were aligned using the Intelligenetics computer program, and the EMBL/ GenBank and PIR data bases were searched for homologies to the DNA sequence and protein sequence of mevalonate kinase.Expression of the Mevalonate Kinase cDNA in Saccharomyces cerevisiae. A 1.3-kilobase...
This paper describes a method, designated "receptor-dependent photosensitization," by which the receptor-mediated endocytosis of low density lipoprotein (LDL) can be used to deliver a photosensitizing agent, pyrene, to cultured human and animal cells. The hydrophobic core of LDL is extracted and replaced with pyrene covalently coupled to cholesteryl oleate. This reconstituted LDL enters cells in significant amounts only when the cells express LDL receptors, resulting in the accumulation of pyrene cholesteryl oleate within lysosomes. Subsequent exposure of the cells to ultraviolet light leads to cell death. Cells killed by this technique include normal and simian virus 40-transformed human fibroblasts, human A431 epidermal carcinoma cells, Chinese hamster ovary cells, and mouse L cells, all of which express LDL receptors. Mutant fibroblasts from a patient with homozygous familial hypercholesterolemia, which lack LDL receptors, do not take up. significant amounts of the pyrene-containing LDL and are not killed by subsequent exposure to light. The current experiments establish the feasibility of receptor-dependent photosensitization as an efficient and selective method for killing cultured human and animal cells.By virtue of the extreme selectivity exhibited by cell surface receptors, the process of receptor-mediated endocytosis theoretically should allow the controlled delivery of biologically active molecules to human and animal cells. Two unique properties of plasma low density lipoprotein (LDL) make it a promising vehicle for this-purpose. First, LDL contains a lipid core composed of about 1500 molecules of cholesteryl ester. This core can be extracted and replaced with biologically active lipids without affecting the functionally active form of the particle (1). Second, LDL enters animal cells by binding to surface receptors that mediate its cellular uptake (2). Once inside the cell, the LDL is carried to lysosomes where the lipoprotein's protein-phospholipid coat is removed and the cholesteryl ester core is exposed. This uptake process normally functions to supply cholesterol to hepatic and extrahepatic cells. In the steady state, LDL uptake proceeds at a constant rate, each receptor carrying one particle of LDL into the cell approximately once every 10 min (3). After each round of uptake, the receptor is recycled and reutilized (4). Thus, large amounts of LDL are transported into the cell even though a cell may have relatively few receptors on its surface.Among the biologically active molecules that can be carried in the core of LDL are toxic compounds. Such toxic LDL particles can be used to kill cells that possess LDL receptors (5, 6). Inasmuch as these LDLs will bind to and enter only those cells that have functional LDL receptors, they permit the survival, and hence the selection, of resistant cells that have acquired mutations that disrupt receptor-mediated endocytosis (6).In order to be effective in killing cells via the LDL receptor pathway, a substance must filfill at least two criteria: (i)...
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