SAS, a gene amplified in human sarcomas, encodes a new member of the transmembrane 4 superfamily of proteins.

Amplification of 12q13-14 occurs in a subset of human sarcomas including malignant fibrous histiocytoma and liposarcoma. This chromosomal region has previously been found to include a number of growth-related genes including the GLI proto-oncogene and the p53-associated protein, MDM2. We now report the characterization of SAS (sarcoma amplified sequence), a novel transcript found in this region. Sequence analysis demonstrates that SAS is a novel member of a transmembrane protein family (transmembrane 4 superfamily or TM4SF) thought to be involved in growth-related cellular processes. This observation adds a TM4SF protein to the cluster of genes at 12q13-14 frequently amplified in human sarcomas.

Inhibition of endothelial cell prostaglandin H synthase gene expression by naproxen.

Naproxen is a potent anti-inflammatory drug whose action is attributed to inhibition of prostaglandin biosynthesis. In view of our recent discovery that aspirin and sodium salicylate are capable of reducing cellular levels of prostaglandin H (PGH) synthase mRNA, we have evaluated the effect of naproxen on PGH synthase protein and mRNA levels in cultured human umbilical vein endothelial cells (HUVEC). PGH synthase mRNA levels were quantified by a competitive polymerase chain reaction (PCR) assay; protein was assessed by Western blotting. Naproxen decreased the PGH synthase protein level in HUVEC in a concentration-dependent manner. It abolished entirely the 70 kDa PGH synthase subunit at 5 micrograms/ml. It appears more effective in blocking interleukin-1 inducible PGH synthase levels. Naproxen also inhibited the synthase mRNA level in a concentration-dependent manner; levels were reduced by 33% at 5 micrograms/ml and 60% at 30 micrograms/ml naproxen. These results indicate that naproxen, like the salicylates, inhibits PGH synthase levels in cultured endothelial cells either by inhibiting transcription of the PGH synthase gene or by destabilizing its messenger RNA.

Estrogen regulation of epidermal growth factor receptor messenger ribonucleic acid.

Previous studies have demonstrated that 17 beta-estradiol (E2) causes a 3-fold increase in epidermal growth factor (EGF) receptors in uterine membranes. We now report that the increase in uterine EGF receptor levels is due to an increase in the steady-state levels of EGF receptor mRNA. After a single E2 injection, EGF receptor mRNA levels, as determined by RNA blots, increase 3- to 4-fold between 1 and 3 h, remain elevated at 6 h, and decline between 12 and 18 h. The effect is specific for E2 since the nonestrogenic hormones progesterone, dexamethasone, 5 alpha-dihydrotestosterone, and the inactive stereoisomer of E2, 17 alpha-estradiol, are without effect. E2-Mediated increases in EGF receptor mRNA levels are blocked by actinomycin D but not by puromycin. Taken together, these results indicate that E2 regulates the level of EGF receptor by increasing the steady-state concentration of EGF receptor mRNA in vivo.

Estrogen regulation of c-fos messenger ribonucleic acid.

Acute administration of 17 beta-estradiol to immature female rats elicits a rapid and striking increase in the size of the uterus. This increase in size to caused by both hypertrophy and hyperplasia in the epithelial, stromal, and myometrial cells in the uterus. Previous studies have shown that induction of mRNA for the epidermal growth factor receptor, the cellular homolog of the erb-B oncogene, occurs early during estrogen-stimulated uterine growth. We report here that estradiol causes a very rapid induction of the mRNA for the cellular oncogene c-fos in immature rat uterus. Steady state levels of c-fos mRNA reach a maximum 3 h after 17 beta-estradiol administration and slowly return to low basal levels in 15 h. Dexamethasone, progesterone, and 5 alpha-dihydrotestosterone had no effect on uterine c-fos mRNA expression. The induction of c-fos mRNA by estrogen was unaffected by the protein synthesis inhibitor puromycin but completely abolished by the RNA synthesis inhibitor actinomycin D.

Steroid hormone-induced expression of oncogene encoded nuclear proteins.

In this article we have attempted to review the literature on the regulation of nuclear protooncogene expression by steroid hormones and other small molecules that interact with receptors of the steroid/thyroid superfamily. Until about 5 years ago, there were relatively few reports of steroidal regulation of cellular oncogenes, but hundreds of papers on this topic have appeared since then. This demonstrates the intense interest in this area that has developed recently. It now been demonstrated that all the major classes of steroid hormones control expression of nuclear protooncogenes in one or more systems. Given the actions of these proteins as transcription factors and their central role in cellular communications systems, it seems likely that they play a key role in mediating the biological effects of steroids on processes such as proliferation and differentiation. To date, most of the work in this general area has focused primarily on the regulation of three genes: c-fos, c-jun, and c-myc. However, a quick glance at the table of nuclear protooncogenes in the introduction of this article indicates that over 40 nuclear protooncogenes are now recognized. For the large majority of these, regulatory effects of steroids and related molecules have not yet been reported. Hence, we predict that reports in this general area of research will continue to appear at a very rapid rate over the next few years. In addition, we have tried to provide enough background information for readers to get an overview of the regulation of nuclear protooncogene expression by nonsteroidal factors. We felt this information was important to emphasize that steroid hormones represent only one of the many classes of regulatory molecules that control expression of nuclear protooncogenes. Thus, an important area for future research will be to understand how these multiple regulatory systems interact to control expression of this important class of cellular oncogenes and the biological processes that they mediate.

Sequence of a 1.4-kb region in the 3'-flanking region of the murine c-fos proto-oncogene which contains an estrogen-response element.

The nucleotide sequence of a 1.4-kb region in the 3'-flanking region of the murine c-fos oncogene has been determined. This region contains an estrogen-response element which is located almost 5 kb downstream from the c-fos promoter.

Cholesterol metabolism in hypercholesterolemia-resistant rabbits.

Normal rabbits typically respond to a diet high in cholesterol with a large increase in the concentration of plasma cholesterol. We have previously described the breeding and partial characterization of a variant rabbit which does not respond to a high cholesterol diet with changes in plasma cholesterol concentration. In the present report we have characterized three components involved in cholesterol homeostasis: the B/E (LDL) receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (HMG-CoA reductase, EC 1.1.1.34) and acyl-coenzyme A: cholesterol acyltransferase activity (ACAT, EC 2.3.1.26) in the livers of the hypercholesterolemia-resistant rabbits. Using normal cholesterol-fed rabbit [125I] beta-VLDL as a ligand, liver membranes prepared from resistant rabbits fed a low-cholesterol diet had 70% higher binding capacity than membranes from normal rabbits fed the same diet. Similar experiments demonstrated that the resistant rabbits had a 240% higher B/E receptor binding capacity compared to normal animals when liver membranes were prepared from animals fed a 0.25% cholesterol-enriched diet. No difference in the binding affinity of [125I]beta-VLDL was detected in membranes prepared from normal or resistant animals. When fed a low-cholesterol diet, the resistant rabbits had approximately 2-fold higher hepatic HMG-CoA reductase activity (97.4 +/- 3.5 pmol product/mg/min in resistant animals compared to 45 +/- 1.1 pmol product/min/mg in normal animals). The difference was exaggerated in animals fed the 0.25% cholesterol-enriched diet, 73.3 +/- 5.5 vs 2.4 +/- 0.56 pmol product/min/mg for resistant and normal membranes respectively. The basal activity of ACAT in hepatic membranes was significantly lower in the resistant rabbits compared to normal rabbits (138 +/- 11 vs 268 +/- 19 pmol cholesteryl ester/min/mg in resistant and normal rabbits respectively); when fed a 0.25% cholesterol-enriched diet, the enzyme was induced 6-fold in normal animals but was increased only 2-fold in the resistant animal. These biochemical data suggested that the resistant rabbit maintained low intracellular cholesterol even when fed a cholesterol-enriched diet. Direct measurement of cellular cholesterol and cholesteryl esters demonstrated that the concentration of these lipids was significantly lower in the resistant animal than in normal animals with the largest differences found in the cytoplasmic rather than the membrane compartment. These studies demonstrate that the resistant rabbit manifests several quantitative differences in cholesterol metabolism and in the regulation of cholesterol metabolism; but these studies do not directly explain the underlying cause of the resistance to hypercholesterolemia in the resistant rabbit.

PET hydroxyephedrine imaging of neuroblastoma.

UNLABELLED: The goals of this investigation were to characterize the uptake of 11C-hydroxyephedrine (HED) in neuroblastoma and to determine the feasibility and potential advantages of utilizing this compound as a tumor imaging agent. METHODS: Seven patients with known or subsequently proven neuroblastoma were studied. Each patient underwent PET scanning with 11C-HED. Six of seven patients underwent scintigraphy with [123I]meta-iodobenzylguanidine (MIBG), and two patients were also studied with [18F]FDG PET. For six patients, CT or MR images were available for comparison. RESULTS: Neuroblastomas were located by PET scanning with 11C-HED in all seven patients. The uptake of HED into neuroblastomas was rapid; tumors were evident on images within 5 min postintravenous injection. Those lesions in the field of view of the PET camera were also identified on [123I]MIBG scintigraphic images. In two patients, tumor deposits in the abdomen were better visualized with MIBG scintigraphy due to relatively less hepatic accumulation of MIBG than HED. CONCLUSION: PET scanning with HED for neuroblastoma results in high quality functional images of the tumors that can be obtained within minutes following injection.

Toxicity of endogenous and environmental estrogens: what is the role of elemental interactions?

Many naturally occurring and man-made chemicals present in the environment possess estrogenic activity. Examples include plant and fungal products, pesticides, plasticizers, and other agricultural and industrial chemicals. These environmental estrogens as well as endogenous ovarian estrogens are thought to initiate their physiological actions in target tissues largely via interactions with a nuclear receptor system. The resultant estrogen-receptor complex in turn affects transcription via its interactions with nucleotide sequences known as estrogen response elements (EREs) present in the regulatory regions of hormone responsive genes. A "consensus" ERE sequence GGTCAnnnTGACC was originally identified in the vitellogenin genes of birds and amphibians, but it is now clear that most naturally occurring EREs differ from this sequence in one or more bases. We and others have obtained both in vivo and in vitro data suggesting a differential interaction of receptor complexes containing different ligands with the multiple EREs present in mammalian systems. This raises the possibility that the toxicity of environmental estrogens may arise in part from a differential pattern of ERE activation by environmental compounds relative to endogenous ovarian estrogens. The experimental basis for such a paradigm and its toxicological implications are discussed in this paper.

Estrogen regulates expression of the jun family of protooncogenes in the uterus.

Treatment of immature female rats with estradiol increases uterine levels of c-jun and jun-B mRNAs approx. 10-fold. This effect is specific for estrogenic steroids. The induction of jun transcripts is blocked by actinomycin D but not puromycin, suggesting that the hormonal effect is due at least in part to transcriptional activation. The hormone effect is rapid and peak levels of jun mRNAs are seen within 3 h after treatment. Inductions of jun and fos transcripts in the uterus by estradiol exhibit similar dose response curves (maximum responses at 4 micrograms/kg). Estradiol also elevates uterine levels of jun-D, and this induction is insensitive to puromycin. In vivo treatment with the phorbol ester TPA rapidly elevates uterine levels of fos, jun, and myc transcripts, indicating that expression of these protooncogenes is under non-estrogenic as well as estrogenic regulation in this target tissue. These results suggest that multiple members of the jun and fos protooncogene families may play a role in amplifying the uterine response to estrogens.

Presence of an estradiol response region in the mouse c-fos oncogene.

We have previously shown that the intracellular content of c-fos mRNA is rapidly induced (within 1 to 3 hours) in ovariectomized rat or mouse uteri following administration of estradiol. This induction is sensitive to actinomycin D but not to protein synthesis inhibitor puromycin, indicating an effect of estradiol at the transcriptional level, possibly mediated by the estrogen receptor. We have used transient transfection assays with defined regions of the mouse c-fos gene ligated to a reporter plasmid expressing chloramphenicol acetyl transferase to study regulation of this gene by estrogens. These recombinants were transfected in two different estrogen-responsive cell lines, GH4 and MCF-7, and stimulated with estradiol. A two- to five-fold induction of chloramphenicol acetyl transferase activity was observed with a construct containing the intact c-fos promoter and 351 bases of 5'-flanking sequence (-351/+44). A similar induction by estrogen is observed with the endogenous c-fos gene in the two cell lines as determined by RNA blot analysis. Estrogen induction is lost when a construct containing -135/+44 region of the c-fos gene is transfected. Plasmid containing the consensus estrogen response element GGTCAnnnTGACC derived from vitellogenin gene is induced 10- to 50-fold in both estrogen-responsive cell lines. Under identical conditions, the oligonucleotide containing the perfect palindrome GGTCTnnnAGACC, present around the -209 region of the c-fos gene, is completely silent when transfected under the control of thymidine kinase promoter. Additional transfection analysis with a number of c-fos promoter constructs has narrowed the estrogen response region to within the -278 to -135 region upstream of the c-fos promoter.

Salt taste preference in baboons.

Dietary salt (NaCl) has been implicated in the etiology of hypertension and atherosclerosis, although its role remains controversial. The human preference for salted foods is well-known and many investigators believe the taste for salt is acquired. An experiment we conducted suggests that the baboon does not have an acquired taste for salt. A sample of 36 baboons from a population of 70 baboons of known sire, sex, and dietary history was used; each had been raised since birth on a diet of fixed salt content in a study of dietary salt and blood pressure. Given this unique group of animals, we decided to test whether baboons raised on one dietary salt level (low, medium, or high) would prefer a different level. After baseline consumption was measured for 9 days, we offered each animal equal amounts of all 3 diets simultaneously in a counterbalanced randomized sequence for 9 days, controlling for tray position preference and color preference. We measured consumption of each diet by weighing the amount of food remaining. Our statistical analyses indicated an overwhelming preference for the lowest dietary salt level, regardless of which diet the animal had been fed since birth (p less than 0.0001).

Flavor preferences, food intake, and weight gain in baboons (Papio sp.).

To evaluate the influence of flavor on ad lib consumption and on associated changes in body weight, female baboons, 7-15 years of age, served in two experiments with seven monkey chows which were identical except for flavor: lemon, orange, apple, sugar, fruit punch, chocolate, and unflavored. In the first experiment, two groups of animals (n=7 and 4) received five of the seven flavors, presented in daily pair-wise combinations. Analysis of quantities consumed demonstrated marked and consistent flavor preferences in individual baboons. Although specific preference varied between animals, total amounts consumed of the various flavors differed significantly, with rank ordering clearly evident. Overall food intake and body weights increased significantly over baseline values obtained with a standard, unflavored chow. In the second experiment, three baboons received chow of a preferred flavor for nine weeks. Amounts consumed and body weights increased significantly over baseline. These results indicate that flavored chows may be useful for producing a nonhuman primate behavioral model of obesity and for inducing animals to eat otherwise unpalatable diets.

Interactions between estrogen and EGF in uterine growth and function.

The rat uterus contains specific, high-affinity EGF receptors which possess a tyrosine kinase activity. As demonstrated autoradiographically, these receptors are present in the epithelial, stromal and myometrial cells of the uterus. Estrogen treatment in vivo produces a 2-3-fold increase in EGF receptor levels in the immature rat, the immature mouse and the ovariectomized adult rat; furthermore, EGF receptor levels vary throughout the estrus cycle in concert with levels of occupied nuclear estrogen receptor. This estrogen-induced increase in EGF receptor is preceded by an increase in the level of EGF receptor mRNA as judged by Northern blot analysis. In general, there is a good correlation between estrogen-induced DNA synthesis and EGF receptor levels in the uterus, although in certain situations EGF receptor levels are elevated without a subsequent increase in DNA synthesis. These observations suggest that an increase in tissue EGF receptor levels is important in estrogen-induced uterine growth, but that this increase in receptor levels alone is not sufficient to stimulate DNA synthesis. In addition to its possible role in tissue growth, we have shown very recently that EGF causes contraction of myometrial smooth muscle in a completely in vitro organ bath system. The qualitative nature of this contractile response is distinct from that produced by other classical uterotonic agents. The physiological significance of this uterine response to EGF remains to be elucidated.

Case report of a newborn with a posterior thoracic midline congenital hemangiopericytoma of the back.

The posterior thoracic midline location is an unusual site for a congenital hemangiopericytoma. The authors report such a case that caused near fatal exsanguination of a newborn after vaginal delivery. Magnetic resonance imaging (MRI) studies of the mass were completed after hemostasis. These studies showed a well-defined border between the tumor and underlying trapezius muscle. The mass was removed successfully surgically and presumed initially to be a teratoma. Pathological diagnosis of the tumor was hemangiopericytoma with low malignant potential. After a 9-day hospital course, the patient was discharged with recovering hepatic and renal function.

Resistance to dietary-induced hypercholesterolemia exhibited by a unique strain of New Zealand white rabbits.

The search for a precise metabolic explanation for the capacity of some individuals to resist the development of dietary-induced hypercholesterolemia, thus avoiding attendant cardiovascular atherosclerotic complications, has long been the focus of our research. From 1 New Zealand white rabbit that failed to show any cholesterolemic response, we have, over the course of 10 years, established a partially inbred strain of strongly cholesterol-resistant rabbits. This achievement has resulted in the production of a large number of cholesterol-resistant animals for study; more importantly, it has shown that a strong genetic factor operates in dietary regulation of plasma cholesterol levels. We have focused our research on the different possibilities associated with this genetic predisposition. Since the cholesterol-resistant rabbits do not accumulate cholesterol or its esters in plasma or in any tissue compartments, we investigated several biochemical pathways involved in cholesterol metabolism. We have recently concentrated on the enzyme cholesterol 7 alpha-hydroxylase and liver bile acid metabolism. We have cloned the complete gene and partial cDNAs for cholesterol 7 alpha-hydroxylase from both normal and cholesterol-resistant rabbits. This has allowed the discovery of changes in the transcription of this gene in the cholesterol-resistant rabbits compared with normal littermates. These cholesterol-resistant rabbits have provided a model demonstrating that there are biological means to prevent large dietary loads of cholesterol from accumulating in plasma or tissues. Our hypothesis is that cholesterol-resistant animals increase cholesterol turnover by increasing bile acid excretion, thus providing a way to reduce plasma cholesterol of either dietary or endogenous origin. The methods and observations of our research are presented chronologically in this review.

Gene therapy for vascular diseases.

Gene transfer by virus- and liposome-mediated vectors has potential for treating genetic diseases, cancer, and cardiovascular diseases. In this article, we discuss the general principle and techniques for gene transfer and the specific issues facing therapy for vascular diseases. We also propose a strategy for using virus-mediated gene transfer to restore the vasoprotective function of the vascular wall, thereby preventing vascular thrombosis. Experimental data from ongoing work in our laboratories are presented to illustrate the importance of this approach in vascular gene transfer therapy.