Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression.

Large granular lymphocyte (LGL) leukemia is characterized by the expansion of antigen-activated cytotoxic T lymphocytes. These leukemic cells are resistant to Fas-mediated apoptosis despite expressing high levels of Fas. We found that leukemic LGL from 19 patients displayed high levels of activated STAT3. Treatment of leukemic LGL with the JAK-selective tyrosine kinase inhibitor AG-490 induced apoptosis with a corresponding decrease in STAT-DNA binding activity. Moreover, using an antisense oligonucleotide approach to diminish STAT3 expression, we found that Fas sensitivity was restored in leukemic LGL. AG-490-induced apoptosis in leukemic LGL was independent of Bcl-xL or Bcl-2 expression. However, we found that the Bcl-2-family protein Mcl-1 was significantly reduced by AG-490 treatment. Activated STAT3 was shown to bind an SIE-related element in the murine mcl-1 promoter. Using a luciferase reporter assay, we demonstrated that v-src overexpression in NIH3T3 induced STAT3-dependent transcriptional activity from the mcl-1 promoter and increased endogenous Mcl-1 protein levels. We conclude that STAT3 activation contributed to accumulation of the leukemic LGL clones. These findings suggest that investigation should focus on novel strategies targeting STAT3 in the treatment of LGL leukemia.

Distinct tumor specific expression of TGFB4 (ebaf)*, a novel human gene of the TGF-beta superfamily.

We recently identified a novel gene of the TGF-beta superfamily, endometrial bleeding associated factor, TGFB4 (ebaf), that, throughout the menstrual cycle, exhibited a defined expression in human endometrium. Here, we report on the expression of TGFB4 (ebaf) in normal and neoplastic human tissues. The expression of this gene was absent in a host of normal tissues including lung, stomach, small bowel, liver, kidney, breast, lymph node, spleen, ovary and fallopian tube. However, a weak expression of the 2.1 kb variant of the TGFB4 (ebaf) mRNA was observed in rectal, ovarian, and testicular tissues and the 2.1 and 2.5 kb TGFB4 (ebaf) mRNAs were observed in the pancreatic tissue. The expression of the mRNA of this gene was absent in sarcomas, Hodgkin's and non-Hodgkin's lymphomas, melanomas, squamous cell carcinomas, hepatocellular carcinomas, renal cell carcinomas, and adenocarcinomas of the breast, endometrium and lung. The expression of the TGFB4 (ebaf) mRNA was observed primarily in adenocarcinomas that exhibited a mucinous differentiation. This included colonic, duodenal, and ovarian adenocarcinomas. The expression of TGFB4 (ebaf) mRNA was absent in non- mucinous colonic, gastric and ovarian adenocarcinomas and adenocarcinomas of colon metastatic to the liver. However, some serous adenocarcinomas of the ovary also exhibited TGFB4 (ebaf) mRNA. The testicular tumors, seminomas and embryonal carcinomas, also expressed TGFB4 (ebaf) mRNA. These findings show that the TGFB4 (ebaf) mRNA has distinct tumor specific expression.

Detection of ebaf, a novel human gene of the transforming growth factor beta superfamily association of gene expression with endometrial bleeding.

Human endometrium is unique since it is the only tissue in the body that bleeds at regular intervals. In addition, abnormal endometrial bleeding is one of the most common manifestations of gynecological diseases, and is a prime indication for hysterectomy. Here, we report on a novel human gene, endometrial bleeding associated factor (ebaf), whose strong expression in endometrium was associated with abnormal endometrial bleeding. In normal human endometrium, this gene was transiently expressed before and during menstrual bleeding. In situ hybridization showed that the mRNA of ebaf was expressed in the stroma without any significant mRNA expression in the endometrial glands or endothelial cells. The predicted protein sequence of ebaf showed homology with and structural features of the members of TGF-beta superfamily. Fluorescence in situ hybridization showed that the ebaf gene is located on human chromosome 1 at band q42.1. Thus, ebaf is a novel member of the TGF-beta superfamily and an endometrial tissue factor whose expression is associated with normal menstrual and abnormal endometrial bleeding.

Cloning and developmental expression of the ecdysone receptor gene from the spruce budworm, Choristoneura fumiferana.

Degenerate oligonucleotides were designed on the basis of conserved amino acid sequences in the DNA and ligand-binding regions of the members of the steroid hormone receptor superfamily. Using these oligonucleotides in RNA-PCR, a cDNA fragment was isolated from the spruce budworm, Choristoneura fumiferana. Comparison of the deduced amino acid sequence of this cDNA fragment with the members of the steroid hormone receptor superfamily suggested that this PCR fragment is a region of the ecdysone receptor from C. fumiferana. Using this cDNA fragment as a probe, 10 clones were isolated from a cDNA library that was constructed using the RNA from 4- and 5-day old embryos of C. fumiferana. Two cDNA clones (1.3 and 3 kb) that overlap and show amino acid identity with Drosophila melanogaster ecdysone receptor B-1 isoform (DmEcR) were characterized and sequenced. The longest open reading frame had 539 codons and covered the complete EcR coding region. The deduced amino acid sequence of this open reading frame had all five of the regions typical for a steroid hormone nuclear receptor. The C domain or DNA binding region showed the highest identity wit EcR proteins from D. melanogaster, Chironomus tendons, Aedes aegypti, Manduca sexta, and Bombyx mori. The A/B region, D domain or hinge region, E domain, or ligand binding region also showed significant amino acid similarity with the EcR proteins from the five insects mentioned above. The C. fumiferana ecdysteroid receptor (CfEcR) cDNA probe detected a 6.0-kb mRNA that was present throughout the development of C. fumiferana. The CfEcR mRNA increases in abundance at the time of the ecdysteroid peak during the molting phase in the embryonic, larval and pupal stages but remains low during the intermolt period. In the 6th instar larvae, the 6-kb CfEcR mRNA was detected in the epidermis, fat body, and midgut and maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CfEcR mRNA was induced in ecdysone treated CF-203 cells as well in the epidermis and midgut of larvae that were fed the nonsteroidal ecdysteroid agonist, RH-5992. The induction occurred within an hour and reached maximum levels around 3 hr, after which it decreased to the basal level by 6 hr. In vitro transcription and translation of the CfEcR cDNA yielded a 67-Kda protein that bound to the ecdysone response element (EcRE) as a heterodimer, along with the ultraspiracle protein.

Effects of long-chain fatty amines on the growth of ras-transformed NIH 3T3 cells.

A number of aliphatic primary amines were tested for their effects on the growth of ras-transformed NIH 3T3 cells (PAP2 cells), as measured by incorporation of tritiated thymidine into DNA. Long-chain, saturated amines (C12 to C18) were growth inhibitory, whereas short-chain amines (C6, C8) were not. Farnesylamine, a branched-chain, unsaturated amine (C15), had an IC50 of 6.9 microM compared to IC50 values of 13.1 to 45.8 microM for straight-chain, saturated amines. Oleylamine, with an IC50 of 0.1 microM, was the most potent inhibitor. The long-chain amines, but not the short-chain amines, were also effective inhibitors of protein kinase C, assayed in vitro in a cell-free system. In addition, studies with indo-1-loaded PAP2 cells showed that long-chain amines induced a reversible rise in intracellular free Ca2+ concentration. Growth inhibition by the amines was positively correlated with this effect, suggesting that factors other than protein kinase C may be involved in the inhibition of growth of PAP2 cells by long-chain amines.

Variations in serum levels of dolichol and its occurrence in human atherosclerotic plaques.

Nonsaponifiable lipids of human atherosclerotic plaques obtained at autopsy from patients ranging in age from 45 to 85 years were analyzed by high pressure liquid chromatography. All plaques contained dolichol, ranging from 125 to 460 micrograms/g wet weight. Dolichol was also present in normal aortic tissue, but the amounts were generally less than in plaques. To investigate the source of the dolichol in plaques, blood serum was analyzed from both volunteer subjects and hypercholesterolemic patients. The levels of dolichol were generally higher in hypercholesterolemic compared with normal subjects, but were not correlated with levels of total or lipoprotein cholesterol. The homologue pattern of dolichol in atherosclerotic plaques differed from that of aorta and blood. The source of dolichol in plaques and its significance remains to be established.

Farnesylamine: an inhibitor of farnesylation and growth of ras-transformed cells.

Farnesylamine, an analogue of farnesol, was shown to inhibit growth of PAP2 cells (ras-transformed NIH 3T3 cells) in a dose-dependent manner. This inhibition was overcome by adding farnesol to the culture medium, but not by adding geranylgeraniol, squalene, cholesterol, dolichol, myristic acid or palmitic acid. Farnesylamine inhibited both farnesyl/protein transferase and geranylgeranyl/protein transferase in whole cell extracts and also inhibited the prenylation of proteins, particularly ras p21, in PAP2 cells. Inhibition of prenylation was associated with increased biosynthesis of other products of the mevalonate biosynthetic pathway. These observations suggest that inhibition of the growth of PAP2 cells by farnesylamine may be due to blocking of ras-mediated signal transduction. This offers a means of investigating mechanisms involved in ras action and raises the possibility of developing novel strategies for anticancer therapy.

Inhibition of scavenger receptor-mediated modified low-density lipoprotein endocytosis in cultured bovine aortic endothelial cells by the glycoprotein processing inhibitor castanospermine.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizidine) is a plant alkaloid that inhibits alpha-glucosidases, including the glycoprotein processing glucosidase I. When endothelial cells were grown for 48 h, or longer, in the presence of this alkaloid, they produced scavenger receptors for modified low-density lipoproteins (LDL) that had mostly Glc3Man7-9(GlcNAc)2 structures rather than the usual complex types of oligosaccharides. Furthermore, growth in the presence of castanospermine resulted in a substantial inhibition in degradation of endocytosed 125I-acetylated LDL, as well as a dose-dependent inhibition of 125I-acetylated LDL binding to these cells. Scatchard analysis of binding curves indicated that the diminished binding was due to a decrease in the number of scavenger receptor molecules at the cell surface rather than to a change in the affinity of the receptors for their ligand. Since castanospermine-treated cells had the same total number of cellular receptor molecules as did controls cells, it seemed likely that castanospermine caused an alteration in receptor targeting, rather than an inhibition in receptor synthesis or a stimulation in receptor degradation. Density gradient fractionation of cell homogenates showed that castanospermine-treated cells did have a much greater percentage of scavenger LDL receptor molecules in the endoplasmic reticulum-Golgi fraction and fewer receptors in the plasma membrane fraction, whereas normal cells showed the opposite distribution.