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Biomedical subjects

M B Sporn

Publications and source records attributed to M B Sporn.

At least 37 records · Page 2Linked to original sources
PubMed1998

Design and synthesis of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, a novel and highly active inhibitor of nitric oxide production in mouse macrophages.

New derivatives with electron-withdrawing substituents at the C-2 position of 3-oxoolean-1-en-28-oic acid were synthesized. Among them, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) was 400 times more potent than previous compounds we have made as an inhibitor of production of nitric oxide induced by interferon-gamma in mouse macrophages (IC50, 0.4 nM). The potency of CDDO was similar to that of dexamethasone, although CDDO does not act through the glucocorticoid receptor.

Animals↗
PubMed1998

Novel triterpenoids suppress inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse macrophages.

We have synthesized more than 80 novel triterpenoids, all derivatives of oleanolic and ursolic acid, as potential anti-inflammatory and chemopreventive agents. These triterpenoids have been tested for their ability to suppress the de novo formation of two enzymes, inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2), using IFN-gamma-stimulated primary mouse macrophages or lipopolysaccharide (LPS)-activated RAW 264.7 macrophages as assay systems. Two synthetic oleananes, 3,12-dioxoolean-1-en-28-oic acid (TP-69) and 3,11-dioxoolean-1,12-dien-28-oic acid (TP-72), were highly active inhibitors of de novo formation of both iNOS and COX-2. Both TP-69 and TP-72 blocked the increase in iNOS or COX-2 mRNA induced by IFN-gamma or LPS. In addition, TP-72 suppressed NF-KB activation in primary macrophages treated with the combination of IFN-gamma and LPS or IFN-gamma and tumor necrosis factor. The 3-alpha(axial)-epimer of ursolic acid suppressed de novo formation of COX-2, in contrast to naturally occurring 3-beta(equatorial)-ursolic acid. Inhibitory effects of TP-69 or TP-72 on iNOS formation were not blocked by the glucocorticoid receptor antagonist RU-486, indicating that these triterpenoids do not act through the glucocorticoid receptor, nor does TP-72 act as an iNOS or COX-2 enzyme inhibitor when added to RAW cells in which synthesis of these two enzymes in response to LPS has already been induced. It may be possible to develop triterpenoids as useful agents for chemoprevention of cancer or other chronic diseases with an inflammatory component.

Animals↗
PubMed1998

The role of transforming growth factor-beta1, -beta2, and -beta3 in androgen-responsive growth of NRP-152 rat prostatic epithelial cells.

We have investigated the role of autocrine/paracrine TGF-beta secretion in the regulation of cell growth by androgens as demonstrated by its inhibition by two androgen response modifiers; the nonsteroidal antiandrogen hydroxyflutamide (OHF), believed to act by inhibiting androgen binding to androgen receptors, or finasteride, an inhibitor of 5alpha-reductase, the enzyme necessary for the conversion of testosterone to 5alpha-dihydrotestosterone (DHT), using the nontumorigenic rat prostatic epithelial cell line NRP-152. Growth of these cells was stimulated three- to sixfold over control by either testosterone or DHT under serum-free culture conditions. This was accompanied by a two- to threefold decrease in the secretion rate of TGF-beta1, -beta2, and -beta3. Finasteride reversed the ability of testosterone but not DHT to stimulate growth and downregulate expression of TGF-beta1, -beta2, and -beta3 in a dose-dependent fashion, suggesting that this activity of testosterone required its conversion to DHT. OHF antagonized the stimulatory effects of DHT on NRP-152 cell growth but could reverse the inhibitory effects of DHT only on TGF-beta2 and TGF-beta3 and not TGF-beta1 secretion. This suggests that either TGF-beta1 regulation by DHT or the androgen antagonism of OHF occurs independent of androgen receptor binding. Neutralizing antibodies to TGF-beta (pantropic and isoform-specific) were able to block the ability of finasteride to antagonize the effects of testosterone nearly completely while only partially inhibiting the antiandrogenic effects of OHF. Thus, the ability of androgens to stimulate growth of NRP-152 cells involves the downregulation of the production of TGF-beta1, -beta2, and -beta3 in addition to other growth-stimulatory mechanisms.

Androgen Antagonists↗
PubMed1998

Mutational analysis of a transforming growth factor-beta receptor binding site.

Transforming growth factor-beta s (TGF-beta 1, -beta 2, -beta 3) are important regulators of cell growth and differentiation which share approximately 70% identical amino acids. Using LS513 colorectal cells, which are growth inhibited by TGF-beta 1 (ED50 of 100 pM), but are refractory to TGF-beta 2 (ED50 of 50,000 to 100,000 pM), we have determined that amino acids 92-98 of TGF-beta specify growth inhibition. The chimeric protein TGF-beta 1/beta 2(92-98), in which amino acids 92-98 of TGF-beta 1 were exchanged for the corresponding amino acids of TGF-beta 2, was indistinguishable from TGF-beta 2 at inhibiting growth of LS513 cells. In contrast, both TGF-beta 1/beta 2(92-95) and TGF-beta 1/beta 2(94-98) inhibited the growth of LS513 cells with an ED50 of approximately 1000 pM. TGF-beta 1/beta 2(95-98), in which amino acids 95-98 of TGF-beta 1 have been replaced with the corresponding amino acids of TGF-beta 2, had full activity and was indistinguishable from TGF-beta 1. Receptor cross-linking experiments demonstrated that binding of the chimeras to the type I and type II receptors of LS513 cells was consistent with their biological activity. TGF-beta 1/beta 2(95-98), TGF-beta 1/beta 2(92-95) and TGF-beta 1/beta 2(94-98) were each similar to TGF-beta 2 in that they failed to bind to the soluble Type II receptor in a solid-phase assay. These results demonstrate that amino acids 92-98 are involved in the interaction between TGF-beta and its signaling receptors and they show that modest changes within this region can substantially alter biological response.

Amino Acid Sequence↗
PubMed1997

The war on cancer: a review.

Twenty-five years ago, then President Nixon "declared war" on cancer. In this personal commentary, the war is reviewed. There have been obvious triumphs, for instance in cure of acute lymphocytic leukemia and other forms of childhood cancer, Hodgkin's disease, and testicular cancer. However, substantial advances in molecular oncology have yet to impinge on mortality statistics. Too many adults still die from common epithelial cancers. Failure to appreciate that local invasion and distant metastasis rather then cell proliferation itself are lethal, obsession with cure of advanced disease rather than prevention of early disease, and neglect of the need to arrest preneoplastic lesions, may all have served to make victory elusive.

Adult↗
PubMed1997

Recent advances in chemoprevention of cancer.

Chemoprevention is the use of pharmacologic or natural agents that inhibit the development of invasive cancer either by blocking the DNA damage that initiates carcinogenesis or by arresting or reversing the progression of premalignant cells in which such damage has already occurred. Recent advances in our understanding of the mechanisms of carcinogenesis have led to the synthesis of new drugs that can inhibit tumor development in experimental animals by selective action on specific molecular targets, such as the estrogen, androgen, and retinoid receptors or inducible cyclooxygenase. Several of these agents (including tamoxifen, 13-cis-retinoic acid, retinyl palmitate, and an acyclic retinoid) are clinically effective in preventing the development of cancer, particularly in patients who are at high risk for developing second primary tumors after surgical removal of the initial tumor.

Animals↗
PubMed1996

Binding affinity of transforming growth factor-beta for its type II receptor is determined by the C-terminal region of the molecule.

Transforming growth factor-beta (TGF-beta) isoforms have differential binding affinities for the TGF-beta type II receptor (TbetaRII). In most cells, TGF-beta1 and TGF-beta3 bind to TbetaRII with much higher affinity than TGF-beta2. Here, we report an analysis of the effect of TGF-beta structure on its binding to TbetaRII by using TGF-beta mutants with domain deletions, amino acid replacements, and isoform chimeras. Examination of the binding of TGF-beta mutants to the recombinant extracellular domain of TbetaRII by a solid-phase TGF-beta/TbetaRII assay demonstrated that only those TGF-beta mutants containing the C terminus of TGF-beta1 (TGF-beta1-(Delta69-73), TGF-beta1-(Trp71), and TGF-beta2/beta1-(83-112)) bind with high affinity to TbetaRII, similar to native TGF-beta1. Moreover, replacement of only 6 amino acids in the C terminus of TGF-beta1 with the corresponding sequence of TGF-beta2 (TGF-beta1/beta2-(91-96)) completely eliminated the high affinity binding of TGF-beta1. Proliferation of fetal bovine heart endothelial (FBHE) cells was inhibited to a similar degree by all of the TGF-beta mutants. However, recombinant soluble TbetaRII blocked the inhibition of FBHE cell proliferation induced by TGF-beta mutants retaining the C terminus of TGF-beta1, consistent with the high binding affinity between these TGF-beta molecules and TbetaRII. It was further confirmed that the TGF-beta2 mutant with its C terminus replaced by that of TGF-beta1 (TGF-beta2/beta1-(83-112)) competed as effectively as TGF-beta1 with 125I-TGF-beta1 for binding to membrane TbetaRI and TbetaRII on FBHE cells. These observations clearly indicate that the domain in TGF-beta1 responsible for its high affinity binding to TbetaRII, both the soluble and membrane-bound forms, is located at C terminus of the molecule.

Amino Acid Sequence↗
PubMed1996

Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimer's disease.

In Alzheimer's disease (AD), affected neurons accumulate beta amyloid protein, components of which can induce mouse microglia to express the high-output isoform of nitric oxide synthase (NOS2) in vitro. Products of NOS2 can be neurotoxic. In mice, NOS2 is normally suppressed by transforming growth factor beta 1 (TGF-beta 1). Expression of TGF-beta 1 is decreased in brains from AD patients, a situation that might be permissive for accumulation of NOS2. Accordingly, we investigated the expression of NOS2 in patients with AD, using three monospecific antibodies: a previously described polyclonal and two new monoclonal antibodies. Neurofibrillary tangle-bearing neurons and neuropil threads contained NOS2 in brains from each of 11 AD patients ranging in age from 47 to 81 years. NOS2 was undetectable in brains from 6 control subjects aged 23-72 years, but was expressed in small amounts in 3 control subjects aged 77-87 years. Thus, human neurons can express NOS2 in vivo. The high-output pathway of NO production may contribute to pathogenesis in AD.

Adult↗
PubMed1996

Transforming growth factor beta 1: three-dimensional structure in solution and comparison with the X-ray structure of transforming growth factor beta 2.

The three-dimensional solution structure of human transforming growth factor beta 1 (TGF-beta 1) has been determined using multinuclear magnetic resonance spectroscopy and a hybrid distance geometry/ simulated annealing algorithm. It represents one of the first examples of a mammalian protein structure that has been solved by isotopic labeling of the protein in a eukaryotic cell line and multinuclear NMR spectroscopy. The solution structure of the 25 kDa disulfide-linked TGF-beta 1 homodimer was calculated from over 3200 distance and dihedral angle restraints. The final ensemble of 33 accepted structures had no NOE or dihedral angle violations greater than 0.30 A and 5.0 degrees, respectively. The RMSD of backbone atoms for the ensemble of 33 structures relative to their mean structure was 1.1 A when all residues were used in the alignment and 0.7 A when loop regions were omitted. The solution structure of TGF-beta 1 follows two independently determined crystal structures of TGF-beta 2 (Daopin et al., 1992, 1993; Schlunegger & Grütter, 1992, 1993), providing the first opportunity to examine structural differences between the two isoforms at the molecular level. Although the structures are very similar, with an RMSD in backbone atom positions of 1.4 A when loop regions are omitted in the alignment and 1.9 A when all residues are considered, there are several notable differences in structure and flexibility which may be related to function. The clearest example of these is in the beta-turn from residues 69-72: the turn type found in the solution structure of TGF-beta 1 falls into the category of type II, whereas that present in the X-ray crystal structure of TGF-beta 2 is more consistent with a type I turn conformation. This may be of functional significance as studies using TGF-beta chimeras and deletion mutants indicate that this portion of the molecule may be important in receptor binding.

Amides↗
PubMed1996

The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.

Animals↗
PubMed1996

The war on cancer.

25 years ago, then President Nixon "declared" War on Cancer. In this personal commentary, the war is reviewed. There have been obvious triumphs, for instance in cure of acute lymphocytic leukaemia and other childhood cancers, Hodgkin's disease, and testicular cancer. However, substantial advances in molecular oncology have yet to impinge on mortality statistics. Too many adults still die from common epithelial cancers. Failure to appreciate that local invasion and distant metastasis rather then cell proliferation itself are lethal, obsession with cure of advanced disease rather than prevention of early disease, and neglect of the need to arrest preneoplastic lesions may all have served to make victory elusive.

Health Policy↗
PubMed1996

Spontaneously increased production of nitric oxide and aberrant expression of the inducible nitric oxide synthase in vivo in the transforming growth factor beta 1 null mouse.

Transforming growth factor beta 1 null mice (TGF-beta 1-/-) suffer from multifocal inflammation and die by 3-4 wk of age. In these mice, levels of nitric oxide (NO) reaction products in serum are elevated approximately fourfold over levels in controls, peaking at 15-17 d of life. Shortterm treatment of TGF-beta 1-/- mice with NG-monomethyl-L-arginine suppressed this elevated production of NO. Expression of inducible NO synthase (iNOS) mRNA and protein is increased in the kidney and heart of TGF-beta 1-/- mice. These findings demonstrate that TGF-beta 1 negatively regulates iNOS expression in vivo, as had been inferred from mechanistic studies on the control of iNOS expression by TGF-beta 1 in vitro.

Amino Acid Sequence↗
PubMed1995

Characterization of the promoter region of the human transforming growth factor-beta type II receptor gene.

Diminished cellular responsiveness to transforming growth factor-beta (TGF-beta) is frequently correlated with decreased transcription of the type II receptor for TGF-beta (TGF-beta RII). We have cloned and characterized the human TGF-beta RII promoter and, using S1 nuclease mapping and 5' rapid amplification of cDNA ends polymerase chain reaction, have identified five alternative transcription start sites within the region -33 to +57. DNA transfection experiments and electrophoretic mobility shift assays have revealed the existence of five distinct regulatory regions including two positive regulatory elements and two negative regulatory elements in addition to the core promoter region. The first positive regulatory element (-219 to -172) interacts with two distinct nuclear protein complexes, at least one of which appears to be a previously unidentified transcription factor. The second positive regulatory element (+1 to +35) also interacts with two separate protein complexes, both of which appear to be novel transcription factors. Deletion of either positive regulatory element markedly decreased expression of the target gene, suggesting that both positive regulatory elements are necessary for basal expression levels of TGF-beta RII.

Base Sequence↗
PubMed1995

Induction of transforming growth factor beta 1 and its receptors during all-trans-retinoic acid (RA) treatment of RA-responsive human neuroblastoma cell lines.

Recent work on a variety of normal and malignant cell lines has shown that induction and secretion of biologically active TGF-beta may occur after exposure to all-trans-retinoic acid (RA), coincident with decreased growth rate and/or differentiation. This study evaluates the expression and regulation of transforming growth factor beta (TGF-beta) and its receptors during RA-induced cell growth arrest and induction of differentiation in the RA-sensitive human neuroblastoma cell line SMS-KCNR and the RA-resistant neuroblastoma cell line SK-N-AS. RA treatment of SMS-KCNR cells results in a 40-fold increase in TGF-beta 1 mRNA after 4 days of RA, a dose-dependent increase in TGF-beta 1 secretion, an increase in types I (TBRI) and III (TBRIII) TGF-beta receptor proteins, and an increase in type II TGF-beta receptor (TBRII) mRNA coincident with RA-responsiveness of the cells. However, in the RA-resistant line SK-N-AS, TGF-beta 1 is constitutively secreted at levels that are unchanged after RA treatment, and although TBRI and TBRIII mRNA is expressed in untreated SK-N-AS cells, levels of TBRI and TBRIII protein and TBRII mRNA decrease after RA treatment. Thus, in RA-sensitive neuroblastoma cells, RA treatment may result in the induction of a negative autocrine TGF-beta 1 growth regulatory loop. These results suggest the hypothesis that: (a) induction of a TGF-beta 1 negative autocrine growth loop may be a necessary component for RA-responsiveness of neuroblastoma cells in vivo; and (b) the inability to induce or maintain this TGF-beta 1 negative autocrine growth loop may be a mechanism of RA resistance in neuroblastoma.

Base Sequence↗