Functional characterization of transforming growth factor beta type II receptor mutants in human cancer

We recently identified missense mutations at amino acid residues 526 and 537 located within the highly conserved subdomain XI of the transforming growth factor beta type II receptor (TbetaR-II) serine-threonine kinase in two human squamous carcinoma cell lines. These cell lines are resistant to transforming growth factor beta-mediated inhibition of growth. Moreover, treatment with transforming growth factor beta fails to increase the levels of type 1 plasminogen activator inhibitor and fibronectin synthesis. To test the effects of the mutations on receptor function, mutant TbetaR-II cDNAs were expressed in TbetaR-II-deficient T47D cells. Cyclin A promoter activity was reduced by 50% in cells expressing wild-type TbetaR-II but increased 2-fold in cells transfected with either of the two mutant receptors. Conversely, plasminogen activator inhibitor type 1 promoter activity was increased 6-fold in cells transfected with wild-type receptor but not with either of the two mutant receptors. Moreover, the activity of both mutant serine-threonine kinases was strongly reduced compared to that of the wild-type receptor. Thus, the amino acid residues at positions 526 and 537 seem to be essential for kinase function and signaling activity of the TbetaR-II.     

Growth inhibition by transforming growth factor beta (TGF-beta) type I is restored in TGF-beta-resistant hepatoma cells after expression of TGF-beta receptor type II cDNA

The growth of human hepatoma Hep 3B cells is potently inhibited by TGF-beta 1 (ID50 = 0.2 ng/ml, 8 pM). A mutant cell line was derived that was not inhibited in growth by TGF-beta 1 at 5 ng/ml (200 pM) and that lacked TGF-beta receptor type II (TGF-beta RII) gene. Transfection of the cloned cDNA for human TGF-beta RII to this mutant cell line restored receptor expression as well as the inhibition in growth by TGF-beta 1. In both wild-type and mutant cells stably transfected with TGF-beta RII cDNA, TGF-beta RII coimmunoprecipitated with TGF-beta receptor type I in the presence of ligand. These experiments provide direct evidence for the role of TGF-beta RII in the inhibitory effect of TGF-beta on growth and suggest that TGF-beta RII acts by means of a heteromeric surface complex with TGF-beta receptor type I.     

Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from delta beta RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.     

Expression of transforming growth factor beta and transforming growth factor beta receptors on AIDS-associated Kaposi's sarcoma

Several humoral growth factors may contribute to the development and growth of AIDS-associated Kaposi's sarcoma (KS). They are either provided by chronically activated cells of the immune system or in an autocrine/paracrine manner by the neoplastic cells themselves. Transforming growth factor beta(TGF-beta) may directly enhance the growth of KS cells and tumor matrix formation. To mediate a signal both TGF-beta receptors type I and type II (TbetaR-I and TbetaR-II) have to be expressed. We investigated the expression of TGF-beta, TGF-beta receptors types I and II, and endoglin, a nonsignaling-type TbetaR-III, by means of immunohistochemistry on skin biopsies from patients with AIDS-related KS. We found that the TGF-beta ligand was expressed by KS cells in 9 of 11 samples. TbetaR-II was strongly expressed in 10 of 12 samples, but none of the investigated tumor samples stained for TbetaR-I. Endoglin was weakly expressed on all KS lesions and stained the endothelium of tumor-associated vessels in 92% of the samples. These findings show that most KS lesions have the ability to produce TGF-beta and that KS cells maintain a high expression of TbetaR-II in the absence of TbetaR-I, which may allow KS to escape growth inhibitory effects of endocrine or paracrine TGF-beta.     

Two different signal transduction pathways can be activated by transforming growth factor beta 1 in epithelial cells

Signal transduction initiated by transforming growth factor beta 1 (TGF beta 1) was studied in two sublines of the same colon carcinoma cell line, which respond in opposite ways to TGF beta 1, by proliferation or by growth inhibition. TGF beta 1 activates ras proteins within 5 min of addition when it acts to inhibit growth but not when it acts as a mitogen. In both cases TGF beta 1 also rapidly modulates the activities of three protein kinases, detected by their in gel kinase activity on the mitogen-activated protein kinase (MAP kinase) substrate, myelin basic protein (MBP). When TGF beta 1 acts as a mitogen for U9 cells, it increases the activity of MBP kinases of 57, 105, and 130 kDa within 10 min of the addition without detectably activating ras proteins. When TGF beta 1 inhibits the growth of HD3 cells, it activates ras proteins and the 57-kDa MBP kinase within 5 min but inhibits the activity of the 105- and 130-kDa MBP kinases. In HD3 cells ras activation occurred in two signal transduction pathways, one from TGF beta 1 leading to growth inhibition and one from epidermal growth factor (EGF) leading to proliferation. In addition to ras proteins, EGF activates a different set of MBP kinases in HD3 cells than does TGF beta 1, MBP kinases of 85, 57, and 44 kDa. The latter is likely to be the 44-kDa MAP kinase extracellular signal-regulated kinase (erk) 1, because EGF treatment of HD3 cells activates erk1 by increasing its phosphotyrosine level. Therefore, in two closely related epithelial cell lines TGF beta 1 activates two different signal transduction pathways, one ras-dependent and one ras-independent, and modulates the activities of a set of MBP kinases.     

A novel protein distinguishes between quiescent and activated forms of the type I transforming growth factor beta receptor

Transforming growth factor beta (TGFbeta) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFbeta receptor (TbetaR-II) phosphorylating type I TGFbeta receptor (TbetaR-I). Because the failure of interaction cloning, thus far, to identify bona fide TbetaR-I substrates might reasonably have been due to the use of inactive TbetaR-I as bait, we sought to identify molecules that interact specifically with active TbetaR-I, employing the triple mutation L193A,P194A,T204D in a yeast two-hybrid system. The Leu-Pro substitutions prevent interaction with FK506-binding protein 12 (FKBP12), whose putative function in TGFbeta signaling we have previously disproved; the charge substitution at Thr204 constitutively activates TbetaR-I. Unlike previous screens using wild-type TbetaR-I, where FKBP12 predominated, none of the resulting colonies encoded FKBP12. A novel protein was identified, TbetaR-I-associated protein-1 (TRAP-1), that interacts in yeast specifically with mutationally activated TbetaR-I, but not wild-type TbetaR-I, TbetaR-II, or irrelevant proteins. In mammalian cells, TRAP-1 was co-precipitated only by mutationally activated TbetaR-I and ligand-activated TbetaR-I, but not wild-type TbetaR-I in the absence of TGFbeta. The partial TRAP-1 protein that specifically binds these mutationally and ligand-activated forms of TbetaR-I can inhibit signaling by the native receptor after stimulation with TGFbeta or by the constitutively activated receptor mutation, as measured by a TGFbeta-dependent reporter gene. Thus, TRAP-1 can distinguish activated forms of the receptor from wild-type receptor in the absence of TGFbeta and may potentially have a functional role in TGFbeta signaling.     

Consistent loss of functional transforming growth factor beta receptor expression in murine plasmacytomas

SPR Online (http:@www.pedrad.org) is a recently developed digital representation of the Society for Pediatric Radiology (SPR) that enables physicians to access pertinent information and services on the Internet. SPR Online was organized on the basis of the five main services of the SPR, which include Administration, Patient Care, Education, Research, and Meetings. For each service, related content from the SPR was digitized and placed onto SPR Online. Usage over a 12-month period was evaluated with server log file analysis. A total of 3,209 users accessed SPR Online, viewing 11,246 pages of information. A wide variety of information was accessed, with that from the Education, Administration, and Meetings services being the most popular. Fifteen percent of users came from foreign countries. As a virtual professional society, SPR Online greatly enhances the power and scope of the SPR and has proved to be a popular resource, meeting the diverse information needs of an international community of pediatric radiologists.     

Reduced levels of transforming growth factor beta receptor type II in human prostate cancer: an immunohistochemical study

In previous studies we demonstrated that the growth of human prostatic adenocarcinoma is associated with aberrant accumulation of transforming growth factor (TGF) beta1, a growth factor that has been shown to be a potent inhibitor of epithelial cell proliferation. We investigated the expression of TGF-beta receptor II (TGFbetaR-II) in benign prostate tissue and in prostate cancer using standard immunohistochemical techniques. Quantitation of immunopositivity for TGFbetaR-II was assessed on a visual analogue scale ranging from 0 (absence of staining) to 4+ (intensely positive staining). All of the benign glandular epithelia stained intensely, either 3+ or 4+, representative of the ubiquitous nature of TGFbetaR-II in normal tissue. Overall, staining was reduced in prostate cancer sections, and there was progressively diminished staining as the histological grade of the cancer increased (P < 0.01, Kruskal-Wallis test). This immunohistochemical study indicates that a decline in the levels of TGFbetaR-II is correlated with advancing histological aggressiveness of the cancer and suggests that aberrant TGFbetaR-II function may play a role in human prostate carcinogenesis.     

Genetic alterations of the transforming growth factor beta receptor genes in pancreatic and biliary adenocarcinomas

Transforming growth factor beta (TGF-beta) is an extracellular ligand that binds to a heterodimeric receptor, initiating signals that regulate growth, differentiation, and apoptosis. Many cancers, including pancreatic cancer, harbor defects in TGF-beta signaling and are resistant to TGF-beta-mediated growth suppression. Genetic alterations of DPC4, which encodes a DNA binding protein that is a downstream component of the pathway, most frequently occur in pancreatic and biliary carcinomas. We searched for other targets of mutation of the TGF-beta pathway in these cancers. We report somatic alterations of the TGF-beta type I receptor gene ALK-5. Homozygous deletions of ALK-5 were identified in 1 of 97 pancreatic and 1 of 12 biliary adenocarcinomas. A germ-line variant of ALK-5, presumably a polymorphism, was identified, but no somatic intragenic mutations were identified upon sequencing of all coding regions of ALK-5. Somatic alterations of the TGF-beta type II receptor gene (TGFBR2) were identified in 4 of 97 (4.1%) pancreas cancers, including a homozygous deletion in a replication error-negative cancer and three homozygous frameshift mutations of the poly(A) tract of the TGF-beta type II receptor in replication error-positive cancers. We also studied other related type I receptors of the TGF-beta superfamily. In a panel of pancreas cancers preselected for loss of heterozygosity at the ALK-1 locus, sequencing of all coding exons of the ALK-1 gene revealed no alterations. No homozygous deletions were detected in the ALK-1, ALK-2, ALK-3, or ALK-6 genes in a panel of 86 pancreatic cancer xenografts and 11 pancreatic cancer and 22 breast cancer cell lines. The rate of genetic inactivation of TGF-beta pathway members was determined in 45 pancreatic cancers. Eighty-two % of these pancreatic cancers had genetic inactivation of the DPC4, p15, ALK-5, or TGFBR2 genes. Our results indicate that the TGF-beta type I and type II receptor genes are selective targets of genetic inactivation in pancreatic and biliary cancers.     

Expression of the transforming growth factor beta isoforms in inflammatory cells of nasal polyps

Patients in chronic dialysis have a higher prevalence of cardiovascular morbidity and mortality, together with higher prevalence of hypertension and valvular diseases. It is not clear whether aortic and mitral defects are linked to the effect of chronic dialysis (for instance hypercalcaemia or hyperparathyroidism) or to hypertension. In order to see whether these factors could independently affect the single valve diseases we studied 48 patients in chronic dialysis. Patients were divided into hypertensive and normotensive. A population of hypertensive and another of normotensive, non-dialyzed patients served as control. The presence of valvular disease was searched by mean of echocardiography. We also investigated the length of dialytic treatment and the levels of parathyroid hormone in order to see if any correlation with the single valve defects existed. Aortic stenosis and insufficiency were not related to hypertension suggesting that circulating factors are likely to be involved in the pathogenesis of this valvulopathy (chi 2 = 6, p < 0.01 between hypertensive and normotensive in dialysis; chi 2 = 6.1, p < 0.01 between patients in dialysis and normotensive non uremic, for aortic stenosis; chi 2 = 12.1, p = 0.02 between non uremic normotensive and dialyzed, for aortic insufficiency). On the contrary for mitral regurgitation we did not find differences between dialyzed patients and controls (chi 2 = 18.2, p < 0.0001 between uremic hypertensive and controls). There was a significant difference in both groups between hypertensive and normotensive subjects suggesting that hypertension plays an important role in this valvulopathy. Mitral and aortic calcifications were more frequent in the uremic patients (55% in hypertensive uremics, 33% in normotensive uremics, 16 and 25% in non uremics).     

Overexpression of transforming growth factor beta type I receptor abolishes malignant phenotype of a rat bladder carcinoma cell line

The disposition of ISIS 2922, a phosphorothioate oligonucleotide for treatment of cytomegalovirus associated retinitis, was evaluated in rabbits. Vitreous humor and retina samples were collected from rabbits that received a single intravitreal injection of 66 microg [14C]-labeled ISIS 2922 and were analyzed using anion exchange HPLC. Four hr postdosing, the concentration of ISIS 2922 in vitreous humor was 3.3 microM. The elimination of ISIS 2922 from the vitreous humor exhibited first-order kinetics with a t1/2 of 62 hr. By 10 days postdosing, the mean concentration of ISIS 2922 in rabbit vitreous humor had decreased to 0.17 microM, which represented 22% of the total radioactivity remaining in the vitreous. The remaining 78% coeluted on anion exchange HPLC with shorter oligonucleotides. In retina, ISIS 2922 accumulated over the first 5 days postdosing, reaching a maximum concentration of 3.5 microM, and then declined thereafter with an estimated t1/2 of 79 hr. By 10 days postdosing when only 24% of the total radioactivity in the retina was parent compound, the concentration of ISIS 2922 remained at 1.6 microM, which was 10 times higher than the concentration in the vitreous humor. Whereas the elimination of full-length ISIS 2922 and total radioactivity from the vitreous humor occurred at nearly equal rates, ISIS 2922 disappeared more rapidly than did total radioactivity from the retina, suggesting a greater role for metabolism in the clearance process from retina than the vitreous. Alternatively, the results are consistent with metabolites being cleared from the vitreous at approximately the same rate as parent compound while in the retina metabolites may be cleared more slowly. The data were analyzed with a user-defined pharmacokinetic model, which was then used to predict the potential for accumulation of ISIS 2922 during clinical dosing.     

Human malignant thyroid tumors displayed reduced levels of transforming growth factor beta receptor type II messenger RNA and protein

Transforming growth factor beta (TGF-beta) is a physiological regulator of thyroid epithelial cell growth and differentiation. This factor signals through a heteromeric complex composed of type I (TGF-beta receptor type I) and type II [TGF-beta receptor type II (TbetaRII)] receptors. Loss of TbetaRII expression has been related to resistance to TGF-beta inhibition of cell proliferation. In the present work, we analyzed the TbetaRII expression in a series of human thyroid tumors, from benign lesions (adenomas) to neoplastic lesions of increasing aggressiveness (papillary and follicular carcinomas) up to the extremely aggressive anaplastic tumors. Results obtained indicated a clear reduced expression of TbetaRII mRNA only in the group of thyroid carcinomas when compared with their relative normal tissues. Immunohistochemical analyses with specific anti-TbetaRII antibodies confirm these observations. These data indicate that loss of expression of TbetaRII can contribute to thyroid cancer progression, inducing cancer cells to escape the growth-inhibitory effect of TGF-beta.     

Mutation and downregulation of the transforming growth factor beta type II receptor gene in primary squamous cell carcinomas of the head and neck

In the present study, we analyzed 28 squamous cell carcinomas of the head and neck (SCCHN) for mutations in the coding region of TbetaR-II using 'Cold' SSCP and automatic DNA sequencing analyses. Twenty-one percent (6/28) of the SCCHN examined contained TbetaR-II mutations compared with patient-matched normal tissues. These alterations included five missense mutations (A:T-->G:C transitions in codons 250, 401, 448 and 488, and a G:C-->T:A transversion in codon 373), and a 38-bp deletion between nucleotides 1825 to 1862. In addition to these code-altering mutations, one case exhibited a silent mutation (A:T-->G:C transition in codon 451) and three cases contained one of two potential population polymorphisms (codons 354 and 389). In contrast to colon and gastric cancers exhibiting microsatellite instability (MI) or replication errors (RER+), no 'indirect' frameshift mutations were identified within a 10-bp polyadenine repeat present in the TbetaR-II coding sequence. All of the mutations in the present study occurred within the highly conserved serine/threonine kinase domain and represent the first report of such 'direct' TbetaR-II mutations in primary human tumors. In addition, we analyzed a subset of SCCHN and corresponding normal samples for TbetaR-II mRNA expression using semi-quantitative multiplex RT-PCR. Expression of TbetaR-II was decreased by 24% to 74% in 20 of 23 SCCHN (87%) compared with patient-matched normal tissues. Taken together, the results from this study suggest that alterations in the nucleic acid sequence and mRNA expression of TbetaR-II are prevalent events in the development of SCCHN, which may deregulate cell cycle control.     

Role of transforming growth factor beta 1 (TGF beta 1) in mediating androgen-induced growth inhibition in human adrenal cortex in vitro

We have previously found that the androgen receptor gene is expressed both in normal and adenomatous human adrenal cortex and in the NCI-H295 human adrenocortical cancer cell line. Furthermore, we have observed that dihydrotestosterone (DHT) at physiological concentrations (10(-11) M) inhibits human adrenocortical cell growth in vitro and slightly decreases c-myc RNA levels in NCI-H295 cells. As c-myc is probably not the main mechanism mediating DHT-induced inhibition of cell growth, other genes controlling cell proliferation may be involved. Transforming Growth Factor beta (TGF beta) is a regulatory peptide that acts by both autocrine and paracrine mechanisms to control proliferation and differentiation, and there is previous evidence that TGF beta may exert an antimitotic effect on human fetal adrenal cells in vitro. This study examines a possible role for TGF beta 1 in mediating the DHT-induced reduction of human adrenocortical cell growth. TGF beta 1 and its receptor (TGF beta RII) are expressed in DHT-treated and nontreated NCI-H295 cells; on Northern blot analysis 24-h treatment with DHT (10(-11) M) produced a small increase in TGF beta RII RNA, and quantitative RT-PCR showed a 1.5-fold increase in TGF beta 1 RNA levels. These findings suggest that TGF beta 1 and its receptor may be involved in DHT-induced inhibition of human adrenocortical cell growth.     

Production of platelet-derived growth factor by interleukin-1 beta and transforming growth factor-beta-stimulated retinal pigment epithelial cells leads to contraction of collagen gels

PURPOSE: In an in vitro model of the later contractile stages of proliferative vitreoretinopathy, interleukin-1 beta (IL-1 beta) and transforming growth factor-beta (TGF-beta) stimulate the contraction of collagen gels by retinal pigment epithelial (RPE) cells. This contraction occurs after a lag period and appears not to be a direct effect of the cytokines but is mediated by another factor produced in the presence of the two cytokines. The nature of this factor has been investigated. METHODS: Human RPE cells were seeded onto collagen gels in the presence of IL-1 beta and TGF-beta. After 24 hours, the conditioned medium was removed and added to new collagen gels seeded with RPE cells, and the diameter of the collagen gels was measured after various intervals. The ability of the conditioned medium to effect contraction was determined after various treatments, including size fractionation, heating, trypsin digestion, and binding to heparin-Sepharose. The involvement of platelet-derived growth factor (PDGF) as a stimulator of contraction was tested with neutralizing antibodies and by polymerase chain reaction analyses of specific mRNAs. RESULTS: IL-1 beta and TGF-beta cause RPE cells to contract after a delay of up to 24 hours, whereas conditioned medium from cytokine-treated cells results in immediate contraction in a manner similar to that of serum. The factor in the conditioned medium causing immediate contraction was found to be heat-stable, trypsin-sensitive, and resistant to extremes of pH. It has a size of between 30 and 50 kDa and binds heparin. The factor in conditioned medium from cytokine-treated cells does not act in the presence of C-kinase inhibitors or cycloheximide, suggesting that signaling is mediated by way of protein kinase C and new protein synthesis. Stimulation of contraction by conditioned medium is inhibited by anti-PDGF antibodies, and contraction is stimulated by human PDGF. CONCLUSIONS: Contraction in the presence of cytokines is mediated by the production of PDGF or a PDGF-like molecule. This factor could have implications in the pathogenesis of proliferative vitreoretinopathy.     

Hepatocyte growth factor releases epithelial and endothelial cells from growth arrest induced by transforming growth factor-beta1

Human lung fibroblasts and Mv1Lu mink lung epithelial cells were used as a model to study the role of extracellular matrix in epithelial-mesenchymal interactions. Extracellular matrices of fibroblasts were found to contain growth promoting activity that reduced the sensitivity of Mv1Lu cells to the growth inhibitory effects of transforming growth factor-beta (TGF-beta). The majority of the activity was identified as hepatocyte growth factor/scatter factor (HGF) by inhibition with specific antibodies and by reconstitution of the effect by recombinant HGF. HGF induced cell proliferation when contact-inhibited Mv1Lu cells were trypsinized and plated in the presence of TGF-beta1. The effect was valid also in assays where Madin-Darby canine kidney epithelial cells or bovine capillary endothelial cells were used. The multiplication of chronically TGF-beta1 inhibited Mv1Lu cells was also induced by HGF. In addition, HGF induced anchorage independent growth of Mv1Lu cells that was refractory to TGF-beta1 growth inhibition. Immunoprecipitation analysis indicated that HGF prevented the suppression of Cdk4 and Cdk2, but not the induction of p21, by TGF-beta1. Since both TGF-beta1 and HGF require proteolysis for activation, the results imply that proteolytic activity of epithelial and endothelial cells directs their responses to signals from mesenchymal-type extracellular matrices, and that during development, matrix-bound growth and invasion promoting and suppressing factors are activated in a coordinated manner.