Inhibition of platelet-derived growth factor autocrine growth stimulation by a monoclonal antibody to the human alpha platelet-derived growth factor receptor

A potent neutralizing monoclonal antibody to the human alpha platelet-derived growth factor (PDGF) receptor (alpha PDGFR) was raised by immunizing BALB/c mice with 32D cells expressing the human alpha PDGFR. This monoclonal antibody, designated alpha R1, immunoprecipitated human, monkey, rabbit, pig, dog, and cat, but not hamster, rat, or mouse alpha PDGFRs. Comparison with PR292, a monoclonal antibody previously generated against the alpha PDGFR, showed that both recognized alpha PDGFR extracellular domains, but neither demonstrated reactivity against the beta PDGFR. In vitro binding studies revealed that alpha R1, but not PR292, detection of the alpha PDGFR was blocked by either PDGF AA or PDGF BB. These results strongly suggest that the receptor ligand-binding domain spatially overlapped with the alpha R1 epitope. Monoclonal antibody alpha R1 also inhibited PDGF stimulation of [3H]thymidine uptake by 32D cells expressing the alpha PDGFR (32D alpha R) as well as autocrine growth stimulation of 32D alpha R cells transfected with and expressing PDGF AA or PDGF BB. Therefore, monoclonal antibody alpha R1 may be useful in the detection and growth inhibition of malignancies in which PDGF autocrine stimulation and/or alpha PDGFR overexpression plays an important role(s).     

Tumor necrosis factor alpha enhances secretion of transforming growth factor beta2 in MCF-7 breast cancer cells

We studied the effect of tumor necrosis factor alpha (TNF-alpha) on transforming growth factor beta (TGF-beta) secretion by human breast cell lines to further characterize the antitumor effects of TNF-alpha. We found that TNF-alpha increased the secretion of TGF-beta in two established breast cancer cell lines (MCF-7 and ZR-75-1) but not in two immortalized human mammary epithelial cell lines (184B5 and MCF-10A). In MCF-7 cells, TNF-alpha increased the secretion of total TGF-beta 6.1-fold within 72 h in a dose-dependent manner. The secretion of both latent and active forms of TGF-beta was increased, and their ratio altered from 25:1 to 12:1 in the medium. TNF-alpha converted the secretory pattern of TGF-beta by MCF-7 cells from the heterodimeric form TGF-beta1.2 to the homodimeric form TGF-beta2. Immunoblot analysis under nonreducing conditions identified four molecular mass species of TGF-beta secreted in the culture media of untreated MCF-7 cells (238, 210, 40-55, and 25 kDa). Under reducing conditions, three molecular mass species of TGF-beta were identified: 88, 44, and 12 kDa. Gel filtration analysis demonstrated that the secreted TGF-beta within the range of 12-88 kDa was biologically active. TNF-alpha treatment did not alter the size of molecular mass species secreted by MCF-7 cells and did not change steady-state levels of mRNA for TGF-beta1 or TGF-beta2. These findings indicate that TNF-alpha may regulate quantitatively and qualitatively TGF-beta secretion by human breast cancer cells in vitro. The diverse biological activities of TGF-beta may also allow TNF-alpha to regulate the growth and metabolism of human mammary epithelial cells and/or stromal cells in a paracrine manner.     

Antiproliferative effect of DNA polymerase alpha antisense oligodeoxynucleotides on breast cancer cells

Antisense oligonucleotides appear to offer considerable promise as sequence-specific inhibitors of gene expression. Different cellular targets for oligodeoxynucleotides with oncologic interest have been identified such as oncogenes, growth factors, and cell cycle-related genes. DNA polymerase alpha (pol alpha) plays a relevant role in DNA synthesis and cell proliferation. Pol alpha gene expression is constitutive throughout the cell cycle and its mRNA content and activity are related to the growth rate and neoplastic phenotype. The effects of a 18-mer pol alpha antisense oligomer on the proliferation of the MDA-MB 231 breast cancer cell line have been investigated. After 48 h in culture with oligomers (10 microM), about 50% growth inhibition was observed in antisense-treated cells, as evaluated by 3-(4,5-dimethythiazol-2yl)-2,5-diphenyltetrazolium bromide assay and cell count. [3H]Thymidine incorporation exhibited a 90% inhibition of DNA synthesis associated to 64% accumulation of cells at the G1-S border of the cycle as by flow cytometry, at 24 h. Northern hybridization and SDS-PAGE of immunoprecipitated MDA-MB 231 cell lysates revealed a decreased expression of pol alpha mRNA and a reduction of the 180-kDa polypeptide, respectively. Collectively, the data further confirm the relevance of pol alpha in the replicative cycle, as well as strengthen the potentiality of the antisense strategy for the control of gene expression and cell growth.     

Regulation of activity levels of glycolipid sulfotransferases by transforming growth factor alpha in renal cell carcinoma cells

Accumulation of sulfolipids associated with markedly elevated levels of glycolipid sulfotransferase activities was previously demonstrated in human renal cell carcinoma cells. To explore the regulation mechanisms of sulfoglycolipid synthesis in renal cancer, effects of various growth factors on the metabolic enzymes of sulfoglycolipids were investigated by using a human renal cell carcinoma cell line, SMKT-R3. Among the growth factors tested, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to increase the sulfotransferase activity markedly (about 300%), but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. The augmented effects of TGF-alpha was abolished by cycloheximide. Since TGF-alpha is known to bind to the same receptor as EGF, SMKT-R3 cells were investigated for the EGF receptor by affinity cross-linking with 125I-EGF. A radiolabeled protein with a molecular mass of 175 kDa corresponding to the ligand-receptor complex was immunoprecipitated with a monoclonal anti-EGF receptor antibody. When production of the growth factors was examined immunochemically, the cells were found to secrete TGF-alpha at a low level and retain it in a membrane-bound form, whereas EGF was not detected. These observations suggest that the sulfotransferase activities are regulated through the autocrine, paracrine, and/or juxtacrine modes of intercellular stimulation by TGF-alpha in human renal cancer cells.     

Stimulation of prostaglandin E2 production by interleukin-1 alpha and transforming growth factor alpha in osteoblastic MC3T3-E1 cells

The mechanism by which interleukin-1 (IL-1) and transforming growth factor alpha (TGF-alpha) regulate prostaglandin synthesis has been examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in DMEM containing 10% fetal calf serum. Prostaglandin E2 (PGE2) production was determined by radioimmunoassay or by prelabeling cells with [3H]arachidonic acid, followed by high-performance liquid chromatography (HPLC) analysis of the labeled products released into the medium. Prostaglandin G/H synthase (PGHS) mRNAs were quantified by northern blot analysis using [32P]labeled cDNA probes. By HPLC, PGE2 was the major prostanoid produced under basal or stimulated conditions. No release of thromboxane or 6-keto-PGF1 alpha into the medium was detected. PGE2 production was stimulated approximately 7- to 14-fold by IL-1 (1 ng/ml) and 3- to 8-fold by TGF-alpha (30 ng/ml) after 24 h. In combination, however, IL-1 and TGF-alpha caused a synergistic 37- to 71-fold increase in PGE2 accumulation. PGHS-1 mRNA levels were maximally increased approximately 2- to 3-fold by IL-1 and 1.5 to 2.5-fold by TGF-alpha after 24 h; the combination of IL-1 and TGF-alpha produced only an additive 3- to 6-fold increase. Western blotting revealed a corresponding 3-fold increase in immunoreactive PGHS-1 protein in response to combined IL-1 and TGF-alpha. PGHS-2 mRNA was increased 1.4-fold by TGF-alpha at 1 h, and the combination of IL-1 and TGF-alpha caused a 1.7-fold increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of c-myc with transforming growth factor alpha and hepatocyte growth factor in hepatocarcinogenesis

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human TGF-alpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. These studies have now been extended to examine the interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c-myc with HGF or TGF-alpha results in profoundly different outcomes of the neoplastic process in the liver.     

Autocrine transforming growth factor alpha provides a growth advantage to malignant cells by facilitating re-entry into the cell cycle from suboptimal growth states

CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor alpha (TGF-alpha) was ectopically expressed by stable transfection of a TGF-alpha cDNA under repressible tetracycline control. The TGF-alpha-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-alpha transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-alpha-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to re-enter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-alpha-transfected cells indicate that autocrine TGF-alpha functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-alpha was associated with increased malignant properties as TGF-alpha transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-alpha expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-alpha. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-alpha.     

Inhibition of anchorage-independent growth of ras-transformed cells on polyHEMA surface by antisense oligodeoxynucleotides directed against K-ras

We examined the effect of antisense oligodeoxynucleotides (AS ODNs) directed against v-Ki-ras on the anchorage-independent growth of v-Ki-ras-transformed rat fibroblasts using plates coated with an antiadhesive polymer, poly(2-hydroxyethyl methacrylate) (polyHEMA). Among AS ODNs tested, 17mer AS ODN centered around codon 12 of v-Ki-ras inhibited the v-Ki-Ras expression and v-Ki-Ras-induced anchorage-independent growth, while its sense sequence did not affect it. Using polyHEMA plates, the direct addition of AS ODNs to the cells growing anchorage-independently and various treatment schedules of AS ODNs are now available to identify the most desirable conditions for AS ODNs treatment.     

Function of the type V transforming growth factor beta receptor in transforming growth factor beta-induced growth inhibition of mink lung epithelial cells

The type V transforming growth factor beta (TGF-beta) is a 400-kDa nonproteoglycan membrane protein that co-expresses with the type I, type II, and type III TGF-beta receptors in most cell types. The type V TGF-beta receptor exhibits a Ser/Thr-specific protein kinase activity with distinct substrate specificity (Liu, Q., Huang, S. S., and Huang, J. (1994) J. Biol. Chem. 269, 9221-9226). In mink lung epithelial cells, the type V TGF-beta receptor was found to form heterocomplexes with the type I TGF-beta receptor by immunoprecipitation with antiserum to the type V TGF-beta receptor after 125I-TGF-beta affinity labeling or Trans35S-label metabolic labeling of the cells. The kinase activity of the type V TGF-beta receptor was stimulated after treatment of mink lung epithelial cells with TGF-beta. TGF-beta stimulation resulted in the growth inhibition of wild-type mink lung epithelial cells and to a lesser extent of the type I and type II TGF-beta receptor-defective mutants, although higher concentrations of TGF-beta were required for the growth inhibition of these mutants. TGF-beta was unable to induce growth inhibition in human colorectal carcinoma cells lacking the type V TGF-beta receptor but expressing the type I and type II TGF-beta receptors. These results suggest that the type V TGF-beta receptor can mediate the TGF-beta-induced growth inhibitory response in the absence of the type I or type II TGF-beta receptor. These results also support the hypothesis that loss of the type V TGF-beta receptor may contribute to the malignancy of certain carcinoma cells.     

Immunocytochemical localization of transforming growth factor alpha, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5-15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell-cell communication, cell fate, and differentiation of conducting airway epithelia.     

Repair phenotype in corneal fibroblasts is controlled by an interleukin-1 alpha autocrine feedback loop

PURPOSE: To explore the role of autocrine interleukin-1 alpha (IL-1 alpha) as a central regulator of the repair phenotype in corneal fibroblasts. METHODS: Disruption of the actin cytoskeleton with cytochalasin B (CB), which mimics changes in shape that occur in repair tissues, was used to stimulate repair gene expression in early-passage fibroblasts. Changes in expression of IL-1 alpha, IL-8, collagenase, and ENA-78 were determined by Northern blot analysis, radioimmunoassay, and an enzyme-amplified sensitivity immunoassay (EASIA). Expression of repair genes was also examined in repair fibroblasts, isolated from healing, penetrating keratectomy wounds in rabbits. RESULTS: Blocking IL-1 alpha activity prevented both constitutive and stimulated increases in synthesis of IL-8 and collagenase in early-passage cultures of corneal fibroblasts, demonstrating the role of IL-1 alpha as a necessary intermediate for expression of these genes. Evidence is also presented that the IL-1 alpha autocrine controls expression of an IL-8 related factor, ENA-78. Unlike early-passage fibroblasts, fibroblasts freshly isolated from the uninjured cornea did not express IL-1 alpha. However, fibroblasts freshly isolated from remodeling corneal repair tissue 3 weeks after injury were found to express substantial levels of IL-1 alpha, regulated through an autocrine feedback loop. Neutralization experiments demonstrated that the IL-1 alpha autocrine is largely responsible for controlling both collagenase and IL-8 synthesis in repair fibroblasts, as it is in early-passage fibroblasts. CONCLUSIONS: These findings provide evidence that activation of an autocrine IL-1 alpha feedback loop is an important mechanism by which fibroblasts adopt a repair phenotype during remodeling of the cornea.     

Inhibition of in vitro proliferation of chronic myelogenous leukemia progenitor cells by c-myb antisense oligodeoxynucleotides

Normal hematopoietic progenitors and acute myelogenous leukemia cells show a differential requirement for the encoded product of c-myb proto-oncogene for proliferation. To determine whether c-myb is also differentially required for the proliferation of hematopoietic progenitors of chronic myelogenous leukemia (CML), mononuclear cells derived from both chronic phase and blast crisis were exposed to c-myb antisense oligodeoxynucleotides and assayed for colony-forming ability. Exposure of CML-BC cells from 12 patients to c-myb antisense oligodeoxynucleotides resulted in significant (p<001) inhibition of leukemia colony formation (average inhibition 63%) and was accompanied by down-regulation of c-myb expression. Colonies derived from CML chronic phase progenitors were virtually unaffected in 10 cases, but down-regulation of c-myb expression was not detected. However, in studies conducted with CD34+ leukemia cells, a subset highly enriched for hematopoietic progenitors, colony formation was inhibited at both disease stages, whereas CFU-GM colony formation derived from normal CD34+ cells was not affected by exposure to c-myb antisense oligodeoxynucleotides. These data suggest that CML chronic phase and blast crisis progenitors are both sensitive to the inhibitory effects of c-myb antisense oligomers, and that the lack of inhibition in partially purified CML-chronic phase progenitors is probably due to inefficient penetration of oligodeoxynucleotides into the clonogenic cells. The preferential effect of c-myb antisense oligodeoxynucleotides on colonies arising from the compartment that includes CML-CD34+ progenitors likely reflects the expansion of a cell population with high proliferative potential and elevated c-myb mRNA levels.     

Trophic actions of transforming growth factor alpha on mesencephalic dopaminergic neurons developing in culture

Transforming growth factor alpha messenger RNA and protein levels are highest in the striatum, the target area of mesencephalic dopaminergic neurons of the substantia nigra, suggesting a role as a target-derived neurotrophic factor for these cells. To test this hypothesis, we characterized the actions of transforming growth factor alpha on fetal rat dopaminergic neurons in culture. Transforming growth factor alpha promoted dopamine uptake in a dose- and time-dependent manner. Administration of transforming growth factor alpha at the time of plating for 2 h produced a significant increase in dopamine uptake after five days of growth in vitro. As cultures aged they became less responsive to transforming growth factor alpha, such that longer times of exposure were required to elicit a similar, but weaker, response. Dopaminergic cell survival was selectively promoted by transforming growth factor alpha, since there was an increase in the number of tyrosine hydroxylase-immunostained cells without a parallel increase in the total number of neuron-specific enolase-immunopositive cells. Neurite length, branch number and soma area of tyrosine hydroxylase-immunopositive cells also were enhanced by transforming growth factor alpha treatment. Increases in each of the dopaminergic parameters due to transforming growth factor alpha were accompanied by a rise in glial cell number, making it possible that these effects were mediated by this cell population. The neurotrophin antagonist, K252b, failed to inhibit the transforming growth factor alpha-induced increase in dopamine uptake, indicating that transforming growth factor alpha's effects were not mediated by neurotrophin mechanisms. The actions of transforming growth factor alpha on the differentiation of dopaminergic neurons only partially overlapped with those of epidermal growth factor. Thus, while transforming growth factor alpha and epidermal growth factor are believed to share the same receptor they differentially affect dopaminergic cell development in vitro. These results indicate that transforming growth factor alpha is a trophic factor for mesencephalic cells in culture and suggests that transforming growth factor alpha plays a physiological role in the development of these cells in vivo.     

Radiation treatment decreases transforming growth factor alpha expression in squamous carcinoma of the tongue

Ionizing radiation (XRT) is often used to treat squamous cell carcinoma of the tongue (SCCT) but little is known of its genetic effects on surviving cancer cells. The effect of XRT on p53, epidermal growth factor receptor (EGFR), and transforming growth factor alpha (TGF alpha) tumor marker expression was evaluated using immunohistochemical analysis in 79 patients with SCCT. Sixty-six patients received no radiation, while 13 received XRT before surgery. Radiation did not influence EGFR or p53 expression. TGF alpha expression, however, was significantly decreased in radiated tumors (15% versus 43%, P = 0.04). These data suggest that XRT either decreases the expression of TGF alpha in SCCT (suggesting a genetic alteration in surviving cancer cells), or does not kill cancer cells with decreased TGF alpha expression. In the latter case, diminished TGF alpha expression may serve as a marker of radioresistance.