Heterozygous inactivation of TGF-beta1 increases the susceptibility to chemically induced mouse lung tumorigenesis independently of mutational activation of K-ras.

Mice heterozygous for deletion of the transforming growth factor beta1 (TGF-beta1) gene show an enhanced rate of lung tumorigenesis following carcinogen treatment. Since the growth inhibitory activity of TGF-beta1 in epithelial cells is associated with K-ras p21, and K-ras mutations commonly occur in chemically-induced mouse lung tumors, we postulated that tumors in heterozygous TGF-beta1 mice might be more likely to have K-ras mutations compared with tumors in wildtype TGF-beta1 mice. Urethane-induced lung tumors in AJBL6 TGF-beta1 +/- and +/+ mice were examined for K-ras mutations by polymerase chain reaction/single strand conformation polymorphism analysis and sequencing. Mutation frequencies were similar in both genotypes: 12/18 +/- tumors (67%) and 10/16 +/+ tumors (62%). Mutations occurred in 80% +/- and 75% +/+ carcinomas, but in only 50% of the adenomas of both TGF-beta1 genotypes. Codon 61 A-->G transition mutations were predominant, occurring in 61% +/- and 44% +/+ tumors. Three +/- (17%) and three +/+ (19%) tumors showed codon 12 mutations, mostly G-->A transitions. Two +/- tumors had both codon 61 and codon 12 mutations. Interestingly, carcinomas with mutations in codon 61 were larger than those with codon 12 changes. It appears that the mechanism of enhanced susceptibility of TGF-beta1+/- mice to urethane-induced lung carcinogenesis does not involve selective development of tumors with K-ras mutations.

Identification of differentially expressed nucleolar TGF-beta1 target (DENTT) in human lung cancer cells that is a new member of the TSPY/SET/NAP-1 superfamily.

The transforming growth factor-beta1 (TGF-beta1) responsive epithelial non-small-cell lung cancer (NSCLC) cell line NCI-H727 was used to identify potential target genes involved in TGF-beta1-mediated responses. Comparative cDNA expression patterns between cells treated with TGF-beta1 and those treated with vehicle were generated by differential mRNA display. One 496-bp fragment, differentially increased threefold by TGF-beta1 and hybridizing to a 2.7-kb mRNA species in NCI-H727 cells by Northern analysis, revealed no significant match to any known gene sequence. The mRNA transcript of this novel gene that we named differentially expressed nucleolar TGF-beta1 target (DENTT) is expressed in several normal human tissues, with the highest level of expression in brain. Human brain cDNA library screening and 5' rapid amplification of cDNA ends yielded full-length DENTT cDNA containing an 1899-bp open reading frame encoding a predicted 633-amino-acid protein with four potential nuclear localization signals (NLSs) and two coiled-coil regions. DENTT contains a conserved 191-residue domain that shows significant identity to, and defines, the TSPY/TSPY-like/SET/NAP-1 superfamily. Enhanced green fluorescent protein (EGFP)-tagged full-length DENTT transfected into COS-7 cells showed nucleolar and cytoplasmic localization. Transfection of EGFP-tagged DENTT NLS deletion constructs lacking the bipartite NLS-1 were excluded from the nucleolus. While NLS-1 is necessary for nucleolar localization of DENTT, it is not sufficient for sole nucleolar localization. Our data show that DENTT mRNA induction by TGF-beta1 correlates with induction of TGF-beta1 mRNA, induction of extracellular matrix gene expression, and inhibition of colony formation in soft agarose in TGF-beta1 responsive NSCLC cells when exposed to TGF-beta1. TGF-beta1 does not induce DENTT mRNA expression in TGF-beta1 nonresponsive NSCLC cells. Our data suggest that this novel TGF-beta1 target gene has distinct domains for direction to different subnuclear locations.

Enhanced tumorigenesis and reduced transforming growth factor-beta type II receptor in lung tumors from mice with reduced gene dosage of transforming growth factor-beta1.

To elucidate the role of transforming growth factor-beta1 (TGF-beta1) and the TGF-beta type II receptor (TGF-beta RII) as tumor-suppressor genes in lung carcinogenesis, we mated C57BL/6 mice heterozygous (HT) for deletion of the TGF-beta1 gene with A/J mice to produce AJBL6 TGF-beta1 HT progeny and their wild-type (WT) littermates. Immunohistochemical staining, in situ hybridization, and northern blot analyses showed lower staining and hybridization for TGF-beta1 protein and mRNA, respectively, in the lungs of normal HT mice versus WT mice. Competitive reverse transcription-polymerase chain reaction (CRT-PCR) amplification showed the level of TGF-beta1 mRNA in the lungs of HT mice to be fourfold lower than the level in WT lung. When challenged with ethyl carbamate, lung adenomas were detected in 55% of HT mice by 4 mo but only in 25% of WT littermates at this time. Whereas all HT mice had adenomas by 6 mo, it was not until 10 mo before all WT mice had adenomas. After 12 mo, the average number of adenomas was fivefold higher in HT lungs than in WT lungs. Most dramatic was the appearance of lung carcinomas in HT mice 8 mo before they were visible in WT mice. Thus, the AJBL6 TGF-beta1 HT mouse provides an excellent model system to examine carcinogen-induced lung tumorigenesis by increasing progressive lesion incidence and multiplicity relative to their WT littermates. Immunohistochemical staining showed expression of the TGF-beta type I receptor (TGF-beta RI) at moderate to strong levels in lung adenomas and carcinomas in HT and WT mice. In contrast, whereas weak immunostaining for TGF-beta RII was detected in 67% of HT carcinomas at 12 mo, only 22% of WT carcinomas showed weak staining for this protein. Individual lung carcinomas showing reduced TGF-beta RII expression and adjacent normal bronchioles were excised from HT lungs using laser capture microdissection, and CRT-PCR amplification of the extracted RNA showed 12-fold less TGF-beta RII mRNA in these carcinomas compared with bronchioles. Decreasing TGF-beta RII mRNA levels occurred with increasing tumorigenesis in lung hyperplasias, adenomas, and carcinomas, with carcinomas having fourfold and sevenfold lower levels of TGF-beta RII mRNA than adenomas and hyperplasias, respectively. These data show enhanced ethyl carbamate-induced lung tumorigenesis in AJBL6 HT mice compared with WT mice, suggesting that both TGF-beta1 alleles are necessary for tumor-suppressor activity. Reduction of TGF-beta RII mRNA expression in progressive stages of lung tumorigenesis in HT mice suggests that loss of TGF-beta RII may play an important role in the promotion of lung carcinogenesis in mice with reduced TGF-beta1 gene dosage when challenged with carcinogen.

Endocervical cancer is associated with an increase in the ligands and receptors for transforming growth factor-beta and a contrasting decrease in p27(Kip1).

OBJECTIVE: The aim of this study was to investigate the relationship between the expression of the TGF-beta ligands and TGF-beta receptors to the expression of p27(Kip1), a TGF-beta-regulated gene, in endocervical cancer. METHODS: To examine the expression of TGF-beta and p27(Kip1) in malignant transformation of the uterine endocervix, a panel of 23 formalin-fixed and paraffin-embedded human cervical specimens, including 8 with benign endocervical glands, 8 with cervical adenocarcinoma in situ, and 7 with cervical adenocarcinomas, was used. Tissues were immunostained with polyclonal antibodies that react specifically with TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta RI, TGF-beta RII, and p27(Kip1). RESULTS: Immunostaining for TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta RI, TGF-beta RII, and p27(Kip1) was detected in normal endocervix, with the TGF-betas showing weak cytoplasmic staining, while p27(Kip1) showed strong nuclear staining. Expression of TGF-beta increased significantly upon neoplastic transformation with the TGF-beta ligands and receptors showing strong cytoplasmic staining in adenocarcinoma in situ compared to normal endocervix. Interestingly, expression of TGF-beta was lower in adenocarcinoma than in adenocarcinoma in situ, but still significantly higher than in normal endocervix. TGF-beta 2 and TGF-beta 3 showed higher levels of immunostaining than TGF-beta 1 in adenocarcinomas. In contrast, p27(Kip1) protein expression decreased with progressive malignancy, with lower p27(Kip1) protein levels detected in adenocarcinoma than in adenocarcinoma in situ, while normal endocervix showed the highest level of p27(Kip1) protein expression. CONCLUSION: Elevated expression of the TGF-beta ligands and receptors is found in both cervical adenocarcinoma in situ and adenocarcinoma compared to normal endocervix. In contrast, a progressive decrease in p27(Kip1) occurs upon neoplastic transformation of the normal endocervix to cervical adenocarcinoma. These results suggest that neoplastic transformation of the endocervix may be related to dysregulation of TGF-beta and p27(Kip1) seen as an elevation of TGF-beta and a reduction of p27(Kip1) expression that may lead to loss of cell cycle control.

Transforming growth factor-beta 1 and its receptors in human lung cancer and mouse lung carcinogenesis.

The transforming growth factor-betas (TGF-beta s) are multifunctional proteins that inhibit the proliferation of many epithelial cells through a set of cell protein receptors that includes the TGF-beta type I (RI) and type II (RII) receptors. Loss of growth inhibition by TGF-beta is thought to contribute to the development of many types of tumors. In the present study, we have examined expression of the proteins and mRNAs for TGF-beta 1, TGF-beta RI, and TGF-beta RII in normal human lung, well-characterized non-small cell lung cancer (NSCLC) cell lines, and primary NSCLC specimens. Immunohistochemical staining for TGF-beta 1, TGF-beta RI, and TGF-beta RII using specific antibodies in normal human lung showed expression of the 3 proteins in the epithelium of bronchi and bronchioles as well as in alveoli. Differential expression of TGF-beta RI and TGF-beta RII proteins was detected in 5 NSCLC cell lines using Western blot analysis, with reduced levels in 3 cell lines. A panel of 45 formalin-fixed and paraffin-embedded NSCLC specimens showed positive immunostaining for TGF-beta 1, TGF-beta RI, and TGF-beta RII, with reduced TGF-beta RII in poorly differentiated adenocarcinomas and squamous cell carcinomas and some moderately differentiated adenocarcinomas. In situ hybridization studies conducted with specific riboprobes for TGF-beta 1, TGF-beta RI, and TGF-beta RII showed corresponding localization of expression of the mRNAs in the specimens that showed positive immunostaining for the proteins. To investigate the roles of TGF-beta 1, TGF-beta RI, and TGF-beta RII in chemically induced mouse lung tumorigenesis, we examined the expression of their proteins and mRNAs in 2 mouse model systems. Whereas expression of the proteins and mRNAs for TGF-beta 1 and TGF-beta RI was comparable in lung adenomas and bronchioles of A/J mice treated with benzo(alpha)pyrene, decreased immunostaining and hybridization for TGF-beta RII protein and mRNA was detected in 50% of lung adenomas in these mice. Interestingly, expression of TGF-beta 1 and the TGF-beta receptor proteins was similar to that of bronchioles in C57B1/6 mice and their littermates heterozygous for deletion of the TGF-beta 1 gene treated with diethylnitrosamine. These data show that reduced levels of expression of TGF-beta RII occur in some, but not all, human and mouse lung tumors. This suggests that different mechanisms of action, some of which may involve the TGF-beta signaling pathway, may contribute to the progression of lung tumorigenesis.

Retinoic acid down-regulates VPAC(1) receptors and TGF-beta 3 but up-regulates TGF-beta 2 in lung cancer cells.

The effects of retinoic acid (RA) on lung cancer cells were investigated. Both all-trans (t-RA) and 13-cis RA (c-RA) decreased specific (125)I-VIP binding to NCI-H1299 cells in a time- and concentration-dependent manner. After 20 hr, 30 microM t-RA decreased specific (125)I-VIP binding by 60%. By Scatchard analysis, the density of VIP binding sites but not the affinity was reduced by 42%. NCI-H1299 VPAC(1) receptor mRNA was reduced by 48%. VIP caused a 3-fold elevation in the NCI-H1299 cAMP, and the increase in cAMP caused by VIP was reduced by 38% if the NCI-H1299 cells were treated with t-RA. Using the MTT assay, 3 microM t-RA and 3 microM c-RA inhibited NCI-H1299 proliferation by 60 and 23% respectively. Also, transforming growth factor (TGF)-beta2 increased after treatment of NCI-H1299 cells with t-RA whereas TGF-beta 1 mRNA was unaffected and TGF-beta 3 mRNA was decreased. These results suggest that RA may inhibit lung cancer growth by down-regulating VPAC(1) receptor and TGF-beta 3 mRNA but up-regulating TGF-beta 2 mRNA.

Human pulmonary acinar aplasia: reduction of transforming growth factor-beta ligands and receptors.

Pulmonary hypoplasia has been found in the human neonatal autopsy population and has been attributed to an alteration in epithelial-mesenchymal interactions during development of the lung. Pulmonary acinar aplasia is a very rare and severe form of pulmonary hypoplasia. The transforming growth factor-betas (TGF-beta) are multifunctional regulatory peptides that are secreted by a variety of normal and malignant cells and are expressed in developing organs including the lung; their tissue distribution patterns have possible significance for signaling roles in many epithelial-mesenchymal interactions. Here, we report our examination of TGF-beta in the lungs of a term female infant diagnosed with pulmonary acinar aplasia whose autopsy revealed extremely hypoplastic lungs with complete absence of alveolar ducts and alveoli. Immunohistochemical and in situ hybridization analyses were used to localize and measure the proteins and mRNA, respectively, for TGF-beta1, TGF-beta2, TGF-beta3, and TGF-beta type I and type II receptors (TGF-beta RI and RII) in formalin-fixed and paraffin-embedded sections of these hypoplastic lungs and normal lungs. Immunostaining for TGF-beta1, TGF-beta2, and TGF-beta RI and RII was significantly lower in the bronchial epithelium and muscle of the hypoplastic lungs than in normal lungs, whereas no difference was detected in staining for other proteins including Clara cell 10-kD protein, adrenomedullin, hepatocyte growth factor/scatter factor, and hepatocyte growth factor receptor/Met in the hypoplastic and normal lungs or in the liver and kidneys of this infant compared with normal liver and kidney. In addition, in situ hybridization showed that TGF-beta1 and TGF-beta RI transcripts were considerably reduced in the bronchial epithelium of the hypoplastic lung compared with normal lung. These results show that there is a selective reduction of TGF-beta in pulmonary acinar aplasia and suggest that the signaling action of TGF-beta in epithelial-mesenchymal interactions in the lungs of this developmental condition may be compromised.

Coordinate expression of transforming growth factor-beta1 and adrenomedullin in rodent embryogenesis.

Transforming growth factor-beta (TGFbeta) and adrenomedullin (AM) are multifunctional regulatory peptides that are secreted by a variety of normal and malignant cells. The TGFbetas are expressed in developing organs and adults, and their tissue distribution pattern has possible significance for signaling roles in many epithelial-mesenchymal interactions. AM is also expressed in a variety of embryonic and adult tissues. The present study reports a comparison of the patterns of expression of the proteins and messenger RNAs (mRNAs) for TGFbeta1 and AM in the developing mouse embryo. Immunohistochemical and in situ hybridization analyses were performed on formalin-fixed paraffin-embedded sections of developing embryonic mouse tissues using specific antibodies and complementary RNA probes for TGFbeta1 and AM. The early placenta, including the giant trophoblastic cells, showed high levels of staining and hybridization for TGFbeta1 and AM proteins and mRNAs. The heart was the first organ that showed expression of TGFbeta1 and AM during embryogenesis. The spatio-temporal patterns of expression of TGFbeta1 and AM in cardiovascular, neural, and skeletal-forming tissues as well as in the main embryonic internal organs showed striking similarities. The lung, kidney, and intestine, in which epithelial-mesenchymal interactions occur, showed similar patterns of TGFbeta1 and AM expression. These data show colocalization of TGFbeta1 and AM in specific cell types associated with several tissues in the developing mouse embryo. Additionally, RT-PCR amplification and Northern blot hybridization showed expression of TGFbeta1 and AM mRNAs in all embryonic and adult mouse and rat tissues examined. Our data show that the expression of TGFbeta1 and AM is regulated in a spatial and temporal manner such that overlapping patterns of expression of TGFbeta1 and AM occur in several tissues at the same stage of development and in the same cellular location in rodent embryogenesis.

Transforming growth factor-beta expression in mouse lung carcinogenesis.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth modulator that inhibits the proliferation of many epithelial cells while stimulating the proliferation of most fibroblasts. To examine the role of TGF-beta in mouse lung chemically induced tumorigenesis, expression of the TGF-beta 1, -beta 2, and -beta 3 proteins was examined in A/J mice treated with the carcinogen urethane to induce lung adenomas using immunohistochemical staining analysis. Immunostaining for the TGF-beta ligands was detected in the epithelium of the bronchioles of untreated A/J mice with immunostaining being more intense for TGF-beta 1 than for TGF-beta 2 and TGF-beta 3; immunostaining for each TGF-beta ligand was also detected in the bronchiolar epithelium of urethane-treated A/J mice at levels similar to untreated mice. Immunostaining for the TGF-beta ligands was also detected in adenomas by 2 months; staining for TGF-beta 1, -beta 2, and -beta 3 in adenomas was detected at levels comparable with bronchioles. Following treatment with urethane for 8 months, immunostaining for TGF-beta s 1, 2, and 3 in bronchioles persisted at levels comparable to that in normal bronchioles and also persisted in adenomas, with staining for the TGF-beta ligands being very prominent on the edge of the tumor. Expression of TGF-beta 1 mRNA was examined in urethane-treated mouse lung tissue using Northern blot hybridization; here, expression of TGF-beta 1 mRNA increased 2-fold in 3-month urethane-treated lung tissue and an additional 2.5-fold by 8 months following urethane administration. Expression of TGF-beta 1 mRNA was also examined in nontumorigenic and tumorigenic mouse lung cells; in these cells, expression of TGF-beta 1 mRNA was higher in the tumorigenic cells than in the nontumorigenic cell line. These data show that there is an increase in expression of TGF-beta 1 during tumorigenesis and suggest that TGF-beta may play an important role in mouse lung carcinogenesis induced by urethane.

Transforming growth factor-beta1 is a new form of tumor suppressor with true haploid insufficiency.

Components of the transforming growth factor-beta (TGF-beta) signal pathway function as classic tumor suppressors, but the role of the TGF-betas themselves is less clear. Here we show that mice heterozygous for deletion of the TGF-beta1 gene express only 10-30% of wild-type TGF-beta1 protein levels. Although grossly normal, these mice have a subtly altered proliferative phenotype, with increased cell turnover in the liver and lung. Treatment of these mice with chemical carcinogens resulted in enhanced tumorigenesis when compared with wild-type littermates. However, tumors in the heterozygous mice did not lose the remaining wild-type TGF-beta1 allele, indicating that the TGF-beta1 ligand is a new form of tumor suppressor that shows true haploid insufficiency in its ability to protect against tumorigenesis.

Reduction in transforming growth factor-beta type II receptor in mouse lung carcinogenesis.

Transforming growth factor-beta (TGF-beta) is a growth modulator that inhibits the proliferation of many epithelial cells through interaction with its receptors, the type I and type II receptors (TGF-beta RI and RII) by activating their serine/threonine kinase activities. Loss of growth inhibition by TGF-beta is thought to contribute to the development of many types of tumors. To examine the roles of TGF-beta1, -beta2, and -beta3 and TGF-beta RI and RII in chemically induced mouse lung tumorigenesis, we used immunohistochemical and in situ hybridization analyses to measure the expression of their proteins and mRNAs in A/J mice treated with the carcinogen urethane to induce lung adenomas. Immunostaining for the TGF-beta ligands and receptors was detected in the epithelia of the bronchioles of untreated and treated A/J mice at similar levels. Immunostaining for the TGF-beta ligands and receptors was also detected in adenomas by 2 mo. While immunostaining for TGF-beta1, -beta2, and -beta3 and TGF-beta RI in adenomas was detected at levels comparable to those in bronchioles, immunostaining for TGF-beta RII was less intense in adenomas than in bronchioles. Decreased immunostaining for TGF-beta RII in adenomas persisted for at least 8 mo after exposure to urethane, whereas immunostaining for TGF-beta1, -beta2, and -beta3 and TGF-beta RI persisted at levels comparable to those in normal bronchioles. In situ hybridization studies conducted with TGF-beta receptor riboprobes showed a corresponding reduction in expression of TGF-beta RII mRNA but not of TGF-beta RI mRNA in adenomas compared with expression in bronchioles. Expression of TGF-beta RII mRNA was also examined in non-tumorigenic and tumorigenic mouse lung cells; expression of TGF-beta RII mRNA was lower in the tumorigenic cells derived from urethane-induced lung tumors. These data suggest that a decrease in expression of TGF-beta RII may contribute to autonomous cell growth and may play an important role in mouse lung carcinogenesis induced by urethane.

Concurrent and distinct transcription and translation of transforming growth factor-beta type I and type II receptors in rodent embryogenesis.

The transforming growth factor-betas (TGF-betas) are multifunctional regulatory polypeptides that play a crucial role in many cell processes and function through a set of cell surface protein receptors that includes TGF-beta type I (RI) and type II (RII). The present study reports a comprehensive comparison of the patterns of expression of TGF-beta RI and RII proteins and mRNAs in the developing mouse embryo using immunohistochemical and in situ hybridization analyses. Although widespread expression of both TGF-beta receptors was detected throughout the embryonic development period so that many similarities occur in localization of the TGF-beta receptors, TGF-beta RI was expressed in a well-defined, non-uniform pattern that was different in many respects from that of TGF-beta RII. Whereas higher levels of TGF-beta RI compared to TGF-beta RII were detected in some tissues of the embryo at the beginning of organogenesis, the level of TGF-beta RII increased more dramatically than that of TGF-beta RI during late organogenesis; this was especially true in many neural structures where TGF-beta RI and RII were comparable by day 16. The lung, kidney and intestine, in which epithelial-mesenchymal interactions occur, showed a complex pattern of TGF-beta RI and Rll expression. Additionally, northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) amplification showed non-uniform expression of the transcripts for TGF-beta RI and RII in embryonic and adult mouse and rat tissues. These data show that regulation of TGF-beta1 RI and RII occurs concurrently, but distinctly, in a spatial and temporal manner in rodent embryogenesis which may allow control of signal transduction of TGF-beta during development.

Piscine (Sparus aurata) alpha subunit of the G-protein transducin is homologous to mammalian cone and rod transducin.

A novel cDNA encoding alpha subunit of the GTP-binding protein, transducin, has been cloned from a marine fish, Sparus aurata. The cDNA contains an open reading frame of 1050 nt (encoding 350 amino acid residues). A high degree of identity was found with known mammalian transducin proteins of cones (Gt2 alpha) or rods (Gt1 alpha): human Gt2 alpha (80.2%), bovine Gt2 alpha (79.3%), mouse Tt1 alpha (78.2%), mouse Gt2 alpha (78%) and bovine Gt1 alpha (77.9%). Northern blot analysis of different tissues revealed a transcript of about 2.5 kb, which is expressed only in the fish eye and not in other tissues from adult fish, supporting its identification as transducin. Ontogeny of transducin mRNA expression during early development of Sparus aurata, determined by Northern blot analysis, showed very low levels in larvae 3 days after hatching but not earlier. Levels increased 3- and 6-fold on days 4 and 6 (respectively) compared with those on day 3 and remained essentially unchanged thereafter, until day 21 after hatching (the last day studied). Our results suggest that in fish only one alpha subunit of transducin is found, which shows similar identity with cone and rod alpha subunits of mammals.

Identification of early growth response gene-1 (Egr-1) as a phorbol myristate acetate-induced gene in lung cancer cells by differential mRNA display.

Cellular regulatory genes including transcription factors may play an important role in the induction, maintenance, and progression of lung cancer. These regulatory genes are inducible by various mitogenic stimuli including phorbol myristate acetate (PMA). The differential mRNA display method was used to identify potential early response genes regulated by PMA in non-small cell lung cancer (NSCLC) cell lines. Using this technique, several cDNA fragments were found to be potentially differentially regulated by PMA in the squamous NSCLC cell line NCI-H157. One of these cDNA fragments of approximately 100 bp was determined to be differentially induced by at least 30-fold by PMA by northern blot analysis and to hybridize to a single 3.4 kb mRNA species. This cDNA fragment was cloned, sequenced, and identified to be identical to a portion of the 3'-untranslated region of the human early growth response gene-1 (Egr-1). Using Egr-1 cDNA as a probe, it was demonstrated that PMA induces Egr-1 mRNA expression in at least three other NSCLC cells as well. In addition, PMA caused a transient increase in expression of the Egr-1 transcript reaching a maximum level by 1 h before decreasing in NCI-H157 and three other types of NSCLC cells. Treatment of these NSCLC cells with TGF-beta1 showed a transient increase in Egr-1 mRNA similar to PMA which also reached a maximum level after 1 h. Normal human bronchial epithelial (NHBE) cells also showed a rapid, transient increase in expression of Egr-1 mRNA after treatment with PMA. In contrast, treatment of NHBE cells with TGF-beta1 showed that expression of Egr-1 mRNA increased by 1 h but reached a maximum level only after 6 h. These results indicate that both PMA and TGF-beta1 can induce Egr- mRNA expression in NSCLC cells and NHBE cells; however, while PMA induces Egr-1 mRNA similarly in both cell types, TGF-beta1 induces Egr-1 mRNA expression more rapidly and more transiently in NSCLC cells than in NHBE cells. Our results suggest that Egr-1 may play different roles in response to mitogens in normal and malignant lung cells.

Differential regulation of protease and extracellular matrix protein expression by transforming growth factor-beta 1 in non-small cell lung cancer cells and normal human bronchial epithelial cells.

In addition to autoregulating its own expression, transforming growth factor-beta 1 (TGF-beta1) also regulates the production of proteases, protease inhibitors and extracellular matrix proteins. To investigate the relationship between plasminogen activator (PA), plasminogen activator inhibitor-1 (PAI-1) and the extracellular matrix in malignant and normal lung epithelial cells and to determine whether malignant lung epithelial cells may be more invasive than normal lung epithelial cells because of differences in expression of these proteins in response to TGF-beta, the regulation of PA, PAI-1, fibronectin, laminin and thrombospondin by TGF-beta1 in human non-small cell lung cancer (NSCLC) cells was examined and compared with normal human bronchial epithelial (NHBE) cells. TGF-beta1 caused a persistent increase in expression of the mRNAs for both PA and PAI-1 in NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. By immunoprecipitation analysis, it was shown that TGF-beta1 also induced a corresponding increase in the amount of PAI-1 protein in these NSCLC cells as well. In contrast, while TGF-beta1 also increased expression of PAI-1 mRNA in NHBE cells, expression of PA mRNA decreased simultaneously. Treatment of NSCLC cells with TGF-beta1 resulted in a persistent increase in expression of the mRNAs for fibronectin, laminin and thrombospondin; expression of fibronectin protein also increased after treatment with TGF-beta1 in these cells. When NHBE cells were similarly cultured in the presence of TGF-beta1, expression of fibronectin mRNA also increased in a persistent manner; however, only an early transient increase in the level of the mRNAs for laminin and thrombospondin was detected in these cells. These data show that there is differential regulation of the genes for PA and PAI-1 and the extracellular matrix protein fibronectin in response to TGF-beta1 not only when NSCLC and NHBE cells are compared, but also when different NSCLC cells are compared with each other.

Transforming growth factor-beta receptors in human cancer cell lines: analysis of transcript, protein and proliferation.

Transforming growth factor-beta (TGF-beta) is a growth regulator which affects multiple cellular functions through the TGF-beta type I and type II receptor (TGF-beta RI and TGF-beta RII) serine/threonine kinases. In this study, the expression of TGF-beta RI and RII was investigated by northern blot, reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses in human breast (MCF-7 and T-47D), ovary (CP70 and OVT2) and prostate (LNCaP, DU145 and PC3) cancer cell lines. Northern blot analysis showed expression of TGF-beta RI and TGF-beta RII mRNAs in all cell lines examined except for MCF-7 and LNCaP, respectively. In contrast, RT-PCR showed that all of the cell lines examined express TGF-beta RI and RII transcripts using specific primer oligonucleotides. Western blot analysis demonstrated that all of the cancer cell lines examined express TGF-beta RI and RII proteins. Southern blot analysis showed there were differences in the restriction enzyme digestion patterns for TGF-beta RI and RII of T47D compared to MCF-7, PC3 and LNCaP. Also, the addition of TGF-beta 1 to the breast and prostate cancer cells inhibited colony formation in soft agarose. Our studies show that in cancer cells, relatively low levels of TGF-beta receptor transcripts may result in protein production and inhibition of cell proliferation by TGF-beta.

Transforming growth factor-beta isoforms in the developing chicken intestine and spleen: increase in transforming growth factor-beta 4 with coccidia infection.

Expression of transforming growth factor-betas 2, 3 and 4 (TGF-beta) in the developing chicken intestine and spleen was investigated using specific cDNA probes and antibodies for the different TGF-beta isoforms. Coordinate expression of the mRNAs for TGF-beta s 2, 3 and 4 was detected in the embryonic intestine by 8 days, with maximal expression of the mRNAs for TGF-beta s 2 and 4 occurring at 12 and 19 days, respectively, while expression of TGF-beta 3 mRNA remained constant during this time. While specific antibodies for TGF-beta s 2, 3 and 4 could detect only weak immunohistochemical staining of the intestinal epithelium in 4-, 12- and 16-day-old embryos, intense staining for TGF-beta s 2, 3 and 4 was detected in the tips of the intestinal villi of 19-day-old embryos. In the spleen, expression of the mRNAs for TGF-beta s 2 and 3 increased in the newly hatched chick compared with the embryo and then decreased in the adult to levels that were lower than in the embryo; expression of TGF-beta 4 mRNA increased progressively with developmental age, with expression in the adult spleen being significantly higher than in the embryonic and hatchling spleen. Immunohistochemical staining of spleens showed a selective increase in the level of reactive TGF-beta 4 with increasing developmental age, while staining for TGF-beta s 2 and 3 was constant during development. After infection of 1-month-old chickens with coccidian parasite, expression of TGF-beta 4 mRNAs increased 5-8-fold in intestinal intra-epithelial lymphocytes and 2.5-fold in spleen cells, while expression of the mRNAs for TGF-beta s 2 and 3 remained constant in these cells. The results of this study suggest that TGF-beta may play a role in development of the intestine and spleen in the chicken and that TGF-beta 4 in particular increases after infection of coccidia in the chicken.

Effects of transforming growth factor-beta 1 and phorbol ester on PAI-1 and PA genes in human lung cells.

Transforming growth factor-beta (TGF-beta) mediates the production of extracellular matrix proteins, proteases and protease inhibitors in epithelial cells. Both TGF-beta and phorbol-12-myristate-13-acetate (PMA) exert both positive and negative effects on mitogenesis in these as well as other cell types. Phorbol esters act through stimulation of protein kinase C (PKC) and are among the most potent tumor promoters known. The present study was conducted to determine whether the effect of TGF-beta in human non-small cell lung cancer (NSCLC) and normal human bronchial epithelial (NHBE) cells parallels that of the phorbol esters and whether this effect of TGF-beta involves PKC. TGF-beta 1 and PMA increased expression of TGF-beta 1 mRNA 24 hr after their addition to both NSCLC and NHBE cells. The effects of these agents on expression of the mRNAs for TGF-beta 2 and TGF-beta 3 were more complex; while TGF-beta 2 and TGF-beta 3 mRNAs increased transiently in response to TGF-beta 1 in NHBE cells and TGF-beta 3 mRNA increased transiently in some NSCLC cells, expression of these mRNAs decreased in most of these cells in response to PMA with the exception of the carcinoid NCI-H727 where TGF-beta 2 mRNA increased dramatically, TGF-beta 1 and PMA both caused a persistent increase in expression of the mRNAs for both plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator (PA) up to 24 hr in most NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. In contrast, while TGF-beta 1 also increased expression of PAI-1 mRNA in NHBE cells, the expression of PA mRNA decreased simultaneously. The effect of PMA on PAI-1 and PA mRNAs was opposite of TGF-beta 1 in these cells, with expression of PAI-1 mRNA decreasing and PA mRNA increasing after addition of PMA. These data show that there is parallel regulation of the genes for TGF-beta 1, PAI-1 and PA by TGF-beta 1 and PMA in NSCLC, but differential regulation of the genes for PAI-1 and PA by these agents in NHBE cells. The responses of the mRNAs and proteins of TGF-beta 1, PAI-1 and PA to TGF-beta 1 and PMA were inhibited by the serine/ threonine kinase inhibitor H7 in NSCLC cells. Treatment of NSCLC cells with TGF-beta 1 and PMA resulted in a persistent increase in the expression of fibronectin mRNA and protein. This response was blocked by the addition of H7. Inhibition of these effects by H7 in NSCLC cells suggests that H7 blocks TGF-beta responses by inhibiting a protein serine/threonine kinase(s). Because the effects of TGF-beta and PMA on the different TGF-beta isoforms, PA, PAI and fibronectin in NHBE and NSCLC cells are complex, our data suggest that there are distinct mechanisms for controlling the different TGF-beta isoforms, PA, PAI and extracellular matrix proteins in normal lung and lung cancer cells.

Molecular cloning of fish alcohol dehydrogenase cDNA.

A cDNA encoding a putative alcohol dehydrogenase class III (ADH) was cloned from a cDNA library constructed from 7-day larvae RNA of the marine teleost Sparus aurata. The full length cDNA is 1350 nucleotides (nt) long and contains an ORF of 1128 nt [encoding 376 amino acid (aa) residues]. Identity of 82% was found with human class III ADH (305 of 372 aa compared), and only 62% identity with a fish (cod) ADH (234 of 375 aa compared). Northern hybridization analysis with the cDNA revealed a transcript of about 1.4-1.5 kb, which is expressed in all tissues from adult fish studied: skeletal muscle, heart muscle, kidney, gill filaments and liver, with the highest levels found in the kidney. The expression of ADH mRNA was determined also during early development of Sparus aurata by Northern blot analysis. ADH transcripts were detected in eggs and in embryos 4, 8 and 12 h after fertilization, as well as on all days post-hatching studied. The levels of expression decreased during early embryonal development, but increased 4-fold from day 1 to day 21 after hatching. The size of the transcript was identical to that of hepatic ADH. Our results suggest that maternal ADH mRNA is present in the eggs and embryos, which decreases as divisions and development occur, while after hatching ADH mRNA is expressed by the larval tissues.

Porcine osteochondrosis: deficiencies in transforming growth factor-beta and insulin-like growth factor-I.

Osteochondrosis and dyschondroplasia are common multifocal disturbances of endochondral ossification in many species of domestic animals, and are characterized by the retention of avascular cartilage. These cartilage disorders are characterized by a failure of chondrocyte differentiation, matrix mineralisation and its replacement by bone. Rabbit polyclonal antibodies to transforming growth factor-beta (TGF-beta) and to insulin-like growth factor-I (IGF-I) were used to detect the two growth factors in normal and osteochondrotic porcine epiphyses. In the normal pig epiphyses IGF-I and TGF-beta were present in the chondrocytes of the epiphyseal hyaline cartilage and IGF-I was readily localised to the hypertrophic chondrocytes in the growth cartilage adjacent to the epiphyseal ossification centre. Both growth factors were found to be deficient in chondrocytes at sites of osteochondrosis. Both these growth factors are thought to be involved in the cascade of events associated with chondrocyte function during endochondral ossification. Deficiencies in TGF-beta and IGF-I demonstrated in porcine osteochondrosis and previously shown in avian dyschondroplasia suggest further similarities in the pathogenesis of these conditions.