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Transforming growth factor-beta (TGF-beta), a regulatory cytokine expressed in the kidney, plays a role in nephrogenic repair. This study utilized a chemical model of renal proximal tubule cellular injury and regeneration to investigate the effects of TGF-beta 1 on regeneration. Confluent monolayers of rabbit renal proximal tubular cells (RPTC) in primary culture exposed to the oxidant t-butylhydroperoxide (800 microM TBHP) for 1.5 hours were 24% confluent after 24 hours. Confluency increased to 50% 4 days after TBHP exposure. Recovery of monolayer confluency was associated with increased monolayer protein but not with DNA content. Daily treatment of injured monolayers with TGF-beta 1 inhibited the recovery of monolayer confluency and inhibited recovery of protein content in a concentration-dependent manner (0.02-1 ng/mL). DNA content of injured monolayers was not altered by TGF-beta 1. A single treatment of injured monolayers with 0.2 ng/mL (8 pM) TGF-beta 1 inhibited recovery of monolayer confluency and protein content without altering monolayer DNA content. These data show that a single 24 hour exposure to a low concentration (8 pM) of TGF-beta 1 inhibits regeneration of renal proximal tubule cell monolayers following oxidative injury by inhibiting, in part, cellular migration/spreading.

The cytotoxicity of transforming growth factor beta 1 (TGF beta 1) was assessed in rat hepatocytes cultured under periportal (PP)-or pericentral (PC)-equivalent conditions. TGF beta 1 induced a 5-fold greater DNA fragmentation and LDH release in PC cultures than in PP cultures. At low exposure level (1 ng/ml TGF beta 1), albumin secretion and mitochondrial activity (rhodamine-123 uptake) were selectively reduced in PP cultures, whereas the incidence of apoptotic cells in PC cultures was about 10-fold higher than that in PP cultures. The time profiles of TGF beta 1-induced apoptotic and necrotic events and the concentration-response relationship differed in PC and PP cultures. In PC cultures the early appearance of cells with apoptotic nuclei was not associated with DNA fragmentation nor with an increase in LDH release or impaired mitochondrial function. At a high exposure level (5 ng/ml TGF beta 1), again cells with apoptotic nuclei were much more strongly induced in PC cultures but DNA fragmentation, LDH release, and impairment of mitochondrial activity all increased in an exposure-time dependent manner in both PP and PC cultures. At this exposure level 48 and 72% of the apoptotic cells detected in PC cultures after continuous exposure for 24 hr were induced within an exposure of 1 and 4 hr, respectively. Aurintricarboxylic acid (50 microM), an inhibitor of endonucleases, significantly inhibited the appearance of apoptotic cells and the progression in apoptosis. Clearly, TGF beta 1 preferentially induced apoptotic cell death in hepatocytes with PC-equivalent metabolism at low exposure levels. High exposure levels or prolonged exposure periods produced both apoptosis and necrosis.

The molecular basis by which transforming growth factor (TGF)-beta 1 protects certain tumor cells from tumor necrosis factor (TNF) cytotoxicity was investigated. When pretreated, with TGF-beta 1, -beta 2, and -beta 3, murine L929S fibroblasts developed resistance to TNF cytotoxicity. Time course experiments revealed that TGF-beta 1 initially induced both cellular protein-tyrosine phosphorylation and simultaneous secretion of a novel extracellular matrix TNF-resistance triggering (TRT) protein(s), which closely preceded the acquisition of TNF-resistance. TGF-beta 2 and -beta 3 also increased tyrosine phosphorylation. However, both molecules failed to stimulate TRT secretion. The increased levels of phosphorylation, particularly to 9 specific protein tyrosine kinase inhibitor-sensitive cellular proteins, appeared to alter the TNF killing pathway. TGF-beta 1-induced TRT secretion required participation of unknown serum factors. TRT adhered strongly to polystyrene plates and resisted treatment with heat (60 degrees C, 30 min), collagenase, alpha 2-macroglobulin, heparin, antibodies against TGF-beta s, and limited trypsin digestion. Notably, TRT promoted TNF-resistance via activation of tyrosine and serine/threonine kinase functions in L929S. Thus, the molecular pathway involves TGF-beta 1-mediated initiation of a rapid tyrosine phosphorylation of cellular protein substrates (which alters TNF cytotoxic pathway), and a simultaneous secretion of TRT, which in turn signals the cells to maintain the levels of phosphorylation, thereby sustaining the TNF-resistance.

The in vitro chemotactic response of human osteoblasts was investigated towards the following growth factors: TGF-beta, PDGFs, FGFs and IGFs. Human osteoblasts grown from trabecular bone after enzymatic digestion were studied. TGF-beta stimulated the migration of human osteoblasts in a dose-dependent manner with a four-fold increase in migrated cells at 100 pg/ml, which was the optimum concentration. PDGF-BB also stimulated migration four-fold in a dose-dependent manner with a maximum response at 10 ng/ml. PDGF-AA, IGF-I and IGF-II stimulated migration two-fold at 100 ng/ml. The results show that TGF-beta and PDGF-BB are important regulators of human osteoblast migration, but other growth factors IGF-I, IGF-II and PDGF-AA may also stimulate osteoblast migration. Our results additionally suggest that TGF-beta and PDGF-BB may participate in the recruitment of osteoblasts during bone remodeling since both TGF-beta and PDGF-BB are found in bone matrix and could be released during osteoclastic bone resorption. They furthermore support a possible use of TGF-beta and PDGF-BB in growth factor-induced osteogenesis.

The direct effect of nicotine on the expression of receptors for the tumor necrosis factor alpha (TNF alpha) and transforming growth factor beta (TGF beta) and the internalization, intracellular distribution and stability of these growth factors in cervical cancer cell line SiHa was studied. Nicotine at concentrations in the range 0.05-0.25% inhibited cell growth and it exerted strong apoptotic and cytolytic effects at concentrations above 0.5%. Nicotine at 0.1% stabilized and protected from degradation [125I]TNF alpha and [125I]TGF beta internalized by cervical cancer SiHa cell line. In the absence of nicotine, [125I]TNF alpha and [125I]TGF beta were internalized during the first hour of incubation and localized mainly in the cytoplasm and in smaller amounts in the nucleus. After 1 day of cell exposure to growth factors, only traces of radioactivity were detected inside the cells, which indicated that both growth factors were rapidly degraded. In the presence of nicotine, both [125I]TNF alpha and [125I]TGF beta were detected in high quantities and in a non-degraded form in the cytoplasm and chromatin during 5 days of incubation. In addition to the lack of growth factor degradation, the presence of nicotine induced a nuclear accumulation of growth factors, with up to 37% of the internalized [125I]TGF beta being in the chromatin. An increased intracellular accumulation of [125I]TNF alpha and [125I]TGF beta in cells exposed to nicotine occurred without changes in expression of the cell surface receptors. Nuclear accumulation of TGF beta was followed by increased RNA synthesis and a switch from the growth-promoting action of TGF beta to the strong growth inhibitory effect. Inhibition of the lysosomal degradation of growth factors by nicotine is discussed as a potential mechanism of tobacco-induced carcinogenesis.

Mutations affecting the p53 gene abrogate its tumor suppressor activity. It is, however, unclear whether such mutations can generate mutant p53 proteins with an intrinsic transforming ability. More importantly, the mechanism(s) by which they exert such activity is unknown. We report here that p53-deficient hepatoma cells (Hep3B) transfected with mutant p53-249ser (codon 249 Arg-->Ser) acquire a new phenotype with an increased in vitro survival and mitotic activity. However, such a phenotypic change is not sufficient to cause a major shift in the poor tumorigenic potential of these cells. This is apparently due to transforming growth factor beta 1-mediated apoptotic death of Hep3B cells which is not affected by the expression of p53-249ser.

To date, apoptosis has been characterized biochemically by the production of 180-200 bp internucleosomal DNA fragments resulting from the activation of an endonuclease(s). The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that this may reflect the oligonucleosomal fragmentation pattern. We have re-examined this dogma by comparing the biochemical and morphological features of cell death in several epithelial cell types (HT-29-I1 colon adenocarcinoma, CC164 mink lung, DU-145 human prostatic carcinoma and MCF-7 human breast adenocarcinoma) and one mesenchymal cell line (H11ras-R3 ras-transformed rat fibroblasts). Cell death was induced either by serum deprivation, TGF-beta 1 or etoposide, or by leaving cells to reach confluence. Cell death was assessed with respect to detachment from monolayers, morphological changes and DNA integrity. The DNA-binding fluorophore Hoechst 33258 revealed chromatin condensation patterns consistent with apoptotic cell death in all cell types except MCF-7 cells. Using field inversion gel electrophoresis in conjunction with conventional 2% agarose gel electrophoresis, cleavage of DNA to 50 kbp fragments was observed in all cases except MCF-7 cells. This preceded the appearance of oligonucleosomal fragments in HT-29-I1, CC164 and H11ras-R3 cells. Although the DNA of DU-145 cells fragmented into 50 kbp units, and although the cells exhibited classical apoptotic morphology, no subsequent internucleosomal cleavage was observed. These results suggest that changes in the integrity of DNA indicative of the release of chromatin loop domains occur before cleavage at internucleosomal sites is initiated and that the latter is not an essential step in the apoptotic process.

The systemic administration of high doses of rHuTGF-beta 1 to rats produced a spectrum of lesions in multiple target tissues, including liver, bone, kidney, heart, thymus, pancreas, stomach, cecum, at the injection vein, and in skeletal muscle at the site of anesthetic injection. The majority of these lesions can be attributed to known biological activities of TGF-beta 1. High-dose dermal application resulted in local effects at the wound sites without systemic toxicity.

Cytokines have been implicated in the pathogenesis of a number of brain diseases in which neurological dysfunction has been attributed to a change in amino acid neurotransmitter metabolism. In the present in vitro study, we investigated the effects of cytokines on astrocyte glutamine synthetase (GS) activity and subsequently on N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity. Proinflammatory cytokines IL-1 alpha, IL-1 beta, and IL-6 at a concentration of 20 ng/ml did not affect GS activity; however, tumor necrosis factor-alpha inhibited this activity by 20% in mixed neuronal/astrocyte cultures. Treatment for 24 h with transforming growth factor (TGF)-beta 1 or -beta 2 inhibited up to 60% GS activity. TGF-beta 2 also inhibited GS in enriched astrocyte cultures with an ED50 of 10 pg/ml. Antibodies specific to TGF-beta 2 blocked this effect. Treatment of astrocytes with TGF-beta 2 (250 pg/ml) resulted in markedly dilated rough endoplasmic reticulum. Since astrocyte GS may play a protective role in NMDA receptor-mediated neurotoxicity, we treated mixed neuronal/astrocyte cultures with TGF-beta 2 (250 pg/ml) and found a threefold potentiation of NMDA receptor-mediated neurotoxicity. These data suggest that TGF-beta impairs astrocyte GS function and enhances neurotoxicity, thus providing insight into understanding one mechanism of cytokine-mediated central nervous system disease.

Transforming growth factor beta (TGF-beta) is a multifunctional homodimeric polypeptide with potent actions upon many target cells, including those of mesenchymal and haemopoietic lineage. The recent reports of high levels of the cytokine in rheumatoid synovium and synovial fluid, prompted this study into the effect of intra-articular injection of TGF beta-2 into rabbit knee-joints. Four daily injections of 1 microgram caused swelling, probably as a consequence of prostaglandin E2 production, synovial fibroblastic hyperplasia and a striking loss of femoral condyle proteoglycan. Using the polymerase chain reaction, no evidence could be obtained for the induction of interleukin-1 alpha gene expression in either synovial tissue or synovial fluid cells. These findings suggest that the TGF-beta present in the rheumatoid joint may contribute directly to the pathogenesis of rheumatoid arthritis.

The present study was conducted to study the interaction between epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in benign human prostatic epithelial cells in culture. Primary cultures of human prostatic epithelial cells were grown in complete WAJC, which consisted of WAJC-404 medium and, in addition to other defined additives, EGF and bovine pituitary extract (BPE). Incomplete WAJC contained the same composition except EGF and BPE were deleted. TGF-beta was added into media at concentrations of 0, 0.1, and 1.0 ng/ml. When cells were grown in complete WAJC, they proliferated rapidly. Cell proliferation was greatly suppressed when incomplete WAJC was used. Addition of TGF-beta to these cultures caused a significant reduction in the final cell number when either complete WAJC or incomplete WAJC was used. In additional experiments, cells were prelabeled with 3H-thymidine for 72 hr prior to treatment with TGF-beta. The percentage of radioactivity released into the medium at the end of a 6-day culture was used as an indication of the extent of cell death. Trypan blue exclusion test was also used to assess the extent of cell death. Addition of TGF-beta into complete WAJC did not significantly affect the extent of cell death beyond what was considered as the result of normal cellular turnover. Addition of TGF-beta into incomplete WAJC, however, caused a significant increase in the percent of cell death in the culture. These results demonstrated an interaction between EGF and TGF-beta in proliferation and cell death in human prostatic epithelia in culture. In the presence of EGF alone in the culture medium, prostatic epithelial cells were stimulated to proliferate. The rate of proliferation was greatly diminished when EGF was deleted from the medium or when TGF-beta was added in the presence of EGF. Finally, cell death was induced when TGF-beta was added into the medium in the absence of EGF.

Transforming growth factor beta 1 (1 ng/ml) caused death of serum-free mouse embryo cells cultured in a medium consisting of a 1:1 mixture of Dulbecco's Modified Eagle's medium and Ham's F12 medium supplemented with fibronectin, insulin, transferrin, epidermal growth factor, and high density lipoprotein. Cell death occurred in the presence of polyunsaturated fatty acids including linoleic acid in the absence of selenium. The death could be reversed by adding alpha-tocopherol to the culture indicating a mechanism involving fatty acid peroxidation. Butylated hydroxytoluene was a poor suppressor of cell death in contrast to alpha-tocopherol. High density lipoprotein and fatty acid-free albumin also suppressed cell death at the level of 20 micrograms/ml and 1 mg/ml, respectively. Transforming growth factor beta 1 also caused a low rate of cell growth after heat treatment of the cells at 45 degrees C.

While stimulating the growth of fibroblasts, transforming growth factor beta 1 (TGF-beta 1) inhibits the growth of various normal and malignant cell lines in vitro. We studied the effects of TGF-beta 1 in vivo. The level of TGF-beta 1 in serum was maximally elevated 2 h after injecting 1 muCi of 125I-TGF-beta 1 into the peritoneal cavity of nude mice. Five h after the i.p. administration of 10 micrograms of unlabeled TGF-beta 1, 20 ng/ml of TGF-beta-like material in serum were detected by a radioreceptor assay on A549 lung carcinoma cells. Trichloracetic acid-precipitable 125I-TGF-beta 1 was taken up by liver, spleen, lungs, kidneys, and tumor tissue but not by the brain. At doses exceeding 2 micrograms/day, TGF-beta 1 induced a generalized interstitial fibrosis and a cachexia, which was not mediated by elevated serum levels of tumor necrosis factor alpha as determined by Western blot analysis and enzyme-linked immunosorbent assay. A total of 200,000 cells of the estrogen receptor-negative human breast cancer line MDA-MB-231, which had been shown to be maximally growth inhibited in vitro by 40 pM TGF-beta 1 and to have high-affinity receptors (9, 11, 12), were injected into the mammary fat pad of each nude mouse. The duration of treatment was 16 days with ten animals in the control group and five animals in the treated groups. The dose ranged from 1 to 4 micrograms per animal daily. The treatment was started 24 h after the injection of the tumor cells. Tumor growth was not significantly affected at either nontoxic or toxic doses of TGF-beta 1. Thus, we have demonstrated that TGF-beta 1, apart from being a local growth factor, has systemic effects, such as cachexia and multiple fibrosis. Its role as an antitumor agent may be limited.