Evidence that transforming growth factor-beta is a hormonally regulated negative growth factor in human breast cancer cells

The hormone-dependent human breast cancer cell line MCF-7 secretes transforming growth factor-beta (TGF-beta), which can be detected in the culture medium in a biologically active form. These polypeptides compete with human platelet-derived TGF-beta for binding to its receptor, are biologically active in TGF-beta-specific growth assays, and are recognized and inactivated by TGF-beta-specific antibodies. Secretion of active TGF-beta is induced 8 to 27-fold under treatment of MCF-7 cells with growth inhibitory concentrations of antiestrogens. Antiestrogen-induced TGF-beta from MCF-7 cells inhibits the growth of an estrogen receptor-negative human breast cancer cell line in coculture experiments; growth inhibition is reversed with anti-TGF-beta antibodies. We conclude that in MCF-7 cells, TGF-beta is a hormonally regulated growth inhibitor with possible autocrine and paracrine functions in breast cancer cells.     

The cell survival signal Akt is differentially activated by PDGF-BB, EGF, and FGF-2 in osteoblastic cells

Stimulation of osteoblast survival signals may be an important mechanism of regulating bone anabolism. Protein kinase B (PKB/Akt), a serine-threonine protein kinase, is a critical regulator of normal cell growth, cell cycle progression, and cell survival. In this study we have investigated the signaling pathways activated by growth factors PDGF-BB, EGF, and FGF-2 and determined whether PDGF-BB, EGF, and FGF-2 activated Akt in human or mouse osteoblastic cells. The results demonstrated that both ERK1 and ERK2 were activated by FGF-2 and PDGF-BB. Activation of ERK1 and ERK2 by PDGF-BB and FGF-2 was inhibited by PD 098059 (100 microM), a specific inhibitor of MEK. Wortmannin (500 nM), a specific inhibitor of phosphatidylinositol 3-kinase ( PI 3-K), inhibited the activation of ERK1 and ERK2 by PDGF-BB but not by FGF-2 suggesting that PI 3-K mediated the activation of ERK MAPK pathway by PDGF-BB but not by FGF-2. Rapamycin, an inhibitor of p70 S6 protein kinase and a downstream target of ERK1/2 and PI 3-K, did not affect the activation of ERK1 and ERK2 by the growth factors. Furthermore, our results demonstrated that Akt, a downstream target of PI 3-K, was activated by PDGF-BB but not by FGF-2. Akt activation by PDGF-BB was inhibited by PI 3-kinase inhibitor LY294002. Rapamycin had no effect on Akt activation. Epidermal growth factor (EGF) also activated Akt in osteoblastic cells which was inhibited by LY294002 but not by rapamycin. Taken together, our data for the first time revealed that the activation of ERK1/2 by PDGF-BB is mediated by PI 3-K, and secondly, Akt is activated by PDGF-BB and EGF but not by FGF-2 in human and mouse osteoblastic cells. These results are of critical importance in understanding the role of these growth factors in apoptosis and cell survival. PDGF-BB and EGF but not FGF-2 may stimulate osteoblast cell survival.     

Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation

The search for novel and more efficient chemo-agents against malignant osteoblastoma is important. In this study, we examined the potential anti-osteoblastoma function of bufotalin, and studied the underlying mechanisms. Our results showed that bufotalin induced osteoblastoma cell death and apoptosis in dose- and time-dependent manners. Further, bufotalin induced endoplasmic reticulum (ER) stress activation in osteoblastoma cells, the latter was detected by the induction of C/EBP homologous protein (CHOP), phosphorylation of inositol-requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), as well as caspase-12 activation. Conversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP depletion by shRNA significantly inhibited bufotalin-induced osteoblastoma cell death and apoptosis. Finally, by using a mice xenograft model, we demonstrated that bufotalin inhibited U2OS osteoblastoma cell growth in vivo. In summary, our results suggest that ER stress contributes to bufotalin-induced apoptosis in osteoblastoma cells. Bufotalin might be investigated as a novel anti-osteoblastoma agent.     

The overexpression of PKCdelta is involved in vascular endothelial growth factor-resistant apoptosis in cultured primary sinusoidal endothelial cells

We have previously reported that primary sinusoidal endothelial cells from the rat liver are highly dependent on VEGF for cell proliferation in in vitro culture. However, even in the presence of VEGF, essentially all the SE cells could not survive longer than 7 days, leading to growth factor-resistant cell death. The death had characteristics typical of apopotosis, such as DNA fragmentation, staining with TUNEL reagent and nuclear condensation. We found that the cell death was blocked by the treatment of TPA in a dose-dependent manner and was preceded by a remarkable increase in PKCdelta at a protein level. Furthermore, PKCdelta-specific inhibitor, Rottrelin, significantly suppressed this VEGF-resistant apoptosis of cultured SE cells, whereas conventional PKC-specific inhibitor, Go6976 could not. TPA was found to downregulate the overexpression of PKCdelta. Thus, we suggest that the VEGF-resistant apoptosis is a new type of endothelial cell death and that PKCdelta is an essential mediator for this process.     

Endoreduplication is not inhibited but induced by aphidicolin in cultured cells of tobacco

Endoreduplication is a common process in plants that allows cells to increase their DNA content. In the tobacco cell cultures studied in this work it can be induced by simple hormone deprivation. Mesophyll protoplast-derived cells cultured in the presence of NAA (auxin) and BAP (cytokinin) keep on dividing, while elongation and concomitant DNA endoreduplication are induced and maintained in a medium containing only NAA. If aphidicolin is given to the two types of culture, no effect is observed on elongating, endoreduplicating cells. However, the cells programmed for division switch to elongation and DNA endoreduplication. Thus aphidicolin, an inhibitor of the replicative DNA polymerases, alpha and delta, does not inhibit endoreduplication, and furthermore actually induces it when the mitotic cell cycle is blocked. DNA duplication and cell growth can only be completely blocked if ddTTP, an inhibitor of DNA polymerase-beta, is given together with aphidicolin. This result implies that an aphidicolin-resistant DNA polymerase, such as the repair-associated DNA polymerase-beta, can mediate DNA synthesis during endoreduplication and can substitute for polymerases-alpha and -delta when the latter are inhibited. Similar results are obtained in cultures of the BY-2 cell line by withdrawing auxins from the culture medium. In this cell line endoreduplication is induced only in a small proportion of the cells. A greater proportion of the cells are blocked in the G(2) phase of the cell cycle.     

Integrin-linked kinase is involved in lactoferrin-induced anchorage-independent cell growth and survival in PC12 cells

AimsBovine lactoferrin (bLf) causes anchorage-independent cell growth in PC12 cells. The present study investigated the mechanisms involved in bLf-induced anchorage-independent cell growth and survival in PC12 cells.Main methodsThe number of adherent cells and suspended cells was estimated separately by using a methyl thiazol tetrazolium (MTT) assay, and the sum of both optical density (O.D.) (570 nm) values was used as a measure of the total number of cells.Key findingsIntegrin-linked kinase (ILK) plays an important role in integrin and growth factor signaling pathways. Stable transfection of PC12 cells with a dominant negative kinase-deficient mutant of ILK (DN-ILK) inhibited bLf-induced anchorage-independent cell growth. The ILK activity in the parental cells was transiently activated after addition of bLf, whereas bLf-induced activation of ILK was blocked in DN-ILK-transfected cells. bLf also activated p38 mitogen-activated protein kinase (MAPK); however, the p38 MAPK activation was inhibited by stable DN-ILK transfection. Moreover, cell viability in the suspended cells by bLf strongly decreased after treatment with SB203580, an inhibitor of p38 MAPK.SignificanceThese results suggest that ILK is involved in bLf-induced anchorage-independent cell growth and viability via activation of p38 MAPK.     

Brain-derived growth factor is a chemoattractant for fibroblasts and astroglial cells

Bovine brain-derived growth factor (BDGF), a 16-17 kDa protein with biochemical properties resembling brain-derived acidic fibroblast growth factor (acidic FGF) and endothelial cell growth factor, was found to have potent chemotactic activity for bovine ligament fibroblasts, human skin fibroblasts and rat astroglial cells, maximal at 100-200 pg/ml. The chemotactic activity was completely blocked by protamine sulfate (5 ug/ml), an inhibitor of receptor-binding and mitogenic activity of BDGF. BDGF did not stimulate migration of human monocytes. These results indicate that the effects of BDGF 'in vivo' might extend to mesenchymal cell recruitment.     

Change in metabolic turnover is an alternate mechanism increasing cell surface epidermal growth factor receptor levels in tumor cells

The biosynthesis and metabolic turnover of the epidermal growth factor (EGF) receptor was examined in a human pancreatic carcinoma cell line, UCVA-1. This cell line has been shown to possess a much higher level of EGF receptors than is expected solely from receptor gene/mRNA dosage. Analysis of the biosynthesis using metabolic labeling, immunological quantitation, and inhibitor treatment revealed that the naked EGF receptor in UCVA-1 cells is a protein of Mr 130,000 that is matured consecutively as a Mr 160,000 and 170,000 glycoprotein through post-translational glycosylation. Analysis of the metabolic turnover using pulse-chase labeling and inhibitor treatment revealed that the rate of EGF receptor synthesis in UCVA-1 cells was similar to that in two squamous cell carcinoma cell lines, NA and Ca9-22, which also have high numbers of EGF receptors, but because of gene amplification. In contrast, the rate of receptor degradation in UCVA-1 cells was significantly slower than in the other two cell lines. These results suggest that the retarded metabolic turnover may constitute a unique mechanism for elevating cell surface EGF receptor levels in some tumor cells independent of gene amplification.     

Lysosomal membrane permeabilization is involved in curcumin-induced apoptosis of A549 lung carcinoma cells

We previously reported that curcumin inhibited lung cancer A549 cells growth and promoted cell apoptosis in vitro. In this study, we further examined the apoptosis-related parameters, including lysosomal damage and cathepsin activation, in A549 cells exposed to curcumin. We found that curcumin caused lysosomal membrane permeabilization (LMP) and cytosolic relocation of cathepsin B (cath B) and cathepsin D (cath D). However, only Z-FA-fmk (a cath B inhibitor) but not pepstatin A (a cath D inhibitor) inhibited curcumin-induced cell apoptosis, mitochondrial membrane potential loss, and cytochrome c release. The antioxidant N-acetylcysteine and glutathione attenuated LMP, suggesting that lysosomal destabilization was dependent on the elevation of reactive oxygen species and which precedes mitochondrial dysfunction. These findings indicated a novel pathway for curcumin regulation of ROS-lysosomal-mitochondrial pathway and provided the key mechanism of regulation of LMP in cell apoptosis, which may be exploited for cancer treatment.     

Lipopolysaccharide-induced expression of the competence gene KC in vascular endothelial cells is mediated through protein kinase C

The KC gene is a cell cycle-dependent competence gene originally identified in platelet-derived growth factor-stimulated BALB/c-3T3 cells. This gene is also induced in murine peritoneal macrophages in response to activation stimuli. We have examined the expression of the KC gene in cultured porcine aortic endothelial cells following treatment with bacterial lipopolysaccharide (LPS) as a first step in defining the early molecular events involved in endothelial cell stimulation by physiologically relevant modulators. LPS markedly elevated the steady-state level of KC mRNA in confluent endothelial cells; maximum induction of KC occurred in the cells following exposure to 10 ng/ml LPS for 2 h. LPS did not increase the growth fraction of the cells, nor was the KC mRNA level changed in dense endothelial cells stimulated to enter the cell cycle with epidermal growth factor. However, KC mRNA expression was elevated by addition of serum to starved, subconfluent endothelial cell cultures. Treatment of endothelial cells with phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-glycerol (OAG) also induced KC gene expression. A maximum response was obtained with 10 nM PMA, the effect decreasing with higher levels of the phorbol ester. The calcium ionophore A23187 exhibited little stimulatory activity alone; however, the ionophore did cause a doubling in the PMA-stimulated KC expression. The increased expression of KC induced by LPS and PMA was inhibited by the presence of 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H7), a protein kinase C inhibitor, but not by HA1004 (an H7 analogue with little protein kinase C inhibitory activity). No cytotoxicity was observed in inhibitor or LPS-treated endothelial cell cultures. These results demonstrate that KC gene expression is stimulated by LPS in vascular endothelial cells in a proliferation-independent process. Second, unlike LPS-induced KC expression in macrophages and platelet-derived growth factor-induced KC expression in 3T3 cells, LPS induction of KC in endothelial cells appears to require activation of protein kinase C.     

Urokinase type plasminogen activator receptor is involved in insulin-like growth factor-induced migration of rhabdomyosarcoma cells in vitro

Urokinase-type plasminogen activator (uPA) binds to its receptor, uPAR, on the surface of cancer cells, leading to the formation of plasmin. Rhabdomyosarcoma (RMS) cell lines secrete high levels of insulin-like growth factor II (IGF-II), suggesting autocrine IGFs play a major role in the unregulated growth and metastasis of RMS. In vitro, IGF-II and IGF-I increased migration of RD cells to 124+/-9% (P<0.01) and 131+/-8% (P<0.05) of control, respectively. IGF-II-induced migration was abolished by insulin-like growth factor binding protein-6 (IGFBP-6) (P<0.01), a relatively specific inhibitor of IGF-II, and by plasminogen activator inhibitor type 1 (PAI-1) (P<0.05). Aprotinin, a plasmin inhibitor, and mannosamine, which inhibits the synthesis of glycosylphosphatidylinositol (GPI), thereby preventing anchorage of GPI-linked proteins such as uPAR to the cell membrane, also decreased IGF-II- (P<0.05 for both) but not IGF-I-induced migration. [Arg54,Arg55]IGF-II and [Leu27]IGF-II, which preferentially bind to the IGF-I and IGF-II/mannose-6-phosphate receptors (IGF-II/M6PR), respectively, both induced RD cell migration to 146+/-8% (P<0.01) and 120+/-7% (P<0.05) of control, respectively. An anti-uPAR anti-serum reduced IGF-II- and IGF-I-induced migration (P<0.05 for both). An anti-low density lipoprotein-related protein (LRP) anti-serum reduced IGF-I-induced migration (P<0.05). IGF-I and -II both increased specific 125I-single chain uPA (scuPA) binding to RD cells in a dose-dependent manner (P<0.01). These results suggest involvement of the PA/plasmin system in IGF-induced migration and indicate important roles these systems may have in RMS metastasis.     

Increased expression of geminin stimulates the growth of mammary epithelial cells and is a frequent event in human tumors

Geminin is a potent inhibitor of origin assembly and re-replication in multicellular eukaryotes and is a negative regulator of DNA replication during the cell cycle. Thus, it was proposed as an inhibitor of cell proliferation and as a potential tumor suppressor gene. However, the protein was found specifically expressed in proliferating lymphocytes and epithelial cells and up-regulated in several malignancies. Therefore, geminin is now regarded as an oncogene but its role in tumor development remains unknown. In this study, we evaluated by Western blot analysis the expression of geminin in a series of human cancer cell lines of various histogenetic origin and in a series of human primary colon, rectal, and breast cancers. Expression of geminin was variable in different cell lines and not related to the expression level of the corresponding mRNA. Moreover, geminin was expressed at higher level in 56% and 58% of colon and rectal cancers, respectively, compared with the corresponding adjacent normal mucosa. A high expression of geminin was also detected by immunohistochemistry in 60% of human primary breast cancers. We also transfected a full-length geminin cDNA in a human non-tumorigenic and a cancer breast cell lines and obtained derivatives expressing high levels of the protein. Geminin overexpression stimulated cell cycle progression and proliferation in both normal and cancer cells and increased the anchorage--independent growth of breast cancer cells. These results demonstrate that expression of geminin is frequently deregulated in tumor cells and might play an important role in the regulation of cell growth in both normal and malignant cells.     

Apoptosis induced by denied adhesion to extracellular matrix (anoikis) in thyroid epithelial cells is p53 dependent but fails to correlate with modulation of p53 expression

An enamine mechanism-based inactivator of mammalian delta-aminobutyric acid aminotransferase, 4-amino 5-fluoropentanoic acid is a potent inhibitor of cell growth and pigment formation in the cyanobacterium Synechococcus PCC 6301. It was demonstrated that 4-amino 5-fluoropentanoic acid inhibits the aminolaevulinate synthesis at glutamate 1-semialdehyde aminotransferase and that in the mutant obtained by exposing cells to 40 microM 4-amino 5-fluoropentanoic acid, this enzyme was insensitive to the inhibitor. The specific activity of glutamate 1-semialdehyde aminotransferase in cell extracts was lower in the mutant, although the cell growth rate was unaffected. The decrease in sensitivity to 4-amino 5-fluoropentanoic acid in the mutant is due to a structural gene mutation, a single base change in the hemL gene resulting in a S162T substitution in the gene product.     

Elastase is a constituent product of T cells

Proteases produced by immune cells have been found to be important components of the immune response to antigen. A protease previously unrecognized as a specific T cell product has been identified which has the gene sequence, serologic crossreactivity, and enzymatic specificity of elastase. T cell elastase, found in combination with the natural elastase inhibitor alpha 1-antitrypsin (alpha 1-protease inhibitor, alpha 1-PI), is produced by both CD4+ and CD8+ T lymphocytes, and is found both in a membrane-bound and in a soluble form in murine T cell lines.