Prolactin isoform 2 as an autocrine growth factor for GH3 cells.

Because PRL has growth factor activities in several tissues, we have asked whether it also has autocrine growth factor activity in pituitary GH3 cells. GH3 cells were grown at increasing densities in the presence or absence of antirat PRL (polyclonal and monoclonal) or nonspecific antibodies. Cell proliferation increased with increasing cell density, as did the concentration of PRL in the medium. Antirat PRL, but not control antibody, markedly inhibited but did not eliminate cell proliferation, and this effect was diminished with increasing PRL concentration in the medium. PRL receptors were demonstrated on 40-50% of the cells by indirect immunofluorescence using a specific antirat PRL receptor monoclonal antibody. Cell surface PRL was colocalized to the same 40-50% of the cells and copatched or cocapped along with the receptors. Absence or presence of PRL receptors did not correlate with stage of the cell cycle, as judged by ethidium bromide dual labeling. Cell surface PRL was found to be on PRL-containing cells. These data have fulfilled four criteria necessary for establishment of a substance as a secreted autocrine growth factor: 1) the factor must be secreted; 2) in log growth phase, increased cell proliferation should occur at increased cell densities; 3) the cells must display a receptor for the factor; and 4) there must be a growth response to the factor. Thus we have established that PRL is an autocrine growth factor for at least 40-50% of the GH3 cell population. This, to our knowledge, is the first example of autocrine growth factor activity of a major hormone normotopically expressed.     

Interleukin-6 functions as an autocrine growth factor in a cholangiocarcinoma cell line

Abstract The tumour cells of a human cholangiocarcinoma cell line, HuCC-T1, were found to express mRNA of interleukin-6 (IL-6) and to secrete a large amount of biologically active IL-6 in the culture medium at the concentration of 22.6 ng/mL. Interleukin-6 was demonstrated in the cytoplasm of the cells by immunohistochemical staining. Furthermore, these cells showed the presence of receptors for IL-6 on the surface, and DNA synthesis of the cells was stimulated by the exogenous addition of recombinant human IL-6 into the culture medium. The cell growth was significantly inhibited in the presence of anti-human IL-6 antibody in the culture medium. These findings indicate that IL-6 is one of the autocrine growth factors of this cell line in vitro.     

Endothelin as an autocrine factor in the regulation of parathyroid cells.

Endothelin, originally purified from porcine aortic endothelial cells, is widely distributed in tissues and is recognized as a product of epithelial cells, glial cells, and neurons in addition to endothelial cells. We found evidence by mRNA content and immunoreactivity that this peptide is synthesized in rat parathyroid epithelial cells (PT-r cells) and bovine parathyroid chief cells. The peptide synthesized by PT-r cells comigrated with synthetic endothelin 1 in reverse-phase HPLC and was diluted out in radioimmunoassay in parallel with the synthetic peptide. Bovine parathyroid endothelial cells (BPE-1 cells) did not express this peptide. Preproendothelin 1 mRNA expression by PT-r cells and endothelin 1 peptide production were regulated by calcium. Shifts in extracellular calcium either from high to low concentrations or vice versa elicited similar evanescent increases in expression of mRNA with a peak at 1 h. Synthesis of the peptide seems to be controlled by mRNA expression, and peptide in the medium appears to be continuously degraded or taken up by cells because its concentration in the medium showed a time course similar to that of mRNA expression. PT-r cells also bear a single class of receptors highly specific for endothelin 1, suggesting an autocrine regulation by endothelin 1 of the parathyroid. The facile regulation of endothelin concentrations in the medium by shifts in extracellular calcium concentration and possible autocrine regulation by endothelin 1 suggest that this peptide may mediate, at least in part, effects of calcium on the parathyroid system.     

Interleukin 1 as an autocrine growth factor for acute myeloid leukemia cells

Production of interleukin 1 (IL-1) by leukemic cells was studied in 13 cases of acute myeloid leukemia. Intracytoplasmic immunofluorescence studies showed that the cells invariably contained the cytokine. Endogenous labeling studies demonstrated that acute myeloid leukemia cells produced either only the 33-kDa propeptide or both the propeptide and the 17-kDa mature form of IL-1 beta. The 33-kDa propeptide IL-1 alpha was always produced but was less frequently released. Involvement of IL-1 in leukemic cell growth was investigated using two antibodies specific for IL-1 subtypes, which inhibited spontaneous cell proliferation in the six cases studied. After acid treatment of the cells, a surface receptor for IL-1 could be demonstrated, which mediated 125I-labeled IL-1-specific uptake by leukemic cells. Furthermore, recombinant IL-1 alpha or IL-1 beta induced significant cell proliferation in 10 of 12 cases. The above findings were uncorrelated with the cytologic type (French-American-British classification) of leukemia. Our studies suggest that IL-1 may act as an autocrine growth factor in most cases of acute myeloid leukemia.     

Vascular endothelial growth factor stimulates rat cholangiocyte proliferation via an autocrine mechanism

BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF) is secreted by several epithelia and modulates cellular functions by autocrine and paracrine mechanisms. The role of VEGF in cholangiocyte pathophysiology is unknown. We evaluated the role of VEGF in the regulation of cholangiocyte proliferation in rats that underwent bile duct ligation. METHODS: The expression of VEGF-A and VEGF-C and their receptors in cholangiocytes from normal and BDL rats was evaluated. Normal or BDL rats were treated with recombinant-VEGF-A or recombinant-VEGF-C or anti-VEGF antibodies, and proliferation of cholangiocytes was evaluated in situ by morphometry and in vitro by proliferating cell nuclear antigen immunoblots and MTS assay. In vitro, normal rat cholangiocyte cultures were stimulated with r-VEGF-A or r-VEGF-C and proliferation and signal transduction were evaluated. RESULTS: We found that (1) cholangiocytes express messenger RNA and protein for VEGF-A, VEGF-C, VEGF receptor 2 (VEGFR-2), and VEGF receptor 3 (VEGFR-3) and secrete VEGF; (2) secretion of VEGF and expression of VEGFR-2 and VEGFR-3 increases in BDL cholangiocytes; (3) blocking VEGF in vivo by anti-VEGF-A or anti-VEGF-C antibodies decreases cholangiocyte proliferation; (4) the in vivo administration of r-VEGF-A or r-VEGF-C induces cholangiocyte proliferation in normal rats; and (5) in vitro, VEGF-A increases normal rat cholangiocyte culture proliferation by activation of inositol 1,4,5-triphosphate/Ca2+/protein kinase C alpha and phosphorylation of Src/ERK1/2. CONCLUSIONS: Cholangiocytes secrete VEGF and express VEGFR-2 and VEGFR-3, all of which are amplified in BDL cholangiocytes. VEGF induces cholangiocyte proliferation by activation of inositol 1,4,5-triphosphate/[Ca2+]i/protein kinase C alpha and phosphorylation of Src/ERK1/2. VEGF mediates the adaptive proliferative response of cholangiocytes to cholestasis.     

Evidence that brain-derived neurotrophic factor acts as an autocrine factor on pituitary melanotrope cells of Xenopus laevis

We have investigated the physiological regulation and functional significance of brain-derived neurotrophic factor (BDNF) in the endocrine melanotrope cells of the pituitary pars intermedia of the amphibian Xenopus laevis, which can adapt its skin color to the light intensity of its environment. In black-adapted animals, melanotrope cells produce and release alpha-melanophore-stimulating hormone (alpha-MSH). In white-adapted animals, the activity of melanotrope cells is inhibited by neuronal input. Using Western blotting and immunocytochemistry at the light and electron microscopical level, we have detected both the BDNF precursor and the mature BDNF protein in Xenopus melanotrope cells. In situ hybridization and RT-PCR revealed the presence of BDNF mRNA in the pituitary pars intermedia, indicating that BDNF is synthesized in the melanotropes. Real-time quantitative RT-PCR showed that levels of BDNF mRNA in melanotrope cells are about 25 times higher in black- than in white-adapted animals. Although there is no difference in the amount of stored mature BDNF, the amount of BDNF precursor protein is 3.5 times higher in melanotropes of black-adapted animals than in those of white-adapted animals. These data indicate that BDNF mRNA expression and BDNF biosynthesis are up-regulated in active melanotrope cells. Because immunoelectron microscopy showed that BDNF is located in melanotrope secretory granules, BDNF is probably coreleased with alpha-MSH via the regulated secretory pathway. Superfusion and (3)H-amino acid incorporation studies demonstrated that BDNF stimulates the release of alpha-MSH and the biosynthesis of its precursor protein, POMC. Our results provide evidence that BDNF regulates the activity of Xenopus melanotrope cells in an autocrine fashion.     

Interleukin-1α as an autocrine growth factor for acute lymphoblastic leukaemia cells

Summary We describe a case of B-lineage acute lymphoblastic leukaemia (ALL) with proliferation of leukaemic cells through an interleukin-1α (IL-lα) autocrine mechanism. Flow cytometric analysis using the IL-1 receptor type II (IL-1Rt II) monoclonal antibody (mAb) demonstrated the expression of the IL-1Rt II on leukaemic cells; this is the first report in IL-1RtII-positive freshly isolated ALL cells from a patient. In accordance with the expression of IL-1RtII, the leukaemic cells proliferated in response to exogenous IL-1α. In addition, anti-IL-1α mAb markedly inhibited the spontaneous cell proliferation. Furthermore, Northern blot analysis detected IL-1α mRNA without any stimulation. These observations suggest that IL-1α may play an important role as an autocrine growth factor in some cases of ALL.     

VEGF促进肝癌SMMC-7721细胞侵袭性的自分泌机制

目的:探讨促血管内皮生长因子(VEGF)对人肝癌细胞SMMC-7721侵袭力以及对该细胞中基质金属蛋白酶9(MMP-9)的影响,初步研究VEGF对肿瘤侵袭和转移的影响及可能的作用机制.方法:使用VEGF体外培养人肝癌SMMC-7721细胞,通过细胞体外侵袭实验检测细胞侵袭能力的改变,再分别使用30μg/L、10μg/LVEGF培养人肝癌SMMC-7721细胞,以正常培养人肝癌SMMC-7721细胞为空白对照组.使用半定量RT-PCR和Western blot法对3组细胞中MMP-9的mRNA和蛋白表达水平进行分析.结果:细胞体外侵袭实验显示,外加VEGF培养后,细胞侵袭力明显增强(P0.01);MMP-9mRNA和蛋白的表达在外加VEGF组中要明显高于空白对照组(0.479±0.025,0.665±0.024vs 0.315±0.022;0.521±0.026,0.662±0.026vs 0.366±0.025,均P<0.01),且高浓度和低浓度组之间也有明显差别.结论:肝癌SMMC-7721细胞系中存在有自分泌机制,VEGF可通过自分泌机制上调肝癌细胞的MMP-9表达,进而促进肿瘤的浸润转移.     

Prolactin as an autocrine/paracrine growth factor in human cancer.

Prolactin (PRL) has a dual function -- as a circulating hormone and as a cytokine. This understanding is based on PRL production and distinct regulation in extrapituitary sites, its binding to membrane receptors of the cytokine receptor superfamily, and activation of signaling pathways that promote cell growth and survival. There is increasing evidence that PRL plays a role in several types of cancer in reproductive and non-reproductive tissues via local production or accumulation. The expression of both PRL and its receptor in human cancer cell lines of diverse origin lends further support to its action as an autocrine/paracrine growth factor. Establishment of PRL as an active participant in tumorigenesis should inspire the development of novel therapies aimed at reducing tumor growth by suppressing PRL production or by blocking its receptors.     

Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor-positive human tumors

Angiogenesis is required for the progression of tumors from a benign to a malignant phenotype and for metastasis. Malignant tumor cells secrete factors such as vascular endothelial growth factor (VEGF), which bind to their cognate receptors on endothelial cells to induce angiogenesis. Here it is shown that several tumor types express VEGF receptors (VEGFRs) and that inhibition of VEGF (VEGF antisense oligonucleotide AS-3) or VEGFRs (neutralizing antibodies) inhibited the proliferation of these cell lines in vitro. Furthermore, this effect was abrogated by exogenous VEGF. Thus, VEGF is an autocrine growth factor for tumor cell lines that express VEGFRs. A modified form of VEGF AS-3 (AS-3m), in which flanking 4 nucleotides were substituted with 2-O-methylnucleosides (mixed backbone oligonucleotides), retained specificity and was active when given orally or systemically in vitro and in murine tumor models. In VEGFR-2-expressing tumors, VEGF inhibition may have dual functions: direct inhibition of tumor cell growth and inhibition of angiogenesis.