Autocrine Secretion Of Growth Factors Research Articles

Neuropeptide G Protein-Coupled Receptors as Oncotargets.

Neuropeptide G protein-coupled receptors (GPCRs) are overexpressed on numerous cancer cells. In a number of tumors, such as small cell lung cancer (SCLC), bombesin (BB) like peptides and neurotensin (NTS) function as autocrine growth factors whereby they are secreted from tumor cells, bind to cell surface receptors and stimulate growth. BB-drug conjugates and BB receptor antagonists inhibit the growth of a number of cancers. Vasoactive intestinal peptide (VIP) increases the secretion rate of BB-like peptide and NTS from SCLC leading to increased proliferation. In contrast, somatostatin (SST) inhibits the secretion of autocrine growth factors from neuroendocrine tumors (NETs) and decreases proliferation. SST analogs such as radiolabeled octreotide can be used to localize tumors, is therapeutic for certain cancer patients and has been approved for four different indications in the diagnosis/treatment of NETs. The review will focus on how BB, NTS, VIP, and SST receptors can facilitate the early detection and treatment of cancer.

Exogenous and autocrine growth factors stimulate human intervertebral disc cell proliferation via the ERK and Akt pathways

Intervertebral disc (IVD) degeneration is accompanied by growth factor-overexpression and increased cell proliferation, probably representing a tissue repair process. Accordingly, we studied the effect of exogenous and autocrine growth factors on the proliferation of human IVD cells. We observed that Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-like Growth Factor-I (IGF-I) stimulate DNA synthesis of human IVD cells, through the activation of the MEK/ERK and the PI-3K/Akt signal transduction pathways. Furthermore, medium conditioned (CM) by IVD cells induced DNA synthesis in the same cells, indicating the secretion of autocrine growth factors. The MEK/ERK and PI-3K/Akt pathways were also induced by CM, while their inhibition reversed in large part the DNA synthesis induction by CM. These responses to the exogenous and autocrine growth factors were qualitatively similar in both nucleus pulposus (NP) and annulus fibrosus (AF) cell cultures. Immunohistochemical studies in human biopsies showed significant activation of both signaling pathways, which was most prominent in the clusters of proliferating cells. These in vitro and in vivo data indicate that the proliferation of human IVD cells is regulated by exogenous and autocrine growth factors mainly via the MEK/ERK and PI-3K/Akt pathways; this may contribute to the design of future interventional approaches.

Cell tension, matrix mechanics, and cancer development

Oncologists often diagnose cancer based on a change of tissue stiffness sensed by palpation, yet cancer researchers generally focus on biochemical signaling mechanisms. Tumors are more rigid because they have a stiffer extracellular matrix. A new study shows that this alteration of matrix mechanics activates integrins, which not only promotes mitogenic signaling through Erk but also cell contractility through Rho, which can further increase matrix stiffness. This establishes a positive feedback loop that switches on the malignant phenotype in mammary epithelial cells. This mechanical "autocrine loop" brings solid-state mechanotransduction on a par with oncogenic signaling pathways in malignant transformation.

Polyomavirus large T antigen induces alterations in cytoplasmic signalling pathways involving Shc activation.

It has been extensively demonstrated that growth factors play a key role in the regulation of proliferation. Several lines of evidence support the hypothesis that for the induction of cell cycle progression in the absence of exogenous growth factors, oncogenes must either induce autocrine growth factor secretion or, alternatively, activate their receptors or their receptor substrates. Cells expressing polyomavirus large T antigen (PyLT) display reduced growth factor requirements, but the mechanisms underlying this phenomenon have yet to be explored. We conducted tests to see whether the reduction in growth factor requirements induced by PyLT was related to alterations of growth factor-dependent signals. To this end, we analyzed the phosphorylation status of a universal tyrosine kinase substrate, the transforming Shc adapter protein, in fibroblasts expressing the viral oncogene. We report that the level of Shc phosphorylation does not decrease in PyLT-expressing fibroblasts after growth factor withdrawal and that this PyLT-mediated effect does not require interaction with protein encoded by the retinoblastoma susceptibility gene. We also found that the chronic activation of the adapter protein is correlated with the binding of Shc to Grb-2 and with defects in the downregulation of mitogen-activated protein kinases. In fibroblasts expressing the nuclear oncoprotein, we also observed the formation of a PyLT-Shc complex that might be involved in constitutive phosphorylation of the adapter protein. Viewed comprehensively, these results suggest that the cell cycle progression induced by PyLT may depend not only on the direct inactivation of nuclear antioncogene products but also on the indirect induction, through the alteration of cytoplasmic pathways, of growth factor-dependent nuclear signals.

Potential Role for Ceramide in Mitogen-activated Protein Kinase Activation and Proliferation of Vascular Smooth Muscle Cells Induced by Oxidized Low Density Lipoprotein

Proliferation of vascular smooth muscle cells (SMC) is a hallmark in the pathogenesis of atherosclerotic lesions. Mildly oxidized low density lipoproteins (UV-oxLDL), which are mitogenic to cultured AG-08133A SMC, activate the sphingomyelin (SM)-ceramide pathway. We report here the following. (i) UV-oxLDL elicited a biphasic and sustained activation of MBP kinase activity, phosphorylation and nuclear translocation of p44/42 mitogen-activated protein kinase (MAPK), and [3H]thymidine incorporation, which were inhibited by PD-098059, a MAPK kinase inhibitor. (ii) The use of preconditioned media (from SMC pre-activated by UV-oxLDL) transferred to native SMC and blocking antibodies against growth factors suggest that UV-oxLDL-induced activation of MAPK and [3H]thymidine incorporation seem to be independent of any autocrine secretion of growth factors. (iii) UV-oxLDL-induced activation of a neutral sphingomyelinase, SM hydrolysis, ceramide production, and [3H]thymidine incorporation were inhibited by two serine-protease inhibitors (serpins), suggesting that a serpin-sensitive proteolytic pathway is involved in the activation of the SM-ceramide signaling pathway. (iv) UV-oxLDL-induced MAPK activation and [3H]thymidine incorporation were mimicked by ceramide generated in the plasma membrane by bacterial sphingomyelinase treatment or by addition of the permeant C2-ceramide. Serpins did not inhibit the MAPK activation and [3H]thymidine incorporation induced by C2-ceramide, indicating that activation of the MAPK and [3H]thymidine incorporation is subsequent to the stimulation of the SM-ceramide pathway. Taken together, these data suggest that mitogenic concentrations of UV-oxLDL are able to stimulate the SM-ceramide pathway through a protease-dependent mechanism and activate p44/42 MAPK, leading to proliferation of vascular SMC.

Nicotinic acetylcholine receptors of muscle and neuronal (alpha7) types coexpressed in a small cell lung carcinoma.

SCC-37 is a small cell lung carcinoma line that aberrantly expresses muscle-type nicotinic acetylcholine receptors (nAChRs). It was established from a patient with a paraneoplastic autoimmune neuromuscular disorder, myasthenia gravis. When grown as a xenograft tumor, SCC-37 cells express plasma membrane receptors that bind 1251-labeled alpha-bungarotoxin (125I-alpha-BTx), cosediment with 9S nAChR pentamers, and bind to a monoclonal antibody (MAb 35) specific for muscle-type (alpha1 subunit) alpha-BTx receptors. The agonist carbamylcholine (carbachol) stimulates influx of 22Na+ in SCC-37 cells; this is inhibited by alpha-BTx and by d-tubocurarine. Long-term cultured SCC-37 cells have functional and ligand-binding evidence for surface coexpression of both alpha1 and neuronal-type (alpha7 subunit) alpha-BTx receptors. A subclone of SCC-37, designated SCC-A9, expresses only the neuronal-type (alpha7 subunit) alpha-BTx receptors on its surface. Carbachol does not stimulate 22Na+ influx in SCC-A9 cells, but cytisine initiates a sustained influx of Ca2+. Activation of this response is inhibited by alpha-BTx and by the alpha7-selective antagonist methyllycaconitine. Addition of Co2+ abrogates the sustained elevation of intracellular free Ca2+ concentration, implying that the cytisine-stimulated influx of Ca2+ is sustained by secondary opening of voltage-sensitive channels in the plasma membrane. Surface receptors for 125I-alpha-BTx are blocked by methyllycaconitine and d-tubocurarine. Solubilized alpha-BTx receptors from plasma membranes of SCC-A9 cells cosediment with 10S neuronal nAChR pentamers and bind to an alpha7-specific monoclonal antibody (MAb P27) but not to the muscle nAChR-reactive MAb 35. However, MAb P27 and MAb 35 both bind to alpha-BTx receptors solubilized from the cytoplasmic compartments of SCC-A9 and the parental SCC-37 line. Reverse transcription-PCR analysis revealed RNA transcripts for alpha7 and alpha1 subunits in both SCC-A9 and SCC-37 cells. The nAChRs that are expressed in these novel human cell lines can regulate cation fluxes directly as well as indirectly by synergizing with the activity of voltage-sensitive Ca2+ channels. These activities may influence the secretion of autocrine growth factors and the transcription of growth regulatory genes and thus be pertinent to the growth and metastasis of malignant neuroendocrine neoplasms.

Herpesvirus saimiri Tip-484 membrane protein markedly increases p56lck activity in T cells.

Herpesvirus saimiri (HVS) is a T-cell-specific transforming and oncogenic virus. A protein encoded by HVS known as Tip-484 (for tyrosine kinase interacting protein from HVS strain 484) is required for this transformation. Tip-484 binds specifically to the nonreceptor protein tyrosine kinase p56lck. By transfecting Tip-484 into T cells, we now show that this interaction leads to a several hundred-fold increase in the kinase activity of p56lck. Tip-484 is part of a protein complex which is dependent on the presence of p56lck and is phosphorylated. We also show that two of the complexed proteins represent two phosphorylated forms of Tip-484. Furthermore, the p56lck kinase activity in HVS-infected human peripheral blood T lymphocytes was at least ninefold higher than that in noninfected control cells and significantly decreased in cells infected with a Tip-484 deletion mutant virus. Finally, we report that Tip-484 is required for oncogenesis in rabbits by the survival of rabbits inoculated with Tip-484 deletion mutant HVS. The data demonstrate dramatic stimulation of the signaling pathway of p56lck. This effect can contribute to the molecular mechanisms that lead to sustained autocrine secretion of growth factors, permanent T-cell growth, and ultimately lymphocytic tumor formation.

Weaning human pancreatic cancer cells from dependence on serum increases autocrine growth factor secretion and alters receptor density

Weaning human pancreatic cancer cells from dependence on serum increases autocrine growth factor secretion and alters receptor density

Does autocrine growth factor secretion form part of a mechanism which paradoxically protects against tumour development?

Autocrine growth factor secretion has classically been considered as a mechanism by which tumour cells achieve autonomous growth. However, there is now considerable evidence that autocrine circuits operate in the growth regulation of normal adult tissues. Here we consider the possible advantages to the normal epithelial cell of utilising such an external growth factor circuit and suggest that autocrine growth factor secretion, when viewed in a multicellular context, could paradoxically form part of a mechanism for preventing tumour development.

Ca 2+ and Ca 2+ channel antagonists in the control of human small cell lung carcinoma cell proliferation

Small cell lung carcinoma cells possess voltage-dependent calcium channels (VDCCs) of the L, ω-conotoxin-sensitive and P-like type. We hypothesized that these VDCCs might regulate the secretion of autocrine growth factors and thus influence the proliferation of these cells. We found that extracellular Ca 2+ plays a stimulatoru role in the proliferation of the GLC8 cell line. L-type calcium channel blockers of the dihydropyridine, phenylalkylamine and benzothiazepine classes inhibited [ 3H]thymidine incorporation in these cells, however at concentrations higher than those required to block L-type channel function. Moreover, the growth of murine Swiss 3T3 fibroblasts which do not possess L-type Ca 2+ channels, was inhibited by the Ca 2+ channel antagonists at the same effective concentrations as in small cell lung carcinoma cells. ω-conotoxin and ω-agatoxin IVA, which block the N- and P-type channel respectively, had no effect on GLC8 cell proliferation. It is concluded that the presence of extracellular Ca 2+ is a positive stimulus for small cell lung carcinoma cell growth. However, under our experimental conditions, the calcium channel blockers inhibited DNA synthesis most probably by a mechanism other than VDCC antagonism.

DEVELOPMENT OF RESISTANCE AGAINST PROTEIN-KINASE INHIBITORS IN LONG-TERM PROTEIN-FREE CULTURE

In two distinct cell lines, the protein kinase inhibitors K-252a and staurosporine did not inhibit the proliferation of two protein-free subclones as compared with their parental serum-dependent clones in a time- or concentration-dependent manner. Resistance to protein kinase inhibitors appeared in the selection of protein-free subclones from serum dependent clones. This concomitant appearance of drug resistance and autonomic proliferation suggests that cell proliferation in a protein-free culture does not depend on signal transduction through the protein kinase system. It is of interest that this growth factor independence is not caused by autocrine growth factor secretion in the protein-free medium, with this phenomenon possibly reflecting the tumor progression from sensitive to resistant in clinical trials of these inhibitors.

Characterization of liver epithelial cells transfected withmyc and/orras oncogenes

While many liver tumors contain activated myc and ras oncogenes, the mechanisms by which these genes contribute to cellular transformation is poorly understood. Activated versions of the cellular oncogenes, c-myc and/or c-H-ras were transfected into normal rat liver epithelial cells to identify cellular pathways that are altered in the cells containing the oncogenes. The results of these and other investigations indicate that the biological properties associated with the transfection of c-myc include immortalization, reduced contact inhibition of growth, activation of phospholipase A2-mediated pathways, increased sensitivity to transformation with a ras gene, and greatly increased sensitivity to growth factors. The biological properties associated with the transfection of the ras gene include morphological transformation, anchorage-independent growth, tumorigenicity, increased phosphatidylinositol metabolism, the induction of growth-factor processing and secretion, which leads to (exogenous) growth factor-independent tumor growth, and a marked resistance to normal inhibitors of growth such as TGF-beta. It is proposed that the complementary actions of the myc and ras genes in cellular transformation may be related to the ras-induced secretion of autocrine growth factors by cells sensitized to their effects by the myc gene. The increased stimulus for growth coupled to a ras-induced insensitivity to growth inhibitors may lead to clonal expansion of these cells and tumor development.

Epidermal growth factor receptors in human epidermal tumours

Immunolocalization studies using the strepavidin/biotin peroxidase system were used to identify epidermal growth factor receptors in 10 samples of normal skin, 13 seborrhoeic warts (SW), five lesions of Bowen's disease (BD), 11 solar keratoses (SK), 22 basal cell carcinomas (BCC) and five squamous cell carcinomas (SCC). The intensity of cell membrane staining was assessed using the 10-cm visual analogue scale technique and the results analysed using non-parametric statistics. The cell membranes in BCC showed significantly less intense staining when compared to normal epidermal cells (P less than 0.001) and the constituent cells of SW (P less than 0.001), SK (P less than 0.001), BD (P less than 0.01), and SCC (P less than 0.001). Classification of the specimens into normal, benign, premalignant and malignant indicated that cell membranes of malignant tumours showed significantly less staining than the other groups (P less than 0.001). The observed reduction in the epidermal growth factor receptors in malignant tumours may be due to occupation and down regulation in response to autocrine secretion of growth factors.

Anti-CD3 antibody induces unresponsiveness to IL-2 in Th1 clones but not in Th2 clones.

Culture of murine T cells with immobilized (platebound) anti-CD3 antibody results in autocrine growth factor secretion in both Th1 (IL-2 producing) and Th2 (IL-4 producing) cells. Using a panel of murine T cell clones, we demonstrate that the IL-2-induced proliferation of Th1 clones is dramatically inhibited by immobilized anti-CD3 antibody, whereas that of Th2 clones is not. This unresponsiveness of Th1 clones to IL-2 is not due to decreases in IL-2R expression. Supernatants from Th1 or Th2 cell cultures fail to alter the effects of anti-CD3 on the two types of clones, suggesting that unresponsiveness induced in Th1 clones or the lack thereof in Th2 clones is not mediated by a stable cytokine(s). Accessory cells enhance the proliferation of Th1 cells exposed to low concentrations of anti-CD3, but the unresponsiveness induced by high concentrations of anti-CD3 is not prevented by accessory cells. Finally, soluble anti-CD4 antibody prevents the induction of the unresponsive state even at high concentrations of anti-CD3. These experiments demonstrate that two subsets of cloned CD4+ T cells differ in their responses to anti-CD3, and that CD4 molecules may play a critical role in regulating the outcome of receptor-mediated stimulation.

Growth factor biology and oncogene activation in human gliomas and their implications for specific therapeutic concepts.

The failure of most available treatment modalities to improve the survival time and the quality of survival in patients with gliomas calls for the intense biological analysis of glial neoplasia. The untimely activation of cellular protooncogenes is often related to the process of neoplasia. Several protein growth factors as well as their cellular receptors have been identified as products of proto-oncogenes. Furthermore, these growth factors have been identified as glial mitogens in normal glial cell cultures as well as in tumors. The analysis of growth factor biology gains additional weight by the demonstration of autocrine growth factor secretion by tumor cells. The role of epidermal growth factor (EGF) and its receptor system in glial cell proliferation and differentiation is presented, with the speculation that the EGF receptor system may integrate the biological actions of many different factors, of which EGF itself may be least important. The literature on platelet-derived growth factor and insulin-like growth factors is reviewed, and the biology of fibroblast growth factor is presented in perspective with such phenomena as neovascularization and cell-matrix interactions.

Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells

Recently intimal thickening was shown to be due to stimulation of proliferation of arterial smooth muscle cells (SMC) by autocrine secretion of growth factor(s). We have reported that cultured rabbit aortic SMC secrete a growth factor (SDGF) distinct from other known growth factors. This paper reports on studies on the biological characteristics of SDGF. Putative “competence” and “progression” growth factors synergistically stimulated DNA synthesis of cultured rabbit aortic SMC. Conditioned medium (CM) from SMC containing SDGF stimulated DNA synthesis in SMC synergistically with either the competence factors or the progression factors. This synergistic effect was also observed in the presence of optimal concentrations of both competence and progression factors. The continuous presence of CM was essential for its stimulation of DNA synthesis whereas the presence of PDGF for only the first 4 h of culture was sufficient to induce maximum stimulation of DNA synthesis. These results suggest that SDGF is a new growth factor distinct from either competence or progression factors and that it stimulates a different pathway in SMC from those stimulated by other known growth factors.

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.

Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells

In attempts to determine the mechanism of proliferation of arterial smooth muscle cells (SMC) in intimal atheromatous lesions, autocrine secretion of growth factors by SMC has recently received much attention. Here we report a new growth factor named smooth muscle cell derived growth factor (SDGF). Cultured rabbit medial SMC secreted SDGF for 1 week during their incubation in serum-free media only after at least 4 passages. SDGF differed from platelet derived growth factor (PDGF) physicochemically, immunologically, and biologically. The properties of SDGF also seemed different from those of other known growth factors that stimulate the proliferation of mesenchymal cells.

Secretion of autocrine growth factors by cultured human lung tumors: Effects on neoplastic lung epithelial cells

Secretion of autocrine growth factors by cultured human lung tumors: Effects on neoplastic lung epithelial cells

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4, insulin 10 micrograms/ml, transferrin 10 micrograms/ml, sodium selenite 10 ng/ml, triiodo-L-thyronine 0.3 nM, phosphoethanolamine 5 microM, epidermal growth factor (20 ng/ml), 17 beta-estradiol 2 nM, and bovine serum albumin 20 micrograms/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 microM), glutathione (20 micrograms/ml), and linoleic acid/bovine serum albumin (150 micrograms/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone.