Autocrine Growth Factor Production Research Articles

Abstract P3-04-04: Estrogen receptor β5 increases aggressiveness of the triple negative breast cancer cell line SUM159

Abstract Recent clinical studies are indicating that the estrogen receptor β variant β5 (ERβ5) expression correlates to worse prognosis. We wanted to know if expression of ERβ5 is changing the growth behavior of the triple negative cell line SUM159. Estrogen receptor β5 is highly similar to estrogen receptor β1 except for a truncated C-terminus making the remaining ligand binding domain incapable of binding to estrogen. In addition a 4 amino acid unique peptide is added to the C-terminal end. Stably expressing ERβ5 using a transposon integrated tetracycline regulated expression system we find that expression of ERβ5 increases proliferation of the triple negative SUM159 cells especially in reduced serum condition compared to control cells. Since SUM159 have been shown to depend on autocrine stimulation for growth we are suggesting that expression of ERβ5 is affecting the production of autocrine growth factors. Citation Format: Faria M, Tin-U C, Dey P, Gustafsson J-A, Strom AM. Estrogen receptor β5 increases aggressiveness of the triple negative breast cancer cell line SUM159. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-04-04.

Autocrine production of IGF-I increases stem cell-mediated neuroprotection.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in motor neuron (MN) loss. There are currently no effective therapies; however, cellular therapies using neural progenitor cells protect MNs and attenuate disease progression in G93A-SOD1 ALS rats. Recently, we completed a phase I clinical trial examining intraspinal human spinal stem cell (HSSC) transplantation in ALS patients which demonstrated our approach was safe and feasible, supporting the phase II trial currently in progress. In parallel, efforts focused on understanding the mechanisms underlying the preclinical benefit of HSSCs in vitro and in animal models of ALS led us to investigate how insulin-like growth factor-I (IGF-I) production contributes to cellular therapy neuroprotection. IGF-I is a potent growth factor with proven efficacy in preclinical ALS studies, and we contend that autocrine IGF-I production may enhance the salutary effects of HSSCs. By comparing the biological properties of HSSCs to HSSCs expressing sixfold higher levels of IGF-I, we demonstrate that IGF-I production augments the production of glial-derived neurotrophic factor and accelerates neurite outgrowth without adversely affecting HSSC proliferation or terminal differentiation. Furthermore, we demonstrate that increased IGF-I induces more potent MN protection from excitotoxicity via both indirect and direct mechanisms, as demonstrated using hanging inserts with primary MNs or by culturing with organotypic spinal cord slices, respectively. These findings support our theory that combining autocrine growth factor production with HSSC transplantation may offer a novel means to achieve additive neuroprotection in ALS.

Pluripotent Fibrosis Initiating Mesenchymal Stromal Cell Linked To Cytopenias In LGL Leukemia Through Genetic Reprogramming

BackgroundLarge Granular Lymphocyte (LGL) leukemia is associated with chronic lymphoproliferation in association with unexplained cytopenias. Discovery of somatic mutations in STAT3 provides new insights into the molecular basis of LGL leukemia, but the precise mechanism leading to cytopenias remains unresolved. To gain insight into this mechanism, bone marrow fibrosis (Fig. 1A) was found to occur in 88% of cases and is significantly associated with splenomegaly, the presence of cytopenias, and co- existence of autoimmune diseases. In this study, we are reporting the identification of the fibrosis initiating cell population. [Display omitted] MethodsPrimary mesenchymal stromal cultures (MSCs) from LGL leukemia BM were isolated and grown under reduced oxygen conditions using undifferentiated and differentiation-specific culture conditions (Fig. 1B). These cultured cells were confirmed to deposit greater amounts of fibrillar Type I, III and V collagen matrix consistent with the heavy reticulin and trichrome positive stains observed in bone marrow biopsies (Fig. 1C). This collagen-producing cell population failed to support normal hematopoietic progenitor proliferation and colony formation in co-culture assays. Microarray analysis of the collagen-producing mesenchymal cell population was conducted and pathway analysis was performed using the GeneGo analysis platform from MetaCore™. Each list of genes was individually grouped and evaluated for membership on the set of GeneGo® pathway maps under the functional ontology enrichment tool. Calculation of p-values using hypergeometric distribution was used to determine pathway statistical significance. Flow cytometry was conducted to further evaluate the mesenchymal lineage. ResultsSupervised hierarchical clustering was performed to determine the overall similarity of LGL MSCs to publicly available, platform-compatible, microarrays from cells of mesenchymal lineage. Clustering showed that both patient and control MSCs display expression patterns distinct from committed mesenchymal lineages including osteoblasts, chondrocytes, and adipocytes. Mesenchymal-derived fibroblast differentiation can be distinguished by down-regulated CD29, CD44, CD105, CD106, CD117, bone morphogenetic protein receptor, and Sca-1 expression, and by up-regulated collagen type I, collagen type III, tenacin C, fibronectin, matrix metalloproteinase 1, fibroblast-specific protein 1, and vimentin. By microarray analysis and flow cytometry, we find no evidence that the cells are differentiated cells aside from collagen over expression. In order to define their pluripotent potential, sub-confluent cultures were grown in three types of differentiating media. Primary MSCs from both LGL leukemia patients and healthy controls were similar in their capacity to undergo trilineage differentiation into osteoblasts (stained with Alizarin Red S), adipocytes (Oil red O), and chondrocytes (Alican blue) (Fig. 1D). Next, pathway analyses revealed significant re-programming of the pluripotent MSCs. ECM production pathways were increased while proliferation pathways were down-regulated. Proliferation of MSCs is governed by production of autocrine growth factors. Both leukemia inhibitor factor (LIF) and basic fibroblast growth factor (FGFb) were significantly reduced compared to control MSCs (p<0.001) and the proliferative capacity of the LGL MSCs was severely diminished. Exogenous FGFb, but not LIF, restored LGLL MSC proliferation comparable to normal MSCs (Fig. 1E), normalized their spindle-shaped morphology, and maintained MSC plasticity. Interestingly, treatment with exogenous FGFb normalized collagen deposition (Fig. 1F) and restored hematopoietic supporting function. ConclusionSTAT-activated LGL leukemia cells, like myeloproliferative neoplasms, share a propensity for the development of bone marrow fibrosis. These results indicate that MSCs from LGL leukemia are pluripotent, but are re-programmed to deposit excessive amounts of collagen. Restoration of FGFb maintains the pluripotency of these MSCs while reversing the fibrosis phenotype. Our data suggests that FGFb-cultured MSCs may be used to reverse cytopenias in LGL leukemia. Disclosures:No relevant conflicts of interest to declare.

Abstract 1572: The role of MIF signaling in melanoma progression

Abstract Melanoma is the most life-threatening form of skin cancer and although highly curable at the early stages, metastatic melanoma is incurable. One of the early events in melanoma progression is the activation of ERK and Akt signalling pathways, involved in proliferation, migration and survival. The causes of such kinase activation include mutations in BRAF, N-RAS and PTEN. Besides gene mutation, it has been shown that autocrine production of growth factors can also contribute to ERK and Akt activation in melanoma. As both pathways are believed to contribute to the chemotherapeutic resistance of melanoma cells, an effective treatment should target both ERK and Akt cascades. One potentially attractive target is the cytokine MIF (migration inhibition factor), since MIF can activate both the ERK and the Akt pathways. Targeting of MIF signalling in tumours producing autocrine MIF is already showing promise in prostate, breast and bladder cancers and although limited, some in vitro evidence suggests that MIF is an autocrine factor involved in the growth and invasion of melanoma cells. We have now extended these findings in an immunohistological study showing the universal in vivo expression of MIF in melanoma with increasing expression levels associated with metastatic tumours. MIF signalling can act through both CXCR4 and CD74/CD44 receptors, the latter molecule acting as a co-receptor. Examination of a panel of 20 melanoma cell lines showed 20/20 to express MIF and CD44 with 7/20 also co-expressing CD74. Only 6/20 cell lines showed heterogenous expression of CXCR4. Inhibition of MIF expression using siRNA-mediated gene knockdown decreased the proliferation and cell migration in a number of melanoma cell lines, an effect attributed to effects on cell cycle with a reduction of the number of cells entering S phase. Conversely, addition of exogenous MIF was shown to increase ERK phosphorylation. Taken together, these results suggest a role for MIF in melanoma progression. Future work will aim to determining the molecular nature of the receptors driving MIF signalling in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1572.

The Phosphoinositide 3-Kinase (PI3K)/AKT Signaling Pathway as a Therapeutic Target for the Treatment of Human Acute Myeloid Leukemia (AML)

The PI3K/Akt signaling network regulates cell growth and apoptosis, and its constitutive activation has been implicated in the pathogenesis of a variety of malignancies. Recent studies suggest that PI3K/Akt signaling is frequently up-regulated in blast cells from AML patients and strongly contributes to proliferation, survival, and drug-resistance of these cells. Up-regulation of this network in AML may due to several reasons, including FLT3 and Ras activating mutations, increased levels of PI3K p110δ catalytic subunit, lack of PTEN lipid phosphatase expression, and autocrine production of growth factors. Small molecules designed to specifically target key components of this signal transduction network have been shown to induce apoptosis and/or increase conventional drug sensitivity of AML blasts in vitro. Therefore, these inhibitory molecules are being developed for clinical use either as single therapeutic agents or in combination with other forms of therapy. Nevertheless, PI3K/Akt blockade in vivo might have detrimental systemic side effects, given the multiple physiological roles played by this pathway, such as insulin-dependent glucose transport. Herein, we summarize our knowledge about PI3K/Akt signaling in AML and we highlight several pharmacological inhibitors which could be used in the future for treating this too often fatal hematological disorder. Keywords: Acute myeloid leukaemia, signal transduction, 3-phosphorylated inositol lipids, apoptosis, drug resistance, pharmacological inhibitors

Protein kinase B/Akt: a nexus of growth factor and cytokine signaling in determining muscle mass

Although the boundaries of skeletal muscle size are fundamentally determined by genetics, this dynamic tissue also demonstrates great plasticity in response to environmental and hormonal factors. Recent work indicates that contractile activity, nutrients, growth factors, and cytokines all contribute to determining muscle mass. Muscle responds not only to endocrine hormones but also to the autocrine production of growth factors and cytokines. Skeletal muscle synthesizes anabolic growth factors such as insulin-like growth factor (IGF)-I and potentially inhibitory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and myostatin. These self-regulating inputs in turn influence muscle metabolism, including the use of nutrients such as glucose and amino acids. These changes are principally achieved by altering the activity of the protein kinase known as protein kinase B or Akt. Akt plays a central role in integrating anabolic and catabolic responses by transducing growth factor and cytokine signals via changes in the phosphorylation of its numerous substrates. Activation of Akt stimulates muscle hypertrophy and antagonizes the loss of muscle protein. Here we review the many signals that funnel through Akt to alter muscle mass.

Autocrine Production of Insulin-like Growth Factor-I (IGF-I) Affects Paracellular Transport Across Epithelial CellsIn Vitro

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.

BCR-ABL Activates IGF-1 Expression and Signaling in Chronic Myelogenous Leukemia Blast Crisis Cell Lines.

CML blast crisis is characterized by the continued presence of the Philadelphia chromosome, which expresses the P210 BCR-ABL fusion protein, and the acquisition of additional molecular and chromosomal alterations. Evolution from CML chronic phase to blast crisis is associated with loss of heterozygosity at chromosome region 1p36, which contains the putative tumor suppressor RIZ1. We found that RIZ1 expression decreases during progression from CML chronic phase to myeloid blast crisis and that forced RIZ1 expression in CML blast crisis (CML-BC) cell lines decreases proliferation, increases apoptosis, and enhances differentiation. Furthermore, we found that RIZ1 represses IGF-1 autocrine production and blocks the activation of the IGF-1 receptor, AKT, and ERK signaling pathways in CML-BC cell lines thereby implicating RIZ1 control of the IGF-1 pathway in the regulation of these phenotypes. As BCR-ABL induces a growth factor independent phenotype due to the activation of autocrine growth factor production, we analyzed IGF-1 expression in CML-BC cell lines following exposure to imatinib. We found that imatinib treatment reduced IGF-1 mRNA and extracellular IGF-1 suggesting that RIZ1 may suppress blast crisis by counteracting the ability of BCR-ABL activate RIZ1 regulated genes. We characterized the signaling pathways used by BCR-ABL to activate IGF-1 expression and found that the HCK inhibitor PP2 and STAT5b shRNA reduced IGF-1 expression whereas the JAK2 inhibitor AG490 increased IGF-1 expression. These results suggest that BCR-ABL activates HCK, which in turn activates STAT5b and induces IGF-1 expression. To confirm the importance of BCR-ABL induced IGF-1 signaling to CML-BC cell line viability, proliferation and apoptosis, we characterized these cellular phenotypes in the presence of IGF-1 receptor blocking antibody and the IGF-1 receptor tyrosine kinase inhibitor AG1024. Blockage of autocrine IGF-1 signaling in CML-BC cell lines using an anti-IGF-1 receptor blocking antibody reduced cell viability and decreased proliferation. AG1024 treatment of CML-BC cell lines decreased proliferation and induced apoptosis. Together these results demonstrate that RIZ1 counteracts the ability of BCR-ABL to induce IGF-1 signaling in CML-BC cell lines, which influences cell proliferation, apoptosis, and differentiation. Our findings highlight RIZ-1 and IGF-1 signaling pathways as potential therapeutic targets for treating CML blast crisis.

Effects of Tamoxifen on Normal Human Dermal Fibroblasts

To evaluate the effects of tamoxifen on the growth and autocrine growth factor production of human dermal fibroblasts from the face. In vitro study of normal adult dermal fibroblast cells developed from surgical specimens in a serum-free model. Cell cultures were exposed to 5-, 8-, 12-, 16-, and 50-microg/mL concentrations of tamoxifen solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, 5, and 7 days after the initial exposure. Population doubling times were calculated, and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF) beta1. Tamoxifen appears to delay cellular proliferation rates in a dose-dependent manner up to a concentration of 12 microg/mL. Higher concentrations, approaching 50 microg/mL, appear to have a toxic effect on cell growth. The analysis of growth factor production revealed decreased levels of bFGF and VEGF but no change in the levels of TGF-beta1. The in vitro findings of delayed cell proliferation and decreased production of VEGF and bFGF in cells exposed to tamoxifen are consistent with previous in vivo reports of delayed wound healing but improved scar formation. The in vitro findings of growth factor modulation by tamoxifen provide cellular and molecular evidence supporting the clinical use of tamoxifen to ultimately improve scar formation.

Effects of Mitomycin-C on Normal Dermal Fibroblasts

To evaluate the effects of mitomycin-C on the growth and autocrine growth factor production of human dermal fibroblasts from the face. In vitro study using normal adult dermal fibroblast cell lines in a serum-free model. Cell cultures were exposed to 4 mg/mL, 0.4 mg/mL, 0.04 mg/mL, 0.004 mg/mL, and 0.0004 mg/mL concentrations of mitomycin-C solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, and 5 days after the initial exposure. Population doubling times were calculated and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta1. Continuous exposure to mitomycin-C caused fibroblast cell death by day 7 at all tested concentrations. A 4 minute exposure to mitomycin-C at 4 mg/mL caused rapid fibroblast cell death. A 4-minute exposure to mitomycin-C at either 0.4 mg/mL or 0.04 mg/mL resulted in decreased fibroblast proliferation. A 4 minute exposure to mitomycin-C at 0.4 mg/mL resulted in a marked increase in the production of both bFGF and TGF-beta1. A clinically ideal concentration of mitomycin-C would slow fibroblast proliferation yet not cause cell death to allow for a wound healing response. Mitomycin-C 0.4 mg/mL for 4 minutes satisfies the above criteria in vitro.

Inactivation of PU.1 in adult mice leads to the development of myeloid leukemia

Genetically primed adult C57BL mice were deleted of exon 5 of the gene encoding the transcription factor PU.1 by IFN activation of Cre recombinase. After a 13-week delay, conditionally deleted (PU.1(-/-)) mice began dying of myeloid leukemia, and 95% of the mice surviving from early postinduction death developed transplantable myeloid leukemia whose cells were deleted of PU.1 and uniformly Gr-1 positive. The leukemic cells formed autonomous colonies in semisolid culture with varying clonal efficiency, but colony formation was enhanced by IL-3 and sometimes by granulocyte-macrophage colony-stimulating factor. Nine of 13 tumors analyzed had developed a capacity for autocrine IL-3 or granulocyte-macrophage colony-stimulating factor production, and there was evidence of rearrangement of the IL-3 gene. Acquisition of autocrine growth-factor production and autonomous growth appeared to be major events in the transformation of conditionally deleted PU.1(-/-) cells to fully developed myeloid leukemic populations.

Exposure of murine cells to pulsed electromagnetic fields rapidly activates the mTOR signaling pathway

Murine pre-osteoblasts and fibroblast cell lines were used to determine the effect of pulsed electromagnetic field (PEMF) exposure on the production of autocrine growth factors and the activation of early signal transduction pathways. Exposure of pre-osteoblast cells to PEMF minimally increased the amount of secreted TGF-beta after 1 day, but had no significant effects thereafter. PEMF exposure of pre-osteoblast cells also had no effect on the amount of prostaglandin E(2) in the conditioned medium. Exposure of both pre-osteoblasts and fibroblasts to PEMF rapidly activated the mTOR signaling pathway, as evidenced by increased phosphorylation of mTOR, p70 S6 kinase, and the ribosomal protein S6. Inhibition of PI3-kinase activity with the chemical inhibitor LY294002 blocked PEMF-dependent activation of mTOR in both the pre-osteoblast and fibroblast cell lines. These findings suggest that PEMF exposure might function in a manner analogous to soluble growth factors by activating a unique set of signaling pathways, inclusive of the PI-3 kinase/mTOR pathway.

Caveolin-1 Deficiency (−/−) Conveys Premalignant Alterations in Mammary Epithelia, with Abnormal Lumen Formation, Growth Factor Independence, and Cell Invasiveness

During breast cancer development, the luminal space of the mammary acinar unit fills with proliferating epithelial cells that exhibit growth factor-independence, cell attachment defects, and a more invasive fibroblastic phenotype. Here, we used primary cultures of mammary epithelial cells derived from genetically engineered mice to identify caveolin-1 (Cav-1) as a critical factor for maintaining the normal architecture of the mammary acinar unit. Isolated cultures of normal mammary epithelial cells retained the capacity to generate mammary acini within extracellular matrix. However, those from Cav-1 (-/-) mice exhibited defects in three-dimensional acinar architecture, including disrupted lumen formation and epidermal growth factor-independent growth due to hyperactivation of the p42/44 mitogen-activated protein kinase cascade. In addition, Cav-1-null mammary epithelial cells deprived of exogenous extracellular matrix underwent a spontaneous epithelial-mesenchymal transition, with reorganization of the actin cytoskeleton, and E-cadherin redistribution. Mechanistically, these phenotypic changes appear to be caused by increases in matrix metalloproteinase-2/9 secretion and transforming growth factor-beta/Smad-2 hyperactivation. Finally, loss of Cav-1 potentiated the ability of growth factors (hepatocyte growth factor and basic fibroblast growth factor) to induce mammary acini branching, indicative of a more invasive fibroblastic phenotype. Thus, a Cav-1 deficiency profoundly affects mammary epithelia by modulating the activation state of important signaling cascades. Primary cultures of Cav-1-deficient mammary epithelia will provide a valuable new model to study the spatial/temporal progression of mammary cell transformation.

Suppression of MEK/ERK signalling by Myc: role of Bin-1

We report for the first time that over-expression of Myc suppresses mitogen-activated ERK kinase (MEK)/extracellular regulated kinase (ERK) signalling in chick embryo fibroblasts (CEF). Myc does not interfere with individual components of the signalling cascade, since efficient signal propagation via MEK and ERK in Myc-infected CEF can be seen. However, using the Myc-binding domain (MBD) of Bin-1, which binds to and negatively regulates the activity of Myc, we selectively suppressed Myc-induced apoptosis, without affecting its transforming properties. This was accompanied by a restoration in MEK/ERK signalling, suggesting a critical role for this pathway in regulating apoptosis in these cells. This was also confirmed using a specific pharmacological inhibitor of MEK. Experiments with conditioned media suggest that over-expression of Myc may inhibit autocrine growth factor production, which can be restored by co-expression of MBD. Although the identity of the growth factor(s) is not known, we propose a feedback mechanism whereby Myc interferes with growth factor signalling.

IGF-I stimulates proliferation of spontaneously immortalized human keratinocytes (HACAT) by autocrine/paracrine mechanisms.

HaCaT keratinocytes are derived from adult human skin and although spontaneously immortalized, remain highly related to their normal counterparts. We observed that HaCaT cells can proliferate in serum-free medium (SFM), in contrast to normal human keratinocytes whose growth in vitro requires a feeder layer and/or the supplementation with hormones and growth factors. Since autocrine production of growth factors has been proposed as the pathway that cells may exploit to escape growth regulation, we have investigated whether this is occurring in HaCaT cultured in SFM. Either epidermal growth factor (EGF) or insulin-like growth factor-1 (IGF-I) was effective and dose-dependently stimulated HaCaT replication. The ability of these keratinocytes to express EGF and IGF-I and their receptors was investigated by northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). We report that HaCaT cells synthesize mRNAs for IGF-I, IGF-II, IGF-IR and EGF-R but not EGF mRNA. Immunoneutralization of IGF-I with specific monoclonal antibodies blocked spontaneous HaCaT proliferation in SFM, as did incubation with antibodies against IGF-IR. These data demonstrate that an autocrine/paracrine loop based on IGF-I may allow HaCaT keratinocytes to proliferate autonomously in culture in contrast to keratinocytes in primary culture. A similar mechanism may be involved in the development of hyperproliferative diseases of human skin and its functional disruption may represent the target for therapeutic approaches.

Effect of hyperbaric oxygen on the growth factor profile of fibroblasts.

Hyperbaric oxygen (HBO) has been used in the clinical setting to heal problem wounds, yet its direct effects on fibroblasts are not clear. The present study evaluates the effects of HBO on the growth and autocrine production of growth factors by fibroblasts grown in an in vitro, serum-free environment. Human dermal fibroblasts were propagated in serum-free media and subjected to daily 90-minute HBO treatments at 1.0, 1.5, 2.0, 2.5, and 3.0 atm of pressure for 7 consecutive days. Cell proliferation and growth-factor assays for basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta1 (TGF-beta1) were performed on days 1, 3, 5, and 7. On day 1, HBO inhibited growth of fibroblasts at all atmospheric pressures compared with control. By day 7, cell proliferation was significantly enhanced only in cells treated with 2.0-atm HBO compared with controls. Secretion of bFGF was significantly increased by HBO-treated fibroblasts on day 1; VEGF levels slightly increased with HBO treatment on day 1, but this effect was not statistically significant; TGF-beta1 levels were detectable on day 1 only for control and HBO-treated cells at 1.0 atm, and not detectable for any cell groups after day 1. These results suggest that daily HBO treatment enhances the growth of fibroblasts when administered to a critical degree. Also, HBO appears to directly effect fibroblast production of autocrine growth factors on initial exposure. We postulate that fibroblasts possess the ability to respond to hyperoxia directly, which causes changes in cell signaling pathways involved in cellular proliferation and growth factor production.

Autocrine-mediated ErbB-2 kinase activation of STAT3 is required for growth factor independence of pancreatic cancer cell lines.

Pancreatic ductal adenocarcinoma (PDAC) cell lines, MIA PaCa-2, and UK Pan-1, were used to investigate the role of ErbB2 in PDAC oncogenesis. Both these cell lines exhibit exogenous growth factor-independent proliferation that was attributed to the production of autocrine growth factors and/or overexpression of growth factor receptors. The exogenous growth factor-independent phenotype displayed by these PDAC cell lines was dependent on ErbB2 kinase activity since treatment of cells with tyrphostin AG879 prevented serum-free media (SFM) induction of cell proliferation. We determined that ErbB2 kinase contributed to aberrant cell cycle regulation in PDAC through the induction of cyclin D1 levels and the suppression of p21(Cip1) and p27(Kip1). Inhibition of ErbB2 kinase led to cell cycle arrest marked by an increased association of p27(Kip1) with cdk2 and reduced levels of phosphorylated pRb. We further observed constitutive STAT3 activation in the PDAC cell lines and an increase in STAT3 activation upon stimulating quiescent cells with SFM. Inhibitors of ErbB2 kinase blocked STAT3 activation, whereas inhibition of EGFR kinase led to a slight reduction of STAT3 activation. STAT3 was coimmunoprecipitated with ErbB2. SFM stimulation caused an increase in the association of ErbB2 and STAT3, which was blocked by inhibition of ErbB2 kinase. Expression of a STAT3 dominant negative prevented SFM-stimulated cell proliferation of MIA PaCa-2 cells, suggesting that activation of STAT3 by ErbB2 is required for a growth factor-independent phenotype of these cells. Consistent with this observation in PDAC cell lines, we found that most PDAC tumor specimens (10 of 11) showed constitutive activation of STAT3 and that ErbB2 was readily detected in most of these tumors (nine of 11). We believe that these findings indicate a novel mechanism of oncogenesis in PDAC and may suggest future therapeutic strategies in the treatment of PDAC.

Constitutive NF-kappaB DNA-binding activity in AML is frequently mediated by a Ras/PI3-K/PKB-dependent pathway.

In the present study, we aimed to elucidate the mechanism responsible for constitutive NF-kappaB DNA-binding activity in AML cells. Intervening in aberrant signaling pathway provides a rational approach for in vivo targeting of AML cells. Constitutive NF-kappaB DNA-binding activity was observed in 16 of 22 (73%) investigated AML cases and was, in general, associated with resistance to spontaneous apoptosis. Indeed, inhibition of NF-kappaB activity by the NF-kappaB inhibitor SN-50 peptide resulted in enhanced chemotherapy-induced apoptosis. In the majority of cases, constitutive NF-kappaB activity was mediated by a Ras/PI3 kinase (PI3-K)/protein kinase B (PKB)-mediated pathway. The PI3-K inhibitor Ly294002 and the Ras inhibitor L-744832 both inhibited PKB phosphorylation and NF-kappaB DNA-binding activity. The constitutive activation of Ras GTP-ase was caused by mutations in the gene encoding for N-Ras in 29% of the cases. The constitutive NF-kappaB activity could so far not be ascribed to the autocrine production of growth factors or to mutations in the Flt3 receptor, since anti-GM-CSF, -IL-1, -IL6, -TNFalpha or the tyrosine kinase inhibitor AG1296 did not affect the NF-kappaB DNA-binding activity. The present study demonstrates that Ras activation is an important pathway for triggering the NF-kappaB pathway in AML cells.

Growth factor profile of irradiated human dermal fibroblasts using a serum-free method.

Radiation therapy for cancer permanently damages tissue in the line of treatment. This study sought to establish a serum-free protocol to evaluate the growth of irradiated fibroblasts and to analyze the levels of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) compared with normal fibroblasts. One irradiated cell line of human dermal fibroblasts was established from an intraoperative specimen obtained from a patient who had undergone radiation therapy for head and neck cancer. Irradiated and normal fibroblasts were then plated in UltraCULTURE (serum and growth factor free), modified Webber's medium (bFGF 50 ng/ml, insulin-like growth factor 100 ng/ml), and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (serum with undefined basal growth factors). Irradiated cells were also seeded in UltraCULTURE with 50 and 100 ng/ml of bFGF. Cell counts were performed at 0, 1, 3, 5, and 7 days, and cell supernatants were assayed for bFGF and TGF-beta. Irradiated and normal fibroblasts exhibited stronger growth in modified Webber's medium than in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum. Growth of irradiated fibroblasts under bFGF modulation was similar to their growth in Webber's medium. Furthermore, irradiated fibroblasts remained viable in a serum-free and growth factor-free environment for at least 7 days; however, their growth and autocrine growth factor production was less than that of normal cells. This confirms the results of previous studies suggesting that cells from irradiated tissue undergo cellular changes. This study provides an effective model for the first-line evaluation of agents to improve wound healing, and it helps to establish standard levels of bFGF and TGF-beta production for irradiated fibroblasts.

Autocrine growth factor production by fetal, keloid, and normal dermal fibroblasts.

To evaluate differences in fibroblast autocrine growth factor production by human fetal, keloid, and normal adult dermal fibroblasts. Serum-free cell lines of fetal, keloid, and normal adult dermal fibroblasts were established. Cell counts were performed and supernatants collected at 4, 24, and 72 hours. Cell-free supernatants were quantitatively assayed for transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF). Population doubling times for fetal, keloid, and normal adult fibroblasts were 120.0, 88.1, and 128.4 hours, respectively. Differences in population doubling times did not reach statistical significance. Statistically significant differences between TGF-beta1 levels from fetal and normal adult fibroblasts were seen at 24 and 72 hours. Significant differences between TGF-beta1 levels from keloid and normal adult fibroblasts were also seen at 24 and 72 hours. Fetal fibroblasts demonstrated higher levels of bFGF than normal adult fibroblasts at each time point, but these differences were not statistically significant. No significant differences were observed between keloid and normal adult bFGF levels. Both fetal and keloid fibroblasts produce significantly more TGF-beta1 than normal adult fibroblasts. Our data and the data of others suggest that fetal fibroblasts produce more bFGF than adult fibroblasts. The serum-free model we describe can be used to quantitatively measure autocrine growth factor production by cells that underlie clinically different types of wound healing. This model provides information that may allow us to better treat and prevent undesirable scarring.