Cultured peritoneal macrophages have previously been shown to release a potent mitogen for mesenchymal cells. Peritoneal macrophages are derived from peripheral blood monocytes, one of the principal inflammatory cells associated with numerous tissue responses to injury. Cultured human monocytes can be activated by endotoxin or concanavalin A to secrete a potent <em>growth</em> <em>factor</em>(s) that is active on human smooth muscle cells, human <em>fibroblasts</em> and 3T3 cells. The optimal conditions for activation of monocyte release of this monocyte derived <em>growth</em> <em>factor</em>(s) MDGF) were to expose 5-day-old monocyte cultures (initially plated at 6.8 X 10(5) cells/ml medium) to 10 microgram/ml endotoxin or 6 microgram/ml concanavalin A for approximately <em>20</em> hr. Monocytes can secrete MDGF into serum-free medium supplemented with 0.15% bovine serum albumin, MDGF stimulates both DNA synthesis and increase in cell number and is trypsin-sensitive, heat labile and nondialyzable. The relationship of MDGF to other monocyte products and its potential importance in wound repair and atherogenesis are discussed.

We report that 10% of melanoma tumors and cell lines harbor mutations in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and <em>20</em> missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer.

Ponatinib (AP24534) is a novel multitargeted kinase inhibitor that potently inhibits native and mutant BCR-ABL at clinically achievable drug levels. Ponatinib also has in vitro inhibitory activity against a discrete set of kinases implicated in the pathogenesis of other hematologic malignancies, including FLT3, KIT, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1), and platelet derived <em>growth</em> <em>factor</em> receptor α (PDGFRα). Here, using leukemic cell lines containing activated forms of each of these receptors, we show that ponatinib potently inhibits receptor phosphorylation and cellular proliferation with IC50 values comparable to those required for inhibition of BCR-ABL (0.3 to <em>20</em> nmol/L). The activity of ponatinib against the FLT3-ITD mutant, found in up to 30% of acute myeloid leukemia (AML) patients, was particularly notable. In MV4-11 (FLT3-ITD(+/+)) but not RS4;11 (FLT3-ITD(-/-)) AML cells, ponatinib inhibited FLT3 signaling and induced apoptosis at concentrations of less than 10 nmol/L. In an MV4-11 mouse xenograft model, once daily oral dosing of ponatinib led to a dose-dependent inhibition of signaling and tumor regression. Ponatinib inhibited viability of primary leukemic blasts from a FLT3-ITD positive AML patient (IC50 4 nmol/L) but not those isolated from 3 patients with AML expressing native FLT3. Overall, these results support the investigation of ponatinib in patients with FLT3-ITD-driven AML and other hematologic malignancies driven by KIT, FGFR1, or PDGFRα.

Intestinal epithelial cells rest on a <em>fibroblast</em> sheath. Thus, <em>factors</em> produced by these <em>fibroblasts</em> may influence epithelial function in a paracrine fashion. We examined modulation of intestinal epithelial function by one such <em>fibroblast</em> product, scatter <em>factor</em>/hepatocyte <em>growth</em> <em>factor</em> (HGF/SF). This effect was studied in vitro by using model T84 intestinal epithelial cells. When applied to confluent T84 monolayers, HGF/SF attenuates transepithelial resistance to passive ion flow in a dose-dependent manner (maximum fall at 300 ng/ml, 28% control monolayer resistance, P < 0.001, ED50 of 1.2 nM), t1/2 of <em>20</em> h. This functional effect of HGF/SF and distribution of its receptor, c-met, are polarized to the basolateral membranes of T84 intestinal epithelial cells. HGF/SF effects on resistance are not attributable to altered transcellular resistance (opening of Cl- and/or basolateral K+ channels), cytotoxicity, or enhanced cell proliferation; they therefore represent specific regulation of paracellular tight junction resistance. Analysis with biochemically purified rodent HGF/SF and Madin-Darby canine kidney cells reveals that effects on paracellular tight junctions also occur in other nontransformed epithelia. Binding of HGF/SF to its receptor in T84 intestinal epithelial cells is accompanied by tyrosine phosphorylation of the receptor. Because loosening of intercellular junctions between cells could facilitate separation, spreading, and migration of epithelial cells during physiologic processes such as wound resealing, we determined the effects of HGF/SF on intestinal epithelial wound resealing using our previously published in vitro model (Nusrat, A., C. Delp, and J. L. Madara. 1992. J. Clin. Invest. 89:1501-1511). HGF/SF markedly enhanced wound closure >> 450% increase in rate, P < 0.001) by influencing the migratory and spreading response in not only cells adjoining the wound but also cells many positions removed from the wound. We thus speculate that HGF/SF may serve as an important cytokine that influences epithelial parameters such as transepithelial resistance and wound resealing. Further pharmacological approaches to manipulate HGF/SF signaling pathways may provide novel therapeutic strategies to enhance repair of intestinal epithelial erosions/ulcerations.

Hepatocyte <em>growth</em> <em>factor</em> (HGF) is a potent mitogen for mature hepatocytes in vitro. The receptor for HGF has recently been characterized as the product of the proto-oncogene c-met. We have examined the possible involvement of HGF in hepatic <em>growth</em> and differentiation in the rat. The experimental systems used were acetylaminofluorene treatment combined with partial hepatectomy to induce proliferation and differentiation of oval cells in adult liver and the pre- and postnatal liver. In the acetylaminofluorene model, Northern blot analysis showed that level of HGF transcripts increased one day after partial hepatectomy, reached a peak by day 6, were maintained at that level until day 13, and then declined, reaching normal level at <em>20</em> days. The expression of c-met also increased gradually, reached a peak around 9 to 13 days after partial hepatectomy, at which time oval cell proliferation was most prominent. In the developing liver, an elevated level of HGF transcripts was found between 4 and 21 days after birth. The expression of c-met also slightly increased at the same time. In situ hybridization showed that the transcripts for HGF were localized in desmin-positive Ito cells, whereas the transcripts for c-met were strongly expressed by oval cells. We have shown earlier that Ito cells and oval cells proliferate simultaneously and exist in close proximity in the acetylaminofluorene model and that Ito cells are a primary source of <em>growth</em> <em>factors</em> such as transforming <em>growth</em> <em>factor</em>-alpha and acidic <em>fibroblast</em> <em>growth</em> <em>factors</em>. The data presented here suggest that HGF is, in combination with other <em>growth</em> <em>factors</em>, involved in the proliferation and differentiation of oval cells via a paracrine mechanism.

Epidemiological studies suggest a causal relationship of dietary polyunsaturated fatty acids (PUFA's) with the morbidity and mortality from breast cancer. In order to reveal possible underlying mechanisms of these findings, we studied the influence of n-3 and n-6 PUFA's in comparison to oleic acid on the proliferation of well characterized estrogen dependent (MCF-7, ZR-75, T-47-D) and estrogen independent (MDA-MB-231, HBL-100) breast cancer cells in culture. The cell <em>growth</em> inhibitory effect was related to the formation of lipid peroxidation products. Normal human skin <em>fibroblasts</em> served as a control. In <em>fibroblasts</em>, the addition of <em>20</em> micrograms/ml of exogenous fatty acids either had no effect or caused an insignificant increase of proliferation. Similar results were obtained with MCF-7 cells. In all other breast cancer cell types, n-3 long-chain PUFA's, eicosapentaenoic and docosahexaenoic acids, were the most effective fatty acids in arresting the cell <em>growth</em>. Alpha-linolenic and gamma-linolenic acid exerted a variable effect on cell proliferation depending on the cell line investigated. Oleic acid significantly stimulated the proliferation of hormone-independent breast cancer cells while it had no effect on the proliferation of hormone-dependent cells. Viability studies by trypan blue excretion indicated that the arrest in cell <em>growth</em> was not due to major cytotoxic effects. The addition of PUFA's to breast cancer cells caused a significant increase in the formation of conjugated dienes and lipid hydroperoxides in the cellular lipids; their content was significantly correlated with the capacity of arresting cell <em>growth</em>. In contrast, the addition of PUFA's to <em>fibroblasts</em> did not increase lipid hydroperoxide formation. The addition of Vitamin E to cancer cells at a concentration of 10 microM to the PUFA-supplemented medium almost completely restored cell <em>growth</em>. Our data indicate that PUFA's significantly interfere with cell proliferation of breast cancer cells in vitro due to the formation of oxidation products. In addition to that, there must be other <em>factors</em> involved, most probably related to the differential metabolism of PUFA's in tumor cells. Our findings may have some impact on treatment and prevention of breast cancer.

Overexpression of Neu (ErbB-2/HER2) is found in approximately <em>20</em>% of breast tumors. Activation of Neu by a point mutation (NeuT) causes constitutive tyrosine kinase activity of this transmembrane receptor and transforming activity in <em>fibroblasts</em>. To identify downstream targets of Neu, we have analyzed the ability of Neu to activate gene expression. Expression of NeuT, but not normal Neu, caused transcriptional activation of Ets, AP-1, or NF-kappaB-dependent reporter genes. Dominant inhibitory Ras or Raf mutants blocked the Neu-mediated transcriptional activation, confirming that Ras signaling pathways were required for this activation. Analysis with Ets2 mutants indicated that activation of Ets2 transcriptional activity mediated by NeuT or oncogenic Ras required phosphorylation of the same Ets2 residue, threonine 72. Cotransfection of dominant inhibitory Ets2 mutants specifically blocked NeuT-mediated activation of Ets-dependent reporter genes. Furthermore, in focus formation assays using NIH 3T3 cells, the transforming activity of NeuT was inhibited 5-fold when NeuT was cotransfected with a dominant negative Ets2 mutant. However, parallel colony formation assays showed that the Ets2 dominant negative mutant did not inhibit the <em>growth</em> of normal cells. Together, these data show that NeuT activates a variety of transcription <em>factor</em> families via the Ras signaling pathway and that Ets activation is required for NeuT-mediated cellular transformation. Thus, downstream targets of Neu, including Ets transcription <em>factors</em>, may be useful points for therapeutic intervention in Neu/ErbB-2-associated cancers.

Human pluripotent (embryonic or induced) stem cells (hPSCs) have many potential applications, not only for research purposes but also for clinical and industrial uses. While culturing these cells as undifferentiated lines, an adherent cell culture based on supportive layers or matrices is most often used. However, the use of hPSCs for industrial or clinical applications requires a scalable, reproducible and controlled process. Here we present a suspension culture system for undifferentiated hPSCs, based on a serum-free medium supplemented with interleukins and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, suitable for the mass production of these cells. The described system supports a suspension culture of hPSC lines, in both static and dynamic cultures. Results showed that hPSCs cultured with the described dynamic method maintained all hPSC features after <em>20</em> passages, including stable karyotype and pluripotency, and increased in cell numbers by 25-fold in 10 d. Thus, the described suspension method is suitable for large-scale culture of undifferentiated hPSCs.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) plays a crucial role in renal phosphate regulation, exemplified by the causal role of PHEX and DMP1 mutations in X-linked hypophosphatemic rickets and autosomal recessive rickets type 1, respectively. Using whole exome sequencing we identified compound heterozygous mutations in family with sequence similarity <em>20</em>, member C (FAM<em>20</em>C) in two siblings referred for hypophosphatemia and severe dental demineralization disease. FAM<em>20</em>C mutations were not found in other undiagnosed probands of a national Norwegian population of familial hypophosphatemia. Our results demonstrate that mutations in FAM<em>20</em>C provide a putative new mechanism in human subjects leading to dysregulated FGF23 levels, hypophosphatemia, hyperphosphaturia, dental anomalies, intracerebral calcifications and osteosclerosis of the long bones in the absence of rickets.

Neural transplantation is developing into a therapeutic alternative in Parkinson's disease. A major limiting <em>factor</em> is that only 3-<em>20</em>% of grafted dopamine neurons survive the procedure. Recent advances regarding how and when the neurons die indicate that events preceding actual tissue implantation and during the first week thereafter are crucial, and that apoptosis plays a pivotal role. Triggers that may initiate neuronal death in grafts include donor tissue hypoxia and hypoglycemia, mechanical trauma, free radicals, <em>growth</em> <em>factor</em> deprivation, and excessive extracellular concentrations of excitatory amino acids in the host brain. Four distinct phases during grafting that can involve cell death have been identified: retrieval of the embryo; dissection and preparation of the donor tissue; implantation procedure followed by the immediate period after graft injection; and later stages of graft maturation. During these phases, cell death processes involving free radicals and caspase activation (leading to apoptosis) may be triggered, possibly involving an increase in intracellular calcium. We review different approaches that reduce cell death and increase survival of grafted neurons, typically by a <em>factor</em> of 2-4. For example, changes in transplantation procedure such as improved media and implantation technique can be beneficial. Calcium channel antagonists such as nimodipine and flunarizine improve nigral graft survival. Agents that counteract oxidative stress and its consequences, such as superoxide dismutase overexpression, and lazaroids can significantly increase the survival of transplanted dopamine neurons. Also, the inhibition of apoptosis by a caspase inhibitor has marked positive effects. Finally, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and members of the transforming <em>growth</em> <em>factor</em>-beta superfamily, such as glial cell line-derived neurotrophic <em>factor</em>, significantly improve the outcome of nigral transplants. These recent advances provide hope for improved survival of transplanted neurons in patients with Parkinson's disease, reducing the need for human embryonic donor tissue and increasing the likelihood of a successful outcome.

BACKGROUND

Murine fibroblast growth factor (FGF) 21 is a nutritionally regulated hormone secreted by the liver principally in response to peroxisome proliferator-activated receptor-alpha (PPAR alpha) activation, which plays a critical role in regulating metabolism during ketosis. FGF21 is also a PPAR gamma target gene in mouse adipose tissue. Little information is available on FGF21 functions in humans.

OBJECTIVE

The aim of the study was to measure plasma FGF21 during fasting, ketogenic diet, and PPAR agonist treatment in humans.

METHODS

We conducted a prospective study involving three patient groups at two university hospitals.

METHODS

Eight healthy male volunteers underwent a 48-h period of starvation followed by 24-h refeeding (group 1); seven obese individuals were allocated to a low-carbohydrate diet for 3 months (group 2); and three groups of healthy, overweight or obese male volunteers received treatment with a PPAR alpha (20 microg/d GW590735) (n=6), PPAR delta (10 mg/d GW501516) (n=6), or PPAR gamma agonist (rosiglitazone) (n=10) for 2 wk (group 3).

METHODS

Fasting plasma FGF21 and serum 3-hydroxybutyrate were measured.

RESULTS

There was no significant variation in human plasma FGF21 during fasting and refeeding. A 3-month ketogenic diet was associated with a 42% decline in plasma FGF21 levels. Circulating FGF21 increased significantly in response to treatment with PPAR alpha (39%) and PPAR delta (32%), but not PPAR gamma agonists.

CONCLUSIONS

FGF21 does not play a major role in regulating the fasting response or ketosis in man. However, plasma FGF21 is elevated in response to pharmacological activation of PPAR alpha and PPAR delta and may contribute to the beneficial metabolic effects observed in response to pharmacotherapy with these compounds.

Neural stem cells (NSCs) can be derived from single mouse embryonic stem cells (ESCs) in the absence of instructive <em>factors</em>. Clonal primitive NSC (pNSC) colonies are formed first, and then give rise to clonal, <em>fibroblast</em> <em>growth</em> <em>factor</em>-dependent definitive neural stem cells (dNSCs). We tested low-oxygen culture as a potential method of alleviating the extensive cell death seen in pNSCs and dNSCs. Culture in low (4%) oxygen promoted survival of pNSCs by inhibiting apoptosis-inducing <em>factor</em> (AIF)-dependent cell death, although pNSCs undergo both AIF- and caspase-mediated cell death in <em>20</em>% oxygen. In contrast, survival of dNSCs in low oxygen was increased by inhibition of caspase-dependent cell death. In normoxia, AIF is implicated in promoting dNSC survival. Neither survival effect was dependent on the main transcriptional effector of hypoxia, hypoxia-inducible <em>factor</em> 1. Low-oxygen concentrations may be involved in expansion of early NSC populations by inhibiting cell death through different pathways in these sequential pNSC and dNSC populations.

Recent advances in nanotechnology and molecular self-assembly may provide novel solutions to current cell transplantation deficiencies. Heparin-binding peptide amphiphiles (HBPAs) self-assemble from aqueous media into nanofibers that bind <em>growth</em> <em>factors</em> through interactions with the bioactive polymer heparin. In this report, we demonstrate that delivery of vascular endothelial <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 from HBPA scaffolds significantly increases blood vessel density in the mouse omentum over control scaffolds without <em>growth</em> <em>factors</em> (P<0.0005) and significantly enhances islet engraftment. Diabetic recipients transplanted with 250 isologous islets and HBPA scaffolds containing vascular endothelial <em>growth</em> <em>factor</em>/<em>fibroblast</em> <em>growth</em> <em>factor</em>-2 achieved normoglycemia at a higher rate (78%) than control animals receiving identical scaffolds without <em>growth</em> <em>factors</em> (30%; P<0.05) or <em>growth</em> <em>factors</em> alone (<em>20</em>%). These data indicate that the enhanced engraftment can be attributed to specific <em>growth</em> <em>factor</em> effects that were made possible by the delivery mechanism of HBPA nanostructures.

The osteogenic <em>factors</em> bone morphogenetic protein (BMP-7), platelet-derived <em>growth</em> <em>factor</em> (PDGF)-BB, and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) regulate the recruitment of osteoprogenitor cells and their proliferation and differentiation into mature osteoblasts. However, their mechanisms of action on osteoprogenitor cell <em>growth</em>, differentiation, and bone mineralization remain unclear. Here, we tested the hypothesis that these osteogenic agents were capable of regulating osteoblast differentiation and bone formation in vitro. Normal human bone marrow stromal (HBMS) cells were treated with BMP-7 (40 ng ml(-1)), PDGF-BB (<em>20</em> ng ml(-1)), FGF-2 (<em>20</em> ng ml(-1)), or FGF-2 plus BMP-7 for 28 days in a serum-containing medium with 10 mM beta-glycerophosphate and 50 microg ml(-1) ascorbic acid. BMP-7 stimulated a morphological change to cuboidal-shaped cells, increased alkaline phosphatase (ALKP) activity, bone sialoprotein (BSP) gene expression, and alizarin red S positive nodule formation. Hydroxyapatite (HA) crystal deposition in the nodules was demonstrated by Fourier transform infrared (FTIR) spectroscopy only in BMP-7- and dexamethasone (DEX)-treated cells. DEX-treated cells appeared elongated and <em>fibroblast</em>-like compared to BMP-7-treated cells. FGF-2 did not stimulate ALKP, and cell morphology was dystrophic. PDGF-BB had little or no effect on ALKP activity and biomineralization. Alizarin Red S staining of cells and calcium assay indicated that BMP-7, DEX, and FGF-2 enhanced calcium mineral deposition, but FTIR spectroscopic analysis demonstrated no formation of HA similar to human bone in control, PDGF-BB-, and FGF-2-treated samples. Thus, FGF-2 stimulated amorphous octacalcium phosphate mineral deposition that failed to mature into HA. Interestingly, FGF-2 abrogated BMP-7-induced ALKP activity and HA formation. Results demonstrate that BMP-7 was competent as a sole <em>factor</em> in the differentiation of human bone marrow stromal cells to bone-forming osteoblasts confirmed by FTIR examination of mineralized matrix. Other <em>growth</em> <em>factors</em>, PDGF, and FGF-2 were incompetent as sole <em>factors</em>, and FGF-2 inhibited BMP-7-stimulated osteoblast differentiation.

Human platelet-derived <em>growth</em> <em>factor</em> (hPDGF) is likely to be important in stimulating tissue repair, based upon its in vivo chemotactic and stimulatory activities for inflammatory cells and <em>fibroblasts</em> and upon the presence of PDGF and related proteins in platelets, macrophages, and activated <em>fibroblasts</em>, cell types that make up the milieu of the healing wound. Recombinant human c-sis (rPDGF-B), homodimers of the B chain of PDGF, were compared with hPDGF in vitro. rPDGF-B was immunologically similar to hPDGF and, at identical concentrations, similar to hPDGF in stimulating <em>fibroblast</em> mitogenesis and chemotaxis of polymorphonuclear leukocytes, monocytes, and <em>fibroblasts</em>. Purified hPDGF and rPDGF-B were also tested in vivo for potency in a model of tissue repair using a linear incision wound through rat dermis. A single application of hPDGF or rPDGF-B (2-<em>20</em> micrograms/wound) in a slow release vehicle at the time of wounding resulted in a dose-dependent, statistically highly significant increase of breaking strength of treated wounds. Wound healing in animals treated with rPDGF-B was 170% stronger and accelerated by 2 d during the first week over control wounds and by 4-6 d over the next 2 wk. Histologic evaluation of <em>growth</em> <em>factor</em>-treated wounds correlated the in vitro chemotactic activity and the accelerated healing of wounds with a striking inflammatory cell infiltrate early after wounding, markedly increased formation of granulation tissue by 4-d, and increased fibrosis by 14 d in comparison to control wounds. The results thus demonstrate that rPDGF-B is fully active in in vitro tests of mitogenesis and chemotaxis and, for the first time, demonstrate directly that PDGF significantly advances wound healing in incisional wounds of experimental animals.

The human beta 2 interferon (IFN-beta 2) gene, a gene that also codes for B cell differentiation <em>factor</em> 2 (BSF-2), plasmacytoma/hybridoma <em>growth</em> <em>factor</em> (HGF), and hepatocyte-stimulating <em>factor</em> (HSF), is expressed in a variety of lymphoid and nonlymphoid tissues. Endotoxin, or bacterial lipopolysaccharide (LPS) preparations derived from the outer membrane of Escherichia coli or Salmonella typhimurium rapidly elevate IFN-beta 2 mRNA level in human skin <em>fibroblasts</em> (FS-4 strain). E. coli-derived LPS enhances IFN-beta 2 mRNA expression in FS-4 <em>fibroblasts</em> at a concentration as low as 0.3 ng/ml; this response is near-maximal in the range of 0.1-1 microgram/ml LPS. The increase in IFN-beta 2 mRNA level caused by LPS in FS-4 cells is detected within 30 min after addition of LPS, is sustained for at least <em>20</em> h thereafter, appears to involve the protein kinase C signal transduction pathway, does not require new protein synthesis, and is inhibited by dexamethasone in a dose-dependent fashion (in the range 10(-6)-10(-8) M). Cultures of LPS-treated FS-4 cells exhibit an antiviral state against vesicular stomatitis virus, which can be prevented by anti-IFN-beta antiserum. Medium obtained from LPS-treated FS-4 cell cultures enhances the number of immunoglobulin-secreting cells in cultures of human B-lymphoblastoid (CESS) cells. Thus, LPS may trigger a number of host defense mechanisms in the course of infection due to Gram-negative bacteria by enhancing IFN-beta 2 production by the ubiquitous <em>fibroblast</em>.

Chondrosarcomas are malignant cartilage-forming tumors arising centrally in bone (central chondrosarcoma) or within the cartilaginous cap of osteochondroma (peripheral chondrosarcoma). For hereditary multiple osteochondromas, two responsible genes, EXT1 and EXT2, have been cloned. Their recently elucidated role in heparan sulfate biosynthesis and Hedgehog diffusion leads to the hypothesis that EXT inactivation affects <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and Indian Hedgehog (IHh)/parathyroid hormone-related peptide (PTHrP) signaling, two important pathways in chondrocyte proliferation and differentiation. The expression of PTHrP, PTHrP-receptor, Bcl-2, FGF2, FGFR1, FGFR3, and p21 is investigated by immunohistochemistry in osteochondromas (n = 24) and peripheral (n = 29) and central (n = <em>20</em>) chondrosarcomas. IHh/PTHrP and FGF signaling molecules are mostly absent in osteochondromas. Although no somatic EXT mutations were found in sporadic osteochondromas, the putative EXT downstream targets are affected similarly in sporadic and hereditary tumors. In chondrosarcomas, re-expression of FGF2, FGFR1, PTHrP, Bcl-2, and p21 is found. Expression levels increase with increasing histological grade. Up-regulation of PTHrP and Bcl-2 characterizes malignant transformation of osteochondroma because PTHrP and Bcl-2 expression is significantly higher in borderline and grade I peripheral chondrosarcomas compared with osteochondromas. In contrast, up-regulation of PTHrP and Bcl-2 seems to be a late event in central cartilaginous tumorigenesis because expression is mainly restricted to high-grade central tumors.

Alternative splicing of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) occurs in a cell-type-specific manner with the mutually exclusive use of exon IIIb or exon IIIc. Specific inclusion of exon IIIb is observed in epithelial cells, whereas exon IIIc inclusion is seen in mesenchymal cells. Epithelium-specific activation of exon IIIb and repression of exon IIIc are coordinately regulated by intronic activating sequence 2 (IAS2) and intronic splicing activator and repressor (ISAR) elements in FGFR2 pre-mRNA. Previously, it has been suggested that IAS2 and a <em>20</em>-nucleotide core sequence of ISAR form a stem structure that allows for the proper regulation of FGFR2 alternative splicing. Replacement of IAS2 and the ISAR core with random sequences capable of stem formation resulted in the proper activation of exon IIIb and repression of exon IIIc in epithelial cells. Given the high degree of phylogenetic conservation of the IAS2-ISAR core structure and the fact that unrelated stem-forming sequences could functionally substitute for IAS2 and ISAR elements, we postulated that the stem structure facilitated the approximation of intronic control elements. Indeed, deletion of the entire stem-loop region and juxtaposition of sequences immediately upstream of IAS2 with sequences immediately downstream of the ISAR core maintained proper cell-type-specific inclusion of exon IIIb. These data demonstrate that IAS2 and the ISAR core are dispensable for the cell-type-specific activation of exon IIIb; thus, the major, if not the sole, role of the IAS2-ISAR stem in exon IIIb activation is to approximate sequences upstream of IAS2 with sequences downstream of the ISAR core. The downstream sequence is very likely a highly conserved GCAUG element, which we show was required for efficient exon IIIb activation.

The ability of prostate-specific antigen (PSA) to predict tumor volume and stage in patients with prostate cancer would be improved if <em>factors</em> regulating its production and clearance were better defined. A thorough understanding of the pharmacokinetics (regulation of production, metabolism, and excretion) of PSA has been precluded, however, by the absence of an in vivo animal model. The purposes of this study are to develop a murine model for evaluating PSA pharmacokinetics in vivo and to assess <em>factors</em> that influence PSA production in vitro. The human prostate cancer cell line, LNCaP, was chosen because it is androgen sensitive and PSA positive. Although LNCaP cells are usually nontumorigenic when inoculated s.c. in athymic mice, coinoculation of 1 x 10(6) LNCaP cells with 1 x 10(6) human bone <em>fibroblasts</em> reliably produces PSA-secreting carcinomas. This LNCaP model provides accurate correlation between tumor volume and serum PSA levels (r = 0.94) and demonstrates that tumor volume and androgens are codeterminants of circulating PSA levels. Following castration, serum PSA levels decrease rapidly up to 8-fold and increase up to <em>20</em>-fold following androgen supplementation, without detectable castration-induced tumor cell death or concomitant changes in tumor volume. Serum PSA levels increase 0.24 ng/ml/mm3 of tumor, which is approximately 5-fold less than that estimated for humans. Most likely this reduced PSA index (PSA:tumor volume ratio) results from a 7-fold faster clearance of PSA in athymic mice than in humans; other than this shorter half-life, PSA elimination in the murine model appears similar to that in humans, with both following first-order kinetics characteristic of a two-compartment model. Interestingly, following prolonged <em>growth</em> (greater than 21 days) in castrate hosts, LNCaP tumors are capable of adapting to an androgen-deprived environment whereby LNCaP tumors regain the ability to secrete PSA in amounts similar to the precastrate state. In LNCaP cells, androgens increase PSA mRNA levels 4-fold in vivo and in vitro. PSA mRNA expression is also altered by various <em>growth</em> <em>factors</em>. Changes in PSA production induced by androgens and <em>growth</em> <em>factors</em> do not always parallel changes in LNCaP cell <em>growth</em> rate induced by these <em>factors</em>, suggesting that PSA production occurs independently of cell <em>growth</em> rate and may be influenced by various interrelated <em>factors</em>, including hormonal and stromal milieu. Observations from this murine model suggest that androgens and tumor volume are independent determinants of serum PSA levels and imply that decreases in circulating PSA following antiandrogen therapy may not always reflect a corresponding reduction in tumor volume.

During angiogenesis, endothelial cells react to stimulation with finely tuned signaling responses. The role of calcium-regulated signaling in angiogenesis has not been defined. This study investigated the calcium dependency of endothelial cell proliferation and invasion by using an inhibitor of ligand-stimulated calcium influx, CAI (carboxy-amidotriazole). Incubation with CAI significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in response to serum (IC50 = 1 microM) or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2; P2 < 0.005 at 10 microM). Statistically significant inhibition of HUVEC adhesion and motility to basement membrane proteins laminin, fibronectin, and type IV collagen was demonstrated (adhesion, P2 < 0.004-0.01; motility, P2 < 0.009-0.018). Marked inhibition of native and FGF2-induced gelatinase activity was shown by zymogram analysis and was confirmed by Northern blot analysis. CAI inhibited HUVEC tube formation on Matrigel and inhibited in vivo angiogenesis in the chicken chorioallantoic membrane assay, 67% at <em>20</em> microM and 56% at 10 microM compared with 16% for an inactive CAI analog or 9% for 0.1% dimethyl sulfoxide control. Incubation of HUVECs with CAI and/or FGF2 followed by immunoprecipitation with anti-phosphotyrosine antibody showed inhibition of FGF2-induced tyrosine phosphorylation of proteins in the range 110-150 kDa. These results suggest that calcium-regulated events are important in native and FGF2-stimulated HUVEC proliferation and invasion, perhaps through regulation of FGF2-induced phosphorylation events, and indicate a role for calcium in the regulation of angiogenesis in vivo.

BACKGROUND

YKL-40 is secreted by macrophages and neutrophils and is a growth factor for vascular endothelial cells and fibroblasts. Elevated serum concentrations of YKL-40 are found in patients with diseases characterized by inflammation or ongoing fibrosis. The aim of this study was to seek association between serum YKL-40 in patients with ulcerative colitis (UC) and Crohn disease (CD) and clinical disease activity.

METHODS

One-hundred-and-sixty-four patients with UC and 173 patients with CD were studied. The Simple Clinical Colitis Activity Index (SCCAI) and the Harvey-Bradshaw (H-B) score were used to assess disease activity. Serum YKL-40 (determined by ELISA) was related to C-reactive protein (CRP) and disease activity.

RESULTS

In patients with UC, the median serum YKL-40 rose with increasing disease activity, and patients with severe active disease had higher serum YKL-40 (median 59 microg/L (95% CI: 26-258 microg/L), P < 0.001) than patients with inactive UC (33 microg/L (19-163)) and age-matched controls (43 microg/L (20-124)). Patients with severe active CD had higher serum YKL-40 (59 microg/L (21-654), P < 0.001) than age-matched controls, but not higher than inactive CD patients (43 microg/L (17-306)). Serum YKL-40 was elevated in 41% of the patients with severe UC, in 10% with inactive UC, in 46% with severe CD and in 30% with inactive CD. Serum YKL-40 correlated with SCCAI in UC patients but not with H-B score in CD patients. In both patient groups, low correlations were found between serum YKL-40 and CRP, albumin and leucocytes.

CONCLUSIONS

Serum YKL-40 is elevated in patients with active IBD and may be complementary to inflammatory markers and clinical characteristics in the assessment of disease activity.

Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid <em>fibroblasts</em> (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing <em>20</em>% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike <em>growth</em> <em>factor</em>-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interpersed nuclear element I (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike <em>growth</em> <em>factor</em>-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and <em>growth</em> from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of diverse genes which may play a role in the senescent arrest of cellular replication and in the genesis of WS, normal biological aging, and attendant diseases.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for>> or = 5 weeks when cocultured with stromal <em>fibroblasts</em>. Some expansion of LTC-ICs in vitro has recently been described, but identification of the <em>factors</em> required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine <em>fibroblasts</em> engineered to produce steel <em>factor</em>, interleukin (IL)-3, and granulocyte colony-stimulating <em>factor</em> showed that approximately <em>20</em>% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel <em>factor</em>, IL-3, IL-6, granulocyte colony-stimulating <em>factor</em>, and nerve <em>growth</em> <em>factor</em> resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included>> or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.

alpha-Thrombin induced a change in the cell morphology of IIC9 <em>fibroblasts</em> from a semiround to an elongated form, accompanied by an increase in stress fibers. Incubation of the cells with phospholipase D (PLD) from Streptomyces chromofuscus and exogenous phosphatidic acid (PA) caused similar morphological changes, whereas platelet-derived <em>growth</em> <em>factor</em> (PDGF) and phorbol 12-myristate 13-acetate (PMA) induced different changes, e.g., disruption of stress fibers and cell rounding. alpha-Thrombin, PDGF, and exogenous PLD increased PA by <em>20</em>-40%, and PMA produced a smaller increase. alpha-Thrombin and exogenous PLD produced rapid increases in the amount of filamentous actin (F-actin) that were sustained for at least 60 min. However, PDGF produced a transient increase of F-actin at 1 min and PMA caused no significant change. Dioctanoylglycerol was ineffective except at 50 micrograms/ml. Phospholipase C from Bacillus cereus, which increased diacylglycerol (DAG) but not PA, did not change F-actin content. Down-regulation of protein kinase C (PKC) did not block actin polymerization induced by alpha-thrombin. H-7 was also ineffective. Exogenous PA activated actin polymerization with a significant effect at 0.01 microgram/ml and a maximal increase at 1 microgram/ml. No other phospholipids tested, including polyphosphoinositides, significantly activated actin polymerization. PDGF partially inhibited PA-induced actin polymerization after an initial increase at 1 min. PMA completely or largely blocked actin polymerization induced by PA or PLD. These results show that PC-derived PA, but not DAG or PKC, activates actin polymerization in IIC9 <em>fibroblasts</em>, and indicate that PDGF and PMA have inhibitory effects on PA-induced actin polymerization.