OBJECTIVE

To determine the clinical and prognostic significance of chromosome 11q13 amplification in squamous cell carcinoma of the head and neck.

METHODS

Retrospective clinical analysis.

METHODS

University and private cancer centers.

METHODS

Fifty-six patients with pathologically confirmed head and neck squamous cell carcinoma whose tumors had been assayed for the presence or absence of chromosome 11q13 amplification.

METHODS

The degree of DNA amplification in each tumor was determined using chromosome 11q13 probes for the bcl-1 major translocation cluster, PRAD1/cyclin D1 (CCND1), the fibroblast growth factor gene HST1, EMS1, and glutathione-S-transferase-pi-1. The presence or absence of amplification in each patient was correlated with primary site, tumor stage, nodal status, presence or absence of distant metastasis, disease recurrence, time to recurrence, clinical outcome (disease status), and overall survival.

RESULTS

Amplification of chromosome 11q13 was identified in 39% (22/56) of patients. Recurrent or persistent disease was identified in 82% (18/22) of cases with amplification and 50% (14/28) of nonamplified cases (P = .04). Mean time to recurrence was shorter in cases with amplification (6.2 months) than those without amplification (10.1 months) (P = .01). Eighteen patients (82%) with amplification and 10 patients (38%) without amplification died of disease or are alive with disease (P = .001). The mean follow-up period was 15.8 months for patients with amplification and 18.6 months for patients without amplification. Overall survival was significantly diminished in patients with amplification (P = .002). Amplification was not related to nodal status, distant metastases, or initial disease stage.

CONCLUSIONS

Amplification of chromosome 11q13 loci may be an important biologic marker indicating poor prognosis, independent of clinical stage in head and neck squamous cell carcinoma, and it should be assessed in prospective trials to determine its utility for stratifying treatment and determining prognosis.

2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-17beta and the oral contraceptive agent 17-ethylestradiol. 2-ME was recently reported to inhibit endothelial cell proliferation. The current study was undertaken to explore the mechanism of 2-ME effects on endothelial cells, especially whether 2-ME induces apoptosis, a prime mechanism in tissue remodeling and angiogenesis. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to 2-ME showed morphological (including ultrastructural) features characteristic of apoptosis: cell shrinkage, cytoplasmic and nuclear condensation, and cell blebbing. 2-ME-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 0.45 +/- 0.09 microM, n = 8). Nucleosomal DNA fragmentation in BPAEC treated with 2-ME was identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end labeling. Under the same experimental conditions, estradiol-17beta and two of its other metabolites, estriol and 2-methoxyestriol (< or =10 microM), did not have an apoptotic effect on BPAEC. 2-ME activated stress-activated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BPAEC in a concentration-dependent manner. The activity of SAPK was increased by 170 +/- 27% and 314 +/- <em>22</em>% over the basal level in the presence of 0.4 and 2 microM 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at 20 min, and returned to basal levels at 60 min after exposure to 2-ME. Inhibition of SAPK/c-Jun amino-terminal protein kinase activation by basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>, or forskolin reduced 2-ME-induced apoptosis. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regulated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPAEC migration (IC50 = 0.71 +/- 0.11 microM, n = 4) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-induced angiogenesis in the chick chorioallantoic membrane model. Taken together, these results suggest that promotion of endothelial cell apoptosis, thereby inhibiting endothelial cell proliferation and migration, may be a major mechanism by which 2-ME inhibits angiogenesis.

The <em>fibroblast</em> <em>growth</em> <em>factor</em>-binding protein (FGF-BP) binds and activates FGF-1 and FGF-2, thereby contributing to tumor angiogenesis. In this study, we identified novel binding partners of FGF-BP, and we provide evidence for a role of this protein in epithelial repair processes. We show that expression of FGF-BP increases after injury to murine and human skin, in particular in keratinocytes. This upregulation is most likely achieved by major keratinocyte mitogens present at the wound site. Most importantly, we demonstrate that FGF-BP interacts with FGF-7, FGF-10, and with the recently identified FGF-<em>22</em>, and enhances the activity of low concentrations of ligand. Due to the important functions of FGF-7 and FGF-10 for repair of injured epithelia, our findings suggest that upregulation of FGF-BP expression after injury stimulates FGF activity at the wound site, thus enhancing the process of epithelial repair.

Focal mechanical injury to the retina substantially increases basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and ciliary neurotrophic <em>factor</em> (CNTF) mRNA expression, accompanied by a transient increase in FGFR-1 mRNA, and this response is thought to protect photoreceptors near the injury site from inherited and light-induced retinal degenerations. We have now examined retinal gene expression of the principal survival <em>factors</em> involved in the response to injury in normal rats as a function of postnatal age both in normal and injured retinas. Sprague-Dawley rats were injured in one eye by needle incision through the retina at postnatal day (P) 10, <em>22</em>, 35, 60, 90, 120 and 180. The other eye was uninjured and served as the control. Retinas were taken 1 day post-injury. Northern blot analysis was performed to determine the mRNA expression of the following <em>factors</em> and receptors: bFGF and acidic <em>fibroblast</em> <em>growth</em> <em>factors</em> (aFGF) and FGF receptor-1 (FGFR-1); CNTF and CNTF receptor alpha (CNTFR-alpha); brain-derived neurotrophic <em>factor</em> (BDNF) and its receptor trk B; and insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) and IGF-I receptor (IGF-IR); glial fibrillary acidic protein (GFAP) and opsin. In the uninjured control eyes, mRNA expression of most of the <em>factors</em> increased with postnatal age, with little expression at P10 and maximal expression levels reached at P<em>22</em> (opsin), P35 (aFGF), P60 (BDNF) or P90 (bFGF, FGFR-1, CNTF and GFAP). In contrast, IGF-1 mRNA rapidly decreased from a high level of expression at P10 to about 55% of that level by P<em>22</em>, reaching a stable 45-50% of the P10 level at P35 and thereafter. The response to injury of bFGF, FGFR-1, CNTF and GFAP mRNAs increased with postnatal age. Unexpectedly, only minimal increases in bFGF, FGFR-1, CNTF and GFAP over those seen in the control eyes were observed before P35. Thereafter, the increase of bFGF mRNA after injury reached a maximum of three-fold at P60, maintained this level to P120, and slightly decreased to 2.5-fold by P180. Expression of FGFR-1 mRNA showed a maximum increase of 2.6-fold at P90. Expression of CNTF and GFAP mRNAs followed a time course similar to that of bFGF. Mechanical injury did not alter the mRNA levels of aFGF, BDNF, IGF-I, and receptors, CNTFR-alpha, trk B and IGF-IR. These data show that the response to injury is minimal at early postnatal ages but increases with age and peaks at P60-90 for most potential survival <em>factors</em>.

BACKGROUND

Plasma and serum biomarkers of angiogenesis and activated endothelial cells were evaluated to assess biological activity of PTK787/ZK 222584 (PTK/ZK), a novel oral angiogenesis inhibitor targeting all known vascular endothelial growth factor (VEGF) receptor tyrosine kinases.

METHODS

Patients with colorectal cancer (CRC) (n=63) were enrolled into two phase I/II dose escalation trials of PTK/ZK in 28-day cycles until discontinuation. Patients with stable disease for>> or =2 months were categorized as 'non-progressors'. Plasma markers of angiogenesis, VEGF-A and basic fibroblast growth factor (bFGF), and the serum markers of activated endothelial cells, sTIE-2 and sE-Selectin, were assessed at baseline, and pre-dose on days 1, 8, 15, 22 and 28 of every cycle, with additional assessments 10 h post-dose on days 1 and 15. The percentage change from baseline was subsequently correlated with AUC and C(max) of PTK/ZK on day 1, cycle 1 and clinical outcome.

RESULTS

A dose-dependent increase in plasma VEGF-A and bFGF was observed in the first cycle of PTK/ZK treatment. The correlation of change in plasma VEGF-A with AUC and C(max) was characterized by an E(max) model, suggesting that a change of>> or =150% from baseline VEGF-A correlated with non-progressive disease. Change from baseline plasma VEGF-A within the first cycle of treatment was significantly correlated with clinical outcome by logistic regression analysis (P=0.027).

CONCLUSIONS

In patients with CRC treated with PTK/ZK, changes in plasma VEGF-A and bFGF demonstrate biological activity of PTK/ZK, may help to establish optimal dose and correlate with outcome.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-1 (FGF-1) is a well characterized <em>growth</em> <em>factor</em> among the <em>22</em> members of the FGF superfamily in humans. It binds to all the four known FGF receptors and regulates a plethora of functions including cell <em>growth</em>, proliferation, migration, differentiation, and survival in different cell types. FGF-1 is involved in the regulation of diverse physiological processes such as development, angiogenesis, wound healing, adipogenesis, and neurogenesis. Deregulation of FGF-1 signaling is not only implicated in tumorigenesis but also is associated with tumor invasion and metastasis. Given the biomedical significance of FGFs and the fact that individual FGFs have different roles in diverse physiological processes, the analysis of signaling pathways induced by the binding of specific FGFs to their cognate receptors demands more focused efforts. Currently, there are no resources in the public domain that facilitate the analysis of signaling pathways induced by individual FGFs in the FGF/FGFR signaling system. Towards this, we have developed a resource of signaling reactions triggered by FGF-1/FGFR system in various cell types/tissues. The pathway data and the reaction map are made available for download in different community standard data exchange formats through NetPath and NetSlim signaling pathway resources.

OBJECTIVE

Synovial sarcoma is a soft tissue sarcoma, the growth regulatory mechanisms of which are unknown. We investigated the involvement of fibroblast growth factor (FGF) signals in synovial sarcoma and evaluated the therapeutic effect of inhibiting the FGF signal.

METHODS

The expression of 22 FGF and 4 FGF receptor (FGFR) genes in 18 primary tumors and five cell lines of synovial sarcoma were analyzed by reverse transcription-PCR. Effects of recombinant FGF2, FGF8, and FGF18 for the activation of mitogen-activated protein kinase (MAPK) and the growth of synovial sarcoma cell lines were analyzed. Growth inhibitory effects of FGFR inhibitors on synovial sarcoma cell lines were investigated in vitro and in vivo.

RESULTS

Synovial sarcoma cell lines expressed multiple FGF genes especially those expressed in neural tissues, among which FGF8 showed growth stimulatory effects in all synovial sarcoma cell lines. FGF signals in synovial sarcoma induced the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38MAPK but not c-Jun NH2-terminal kinase. Disruption of the FGF signaling pathway in synovial sarcoma by specific inhibitors of FGFR caused cell cycle arrest leading to significant growth inhibition both in vitro and in vivo. Growth inhibition by the FGFR inhibitor was associated with a down-regulation of phosphorylated ERK1/2 but not p38MAPK, and an ERK kinase inhibitor also showed growth inhibitory effects for synovial sarcoma, indicating that the growth stimulatory effect of FGF was transmitted through the ERK1/2.

CONCLUSIONS

FGF signals have an important role in the growth of synovial sarcoma, and inhibitory molecules will be of potential use for molecular target therapy in synovial sarcoma.

OBJECTIVE

Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel.

METHODS

Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers.

RESULTS

A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified.

CONCLUSIONS

Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFβ (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) plays a vital role in the <em>growth</em> and differentiation of cardiac myocytes. It exists in high and low molecular weight forms because of the use of alternative initiation codons in the same mRNA. Higher levels of high molecular weight forms (molecular mass of <em>22</em> and 21.5 kD) are present in the rat heart during the neonatal stage, whereas the low molecular weight form (molecular mass of 18 kD) is predominant in the adult heart, suggesting different roles in development. Rat FGF-2 cDNAs that can preferentially express high or low molecular weight forms were introduced into neonatal rat ventricular myocyte cultures. Significant and comparable increases in overall cardiac myocyte DNA synthesis and proliferation were seen with <em>22</em>/21.5- and 18-kD FGF-2 expression. A significantly higher mitotic index was seen in the vicinity of cardiac myocytes overexpressing high or low molecular weight forms of FGF-2 compared with nonoverexpressing cells. This increase was inhibited in the presence of neutralizing antibodies to FGF-2, pointing to a proximity-dependent paracrine effect of <em>22</em>/21.5- and 18-kD FGF-2 on mitosis. By contrast, overexpression of high but not low molecular weight FGF-2 was associated with a significant increase in binucleation (approximately 36% of cardiac myocytes overexpressing <em>22</em>/21.5-kD FGF-2 were binucleated compared with 9% of cardiac myocytes overexpressing 18-kD FGF-2), which was not affected by neutralizing antibodies to FGF-2. These results suggest that <em>22</em>/21.5-kD FGF-2 and 18-kD FGF-2 have similar paracrine effects on proliferation but that <em>22</em>-21.5-kD FGF-2 exerts a distinct intracrine effect on binucleation.

TNF-like weak inducer of apoptosis (TWEAK) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-inducible 14 (Fn14) are a TNF superfamily ligand-receptor pair involved in many cellular processes including proliferation, migration, differentiation, inflammation, and angiogenesis. The Fn14 receptor is expressed at relatively low levels in normal tissues, but it is known to be dramatically elevated in a number of tumor types, including brain and breast tumors. Thus, it seems to be an excellent candidate for therapeutic intervention. We first analyzed Fn14 expression in human tumor cell lines. Fn14 was expressed in a variety of lines including breast, brain, bladder, skin, lung, ovarian, pancreatic, colon, prostate, and cervical cancer cell lines. We then developed an immunoconjugate containing a high-affinity anti-Fn14 monoclonal antibody (ITEM-4) conjugated to recombinant gelonin (rGel), a highly cytotoxic ribosome-inactivating N-glycosidase. Both ITEM-4 and the conjugate were found to bind to cells to an equivalent extent. Confocal microscopic analysis showed that ITEM4-rGel specifically and rapidly (within 2 hours) internalized into Fn14-positive T-24 bladder cancer cells but not into Fn14-deficient mouse embryonic <em>fibroblasts</em>. Cytotoxicity studies against <em>22</em> different tumor cell lines showed that ITEM4-rGel was highly cytotoxic to Fn14-expressing cells and was 8- to 8 × 10(4)-fold more potent than free rGel. ITEM4-rGel was found to kill cells by inducing apoptosis with high-mobility group box 1 protein release. Finally, ITEM4-rGel immunoconjugate administration promoted long-term tumor <em>growth</em> suppression in nude mice bearing T-24 human bladder cancer cell xenografts. Our data support the use of an antibody-drug conjugate approach to selectively target and inhibit the <em>growth</em> of Fn14-expressing tumors.

We and others have previously reported linkage to schizophrenia on chromosome 10q25-q26 but, to date, a susceptibility gene in the region has not been identified. We examined data from 3606 single-nucleotide polymorphisms (SNPs) mapping to 10q25-q26 that had been typed in a genome-wide association study (GWAS) of schizophrenia (479 UK cases/2937 controls). SNPs with P<0.01 (n=40) were genotyped in an additional 163 UK cases and those markers that remained nominally significant at P<0.01 (n=<em>22</em>) were genotyped in replication samples from Ireland, Germany and Bulgaria consisting of a total of 1664 cases with schizophrenia and 3541 controls. Only one SNP, rs17101921, was nominally significant after meta-analyses across the replication samples and this was genotyped in an additional six samples from the United States/Australia, Germany, China, Japan, Israel and Sweden (n=5142 cases/6561 controls). Across all replication samples, the allele at rs17101921 that was associated in the GWAS showed evidence for association independent of the original data (OR 1.17 (95% CI 1.06-1.29), P=0.0009). The SNP maps 85 kb from the nearest gene encoding <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) making this a potential susceptibility gene for schizophrenia.

Most normal somatic cells enter a state called replicative senescence after a certain number of divisions, characterized by irreversible <em>growth</em> arrest. Moreover, they express a pronounced inflammatory phenotype that could contribute to the aging process and the development of age-related pathologies. Among the molecules involved in the inflammatory response that are overexpressed in senescent cells and aged tissues is intercellular adhesion molecule-1 (ICAM-1). Furthermore, ICAM-1 is overexpressed in atherosclerosis, an age-related, chronic inflammatory disease. We have recently reported that the transcriptional activator p53 can trigger ICAM-1 expression in an nuclear <em>factor</em>-kappa B (NF-kappaB)-independent manner (Gorgoulis et al, EMBO J. 2003; <em>22</em>: 1567-1578). As p53 exhibits an increased transcriptional activity in senescent cells, we investigated whether p53 activation is responsible for the senescence-associated ICAM-1 overexpression. To this end, we used two model systems of cellular senescence: (a) human <em>fibroblasts</em> and (b) conditionally immortalized human vascular smooth muscle cells. Here, we present evidence from both cell systems to support a p53-mediated ICAM-1 overexpression in senescent cells that is independent of NF-kappaB. We also demonstrate in atherosclerotic lesions the presence of cells coexpressing activated p53, ICAM-1, and stained with the senescence-associated beta-galactosidase, a biomarker of replicative senescence. Collectively, our data suggest a direct functional link between p53 and ICAM-1 in senescence and age-related disorders.

BACKGROUND

Chronic inflammation is a well-known corollary of the aging process and is believed to significantly contribute to morbidity and mortality of many age-associated chronic diseases. However, the mechanisms that cause age-associated inflammatory changes are not well understood. Particularly, the contribution of cell stress responses to age-associated inflammation in 'non-inflammatory' cells remains poorly defined. The present cross-sectional study focused on differences in molecular signatures indicative of inflammatory states associated with biological aging of human <em>fibroblasts</em> from donors aged <em>22</em> to 92 years.

RESULTS

Gene expression profiling revealed elevated steady-state transcript levels consistent with a chronic inflammatory state in fibroblast cell-strains obtained from older donors. We also observed enhanced NF-kappaB DNA binding activity in a subset of strains, and the NF-kappaB profile correlated with mRNA expression levels characteristic of inflammatory processes, which include transcripts coding for cytokines, chemokines, components of the complement cascade and MHC molecules. This intrinsic low-grade inflammatory state, as it relates to aging, occurs in cultured cells irrespective of the presence of other cell types or the in vivo context.

CONCLUSIONS

Our results are consistent with the view that constitutive activation of inflammatory pathways is a phenomenon prevalent in aged fibroblasts. It is possibly part of a cellular survival process in response to compromised mitochondrial function. Importantly, the inflammatory gene expression signature described here is cell autonomous, i.e. occurs in the absence of prototypical immune or pro-inflammatory cells, growth factors, or other inflammatory mediators.

Serum FGF-23 regulation was studied in patients with hypoparathyroidism or pseudohypoparathyroidism treated with calcitriol. Serum FGF-23 levels changed in parallel in response to changes in serum 1,25-D, suggesting that FGF-23 may be regulated by 1,25-D. In addition, the phosphaturic effect of FGF-23 may be diminished in the absence of PTH action on the kidney.

BACKGROUND

Fibroblast growth factor (FGF)-23 is a recently described hormone that has been shown to be involved in the regulation of phosphate and vitamin D metabolism. The physiologic role of FGF-23 in mineral metabolism and how serum FGF-23 levels are regulated have yet to be elucidated. Three patients with mineral metabolism defects that allowed for the investigation of the regulation of FGF-23 were studied.

METHODS

Patient 1 had postsurgical hypoparathyroidism and Munchausen's syndrome and consumed a pharmacologic dose of calcitriol. Patient 2 had postsurgical hypoparathyroidism and fibrous dysplasia of bone. She was treated with increasing doses of calcitriol followed by synthetic PTH(1-34). Patient 3 had pseudohypoparathyroidism type 1B and tertiary hyperparathyroidism. She underwent parathyroidectomy, which was followed by the development of hungry bone syndrome and hypocalcemia, requiring treatment with calcitriol. Serum FGF-23 and serum and urine levels of mineral metabolites were measured in all three patients.

RESULTS

Patient 1 had an acute and marked increase in serum FGF-23 (70 to 670 RU/ml; normal range, 18-108 RU/ml) within 24 h in response to high-dose calcitriol administration. Patient 2 showed stepwise increases in serum FGF-23 from 117 to 824 RU/ml in response to increasing serum levels of 1alpha,25-dihydroxyvitamin D (1,25-D). Finally, before parathyroidectomy, while hypercalcemic, euphosphatemic, with low levels of 1,25-D (10 pg/ml; normal range, 22-67 pg/ml), and with very high serum PTH (863.7 pg/ml; normal range, 6.0-40.0 pg/ml), patient 3 had high serum FGF-23 levels (217 RU/ml). After surgery, while hypocalcemic, euphosphatemic, and with high serum levels of serum 1,25-D (140 pg/ml), FGF-23 levels were higher than preoperative levels (305 RU/ml). It seemed that the phosphaturic effect of FGF-23 was diminished in the absence of PTH or a PTH effect.

CONCLUSIONS

Serum FGF-23 may be regulated by serum 1,25-D, and its phosphaturic effect may be less in the absence of PTH.

A 26-kDa protein, originally described in human <em>fibroblasts</em> superinduced for interferon beta (IFN-beta) production, and termed IFN-beta 2 by other investigators, is induced by cycloheximide and by a <em>22</em>-kDa, interleukin 1 (IL-1)-related <em>factor</em>. Although the structure and sequence of the corresponding gene show nonhomology with the IFN-beta gene, the gene is identical to that of B-cell stimulatory <em>factor</em> 2, a human interleukin, and displays a very potent <em>growth</em> and differentiation <em>factor</em> activity for B lymphocytes. In this work we show that IL-1 beta and tumor necrosis <em>factor</em> (TNF) strongly induce the 26-kDa protein in FS-4 <em>fibroblasts</em> and in some transformed cell lines. Addition of cycloheximide to recombinant (r)IL-1 beta and rTNF further enhances the level of 26-kDa-protein mRNA. We determined the kinetics of induction and the amounts of rTNF and rIL-1 beta required for optimal induction of this mRNA in FS-4 cells and in HeLa H21 cells and found that rIL-1 beta is a more efficient inducer of 26-kDa protein mRNA than is TNF. By analyzing the inducibility of the 26-kDa protein gene by rTNF and rIL-1 beta in a series of transformed cell lines that differ in their sensitivity to the cytotoxic action of TNF, we report a direct correlation between the 26-kDa protein mRNA expression and the resistance of these cells to the cytotoxic effect of TNF.

The <em>22</em> members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family have been implicated in cell proliferation, differentiation, survival, and migration. They are required for both development and maintenance of vertebrates, demonstrating an exquisite pattern of affinities for both protein and proteoglycan receptors. Recent crystal structures have suggested two models for the complex between FGFs, FGF receptors (FGFRs) and the proteoglycan heparan sulphate that mediates signalling, and have provided insight into how FGFs show differing affinities for the range of FGFRs. However, the physiological relevance of the two different models has not been made clear. Here, we demonstrate that the two complexes can be prepared from the same protein components, confirming that neither complex is the product of misfolded protein samples. Analyses of the complexes with mass spectrometry and analytical ultracentrifugation show that the species observed are consistent with the crystal structures formed using the two preparation protocols. This analysis supports the contention that both of the crystal structures reflect the state of the molecules in solution. Mass spectrometry of the complexes suggests that the stoichiometry of the complexes is 2 FGF1:2 FGFR2:1 heparin, regardless of the method used to prepare the complexes. These observations suggest that the two proposed complex architectures may both have relevance to the formation of an in vivo signalling complex, with a combination of the two interactions contributing to the formation of a larger focal complex.

EGFRvIII is a mutant epidermal <em>growth</em> <em>factor</em> receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 x 10(6) members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of <em>22</em> nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse <em>fibroblasts</em> transfected with EGFRvIII and an IC50 of 7-10 ng/ml (110-160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37 degrees C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

OBJECTIVE

Circulating fibroblast growth factor 23 (FGF23) is associated with adverse cardiovascular outcomes in CKD. Whether FGF23 predicts cardiovascular mortality after kidney transplantation, independent of measures of mineral metabolism and cardiovascular risk factors, is unknown.

METHODS

The association between plasma C-terminal FGF23 and cardiovascular mortality was analyzed in a single-center prospective cohort of 593 stable kidney transplant recipients (mean age ± SD, 52 ± 12 years; 54% male; estimated GFR, 47 ± 16 ml/min per 1.73 m(2)), at a median of 6.1 (interquartile range, 2.7-11.7) years after transplantation. Multivariate Cox regression models were built, adjusting for measures of renal function and mineral metabolism; Framingham risk factors; the left ventricular wall strain markers midregional fragment of pro-A-type natriuretic peptide (MR-proANP) and N-terminal-pro brain natriuretic peptide (NT-proBNP); and copeptin, the stable C-terminal portion of the precursor of vasopressin.

RESULTS

In multivariate linear regression analysis, MR-proANP (β=0.20, P<0.001), NT-proBNP (β=0.18, P<0.001), and copeptin (β=0.26, P<0.001) were independently associated with FGF23. During follow-up for 7.0 (interquartile range, 6.2-7.5) years, 128 patients (22%) died, of whom 66 (11%) died due to cardiovascular disease; 54 (9%) had graft failure. FGF23 was associated with an higher risk of cardiovascular mortality in a fully adjusted multivariate Cox regression model (hazard ratio [HR], 1.88 [95% confidence interval (CI), 1.11 to 3.19]; P=0.02). FGF23 was also independently associated with all-cause mortality (full model HR, 1.86 [95% CI, 1.27 to 2.73]; P=0.001). Net reclassification improved for both cardiovascular mortality (HR, 0.07 [95% CI, 0.01 to 0.14]; P<0.05) and all-cause mortality (HR, 0.11 [95% CI, 0.05 to 0.18]; P<0.001).

CONCLUSIONS

Plasma FGF23 is independently associated with cardiovascular and all-cause mortality after kidney transplantation. The association remained significant after adjustment for measures of mineral metabolism and cardiovascular risk factors.

OBJECTIVE

Fibrotic sequelae remain the most important dose-limiting toxicity of radiation therapy to soft tissue. Functionally, this is reflected in loss of range of motion and muscle strength and the development of limb edema and pain. Tumor necrosis factor alpha and fibroblast growth factor 2 (FGF2), which are abnormally elevated in irradiated tissues, may mediate radiation fibrovascular injury.

METHODS

In an open label drug trial, we studied the effects of pentoxifylline (400 mg orally tid for 8 weeks) on 30 patients who displayed late, radiation-induced fibrosis at 1 to 29 years posttreatment (40 to 84 Gy). The primary outcome measurement was change in physical impairments thought to be secondary to radiation, including active and passive range of motion (AROM and PROM), muscle strength, limb edema, and pain. Plasma levels of cytokines (tumor necrosis factor alpha and FGF2) also were measured. Twenty-seven patients completed baseline and 8-week assessments, and 24 patients completed baseline, 8-week, and 16-week assessments.

RESULTS

After 8 weeks of pentoxifylline intervention, 20 of 23 patients with impaired AROM and 19 of 22 with impaired PROM improved; 11 of 19 patients with muscle weakness showed improved motor strength; five of seven patients with edema had decreased limb girth; and nine of 20 patients had decreased pain. Pretreatment FGF2 levels dropped from an average of 44.9 pg/mL to 24.0 pg/mL after 8 weeks of treatment.

CONCLUSIONS

Patients receiving pentoxifylline demonstrated improved AROM, PROM, and muscle strength and decreased limb edema and pain. Reversal of these delayed radiation effects was associated with a decrease in circulating FGF2.

The human <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family contains <em>22</em> proteins that regulate a plethora of physiological processes in both developing and adult organism. The mutations in the FGF genes were not known to play role in human disease until the year 2000, when mutations in FGF23 were found to cause hypophosphatemic rickets. Nine years later, seven FGFs have been associated with human disorders. These include FGF3 in Michel aplasia; FGF8 in cleft lip/palate and in hypogonadotropic hypogonadism; FGF9 in carcinoma; FGF10 in the lacrimal/salivary glands aplasia, and lacrimo-auriculo-dento-digital syndrome; FGF14 in spinocerebellar ataxia; FGF20 in Parkinson disease; and FGF23 in tumoral calcinosis and hypophosphatemic rickets. The heterogeneity in the functional consequences of FGF mutations, the modes of inheritance, pattern of involved tissues/organs, and effects in different developmental stages provide fascinating insights into the physiology of the FGF signaling system. We review the current knowledge about the molecular pathology of the FGF family.

BACKGROUND

Many fibroblast growth factor family proteins (FGFs) bind to the heparan sulfate/heparin (HP) subtypes of sulfated glycosaminoglycans (GAGs), and a few have recently been reported to also interact with chondroitin sulfate (CS), another sulfated GAG subtype.

METHODS

To gain additional insight into this interaction, we prepared all currently known FGFs (i.e., FGF1-FGF23) and assessed their affinity for HP, CS-B, CS-D and CS-E. In addition, midkine, hepatocyte growth factor and pleiotrophin were studied as other known HP-binding proteins.

RESULTS

We found that members of the FGF19 subfamily (i.e., FGF15, 19, 21 and 23) had little or no affinity for HP; all of the other secretable growth factors tested had strong affinities for HP, as was indicated by the finding that their elution from HP-Sepharose columns required 1.0-1.5 M NaCl. We also found that FGF3, 6, 8 and 22 had strong affinities for CS-E, while FGF5 had a moderate affinity for CS-D. The interactions between FGFs and GAGs thus appear to be more diverse than previously understood.

CONCLUSIONS

This is noteworthy, as the differential interactions of these growth factors with GAGs may be key determinants of their specific biological activities.

BACKGROUND

Angiogenesis is a target in the treatment of ovarian cancer. Nintedanib, an oral triple angiokinase inhibitor of VEGF receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, has shown activity in phase 2 trials in this setting. We investigated the combination of nintedanib with standard carboplatin and paclitaxel chemotherapy in patients with newly diagnosed advanced ovarian cancer.

METHODS

In this double-blind phase 3 trial, chemotherapy-naive patients (aged 18 years or older) with International Federation of Gynecology and Obstetrics (FIGO) IIB-IV ovarian cancer and upfront debulking surgery were stratified by postoperative resection status, FIGO stage, and planned carboplatin dose. Patients were randomly assigned (2:1) via an interactive voice or web-based response system to receive six cycles of carboplatin (AUC 5 mg/mL per min or 6 mg/mL per min) and paclitaxel (175 mg/m(2)) in addition to either 200 mg of nintedanib (nintedanib group) or placebo (placebo group) twice daily on days 2-21 of every 3-week cycle for up to 120 weeks. Patients, investigators, and independent radiological reviewers were masked to treatment allocation. The primary endpoint was investigator-assessed progression-free survival analysed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT01015118.

RESULTS

Between Dec 9, 2009, and July 27, 2011, 1503 patients were screened and 1366 randomly assigned by nine study groups in 22 countries: 911 to the nintedanib group and 455 to the placebo group. 486 (53%) of 911 patients in the nintedanib group experienced disease progression or death compared with 266 (58%) of 455 in the placebo group. Median progression-free survival was significantly longer in the nintedanib group than in the placebo group (17·2 months [95% CI 16·6-19·9] vs 16·6 months [13·9-19·1]; hazard ratio 0·84 [95% CI 0·72-0·98]; p=0·024). The most common adverse events were gastrointestinal (diarrhoea: nintedanib group 191 [21%] of 902 grade 3 and three [<1%] grade 4 vs placebo group nine [2%] of 450 grade 3 only) and haematological (neutropenia: nintedanib group 180 [20%] grade 3 and 200 (22%) grade 4 vs placebo group 90 [20%] grade 3 and 72 [16%] grade 4; thrombocytopenia: 105 [12%] and 55 [6%] vs 21 [5%] and eight [2%]; anaemia: 108 [12%] and 13 [1%] vs 26 [6%] and five [1%]). Serious adverse events were reported in 376 (42%) of 902 patients in the nintedanib group and 155 (34%) of 450 in the placebo group. 29 (3%) of 902 patients in the nintedanib group experienced serious adverse events associated with death compared with 16 (4%) of 450 in the placebo group, including 12 (1%) in the nintedanib group and six (1%) in the placebo group with a malignant neoplasm progression classified as an adverse event by the investigator. Drug-related adverse events leading to death occurred in three patients in the nintedanib group (one without diagnosis of cause; one due to non-drug-related sepsis associated with drug-related diarrhoea and renal failure; and one due to peritonitis) and in one patient in the placebo group (cause unknown).

CONCLUSIONS

Nintedanib in combination with carboplatin and paclitaxel is an active first-line treatment that significantly increases progression-free survival for women with advanced ovarian cancer, but is associated with more gastrointestinal adverse events. Future studies should focus on improving patient selection and optimisation of tolerability.

BACKGROUND

Boehringer Ingelheim.

The p53 tumor suppressor protein induces transient <em>growth</em> arrest or apoptosis in response to genotoxic stress and mediates the irreversible <em>growth</em> arrest of cellular senescence. We present evidence here that p53 also contributes to the reversible, <em>growth</em> <em>factor</em>-dependent arrest of quiescence (G(0)). Microinjection of expression vectors encoding either MDM2 or a pRb-binding mutant of SV40 T antigen, both of which abrogate p53 function, stimulated quiescent normal human <em>fibroblasts</em> to initiate DNA synthesis and were 40-70% as effective as wild-type T antigen. Electrophoretic mobility shift and p53 transactivation assays showed that p53 activity was higher in quiescent and senescent cells compared with proliferating cells. As proliferating cells entered G(0) after <em>growth</em> <em>factor</em> withdrawal, the p53 mRNA level increased, followed by transient accumulation of the protein. Shortly thereafter, the expression (mRNA and protein) of p21, a p53 target gene and effector of cell cycle arrest, increased. Finally, stable expression of the HPV16 E6 oncogene or dominant negative p53 peptide, GSE-<em>22</em>, both of which inhibit p53 function, delayed entry into quiescence following <em>growth</em> <em>factor</em> withdrawal. Our data indicate that p53 is activated during both quiescence and senescence. They further suggest that p53 activity contributes, albeit not exclusively, to the quiescent <em>growth</em> arrest.

Hepatitis B virus infection is still a major global health problem, despite decades of research. Interleukin (IL)-<em>22</em> induces acute phase reactants and chemokines, favors anti-microbial defence and protects tissues from damage. IL-<em>22</em> is important in chronic skin inflammation, but its role in chronic hepatitis B (CHB) is unclear. This study explores the association between intra-hepatic IL-<em>22</em> expression, its relevant associated cytokines and the severity of liver inflammation/fibrosis in CHB patients. IL-<em>22</em>, IL-17, IL-10, IL-6, non-ELR-CXC chemokines (CXCL-9, CXCL-10, CXCL-11), <em>fibroblast</em> <em>growth</em> <em>factors</em> and Kupffer cell (KC) numbers were measured in patients with CHB (n=65), acute hepatitis B (AHB; n=4), chronic hepatitis C (CHC; n=14) and non-viral hepatitis (n=23), using immunohistochemistry. Expression of IL-<em>22</em>, IL-17, IL-10, IL-6, non-ELR-CXC chemokines and number of KCs in liver tissues were substantially higher in AHB patients than others. In CHB patients, the expression of IL-<em>22</em>, IL-6, CXCL-9 and CXCL-10 were significantly higher with alanine aminotransferase (ALT) levels ≤ twice the upper limit of normal (ULN), compared with those with ALT levels>>twice the ULN, whereas IL-10 and IL-17 showed a reverse pattern. IL-<em>22</em> was inversely (P<0.01), but IL-17 was positively (P<0.05), correlated with the histological activity index) in these patients, and a significant negative correlation between the fibrosis stage and IL-<em>22</em> or non-ELR-CXC chemokines was observed. Furthermore, immunofluorescent labeling demonstrated a close spatial association of IL-<em>22</em>, CXCL-9, -10 or -11 in the CHB liver. We speculate that IL-<em>22</em> and non-ELR-CXC chemokines synergistically may provide protection in liver inflammation/fibrosis during CHB infection.