BACKGROUND

Mitomycin C (MMC), an antitumoral antibiotic, has been described inhibiting the proliferation of different cell types in vitro. Since irradiation is commonly used to stop the cell growth of adherent cells in several experimental models, we aimed to define the optimal dose and incubation time of MMC capable of inhibiting the growth of murine fibroblasts, used as an adherent feeder layer in long-term hematopoietic culture assay.

METHODS

M2 10B4 (both parental and engineered to produce human IL-3 and G-CSF) and Sl/Sl (engineered to produce human IL-3 and steel factor) murine fibroblast cell-lines, frequently used in LTC-IC assay, were incubated with increasing doses of MMC for either a short (3 h) or a long (16 h) period. The efficiency of MMC in stopping the cell growth was evaluated for 5 days following MMC removal. The effects of MMC treatment on human hematopoietic cells were studied using both LTC-IC and limiting dilution (CAFC) assays.

RESULTS

The growth of M2 10B4 cells was stopped at 3 and 16 h in the presence of 20 microg/mL and 2 microg/mL of MMC, respectively while Sl/Sl fibroblasts required a lower dose of drug (2 and 0.2 microg/mL, respectively). No significant difference was found between the number of LTC-IC or CAFC obtained from cultures containing irradiated or MMC-treated feeder cells.

CONCLUSIONS

MMC inhibits the growth of murine fibroblasts used as adherent feeder cells in long-term culture assays, without interfering with the subsequent growth of co-cultured hemopoietic cells. Different cell types might present a different sensitivity to MMC and therefore a dose-response curve to MMC has to be obtained for each cell type of interest.

OBJECTIVE

To examine the roles of oxygen, basic fibroblast growth factor (bFGF), and photoreceptor debris in the photoreceptor dystrophy of the Royal College of Surgeons (RCS) rat.

METHODS

Pups were exposed during the critical period of their development (postnatal day [P] 16-24) and for some days thereafter to hypoxia and hyperoxia. The effects of these exposures on photoreceptor death, debris accumulation in the subretinal space, and the expression of bFGF protein and mRNA by surviving cells were studied.

RESULTS

During the critical period hyperoxia slowed photoreceptor death in a dose-related fashion and decreased bFGF protein levels, whereas hypoxia accelerated death and increased bFGF levels. At the edges of the retina, where photoreceptors survive longest in normoxia, hypoxia had little effect on either photoreceptor death or bFGF protein levels. Oxygen-induced modulation of rates of death could not be related to the accumulation of debris in the subretinal space. After P27, the relationship between oxygen and photoreceptor death changed markedly, hyperoxia no longer delaying and hypoxia no longer accelerating death.

CONCLUSIONS

The death of RCS rat photoreceptors in the period P16 to P27 is precipitated by hypoxia that may result from the accumulation of photoreceptor debris in the subretinal space. This debris, the result of the phagocytotic failure of the retinal pigment epithelium in this strain, lies in the normal pathway of oxygen diffusing to the photoreceptors from the choriocapillaris. During this period the retina responds to hypoxia by increasing expression of a potentially protective protein (bFGF), but hypoxia-induced damage overwhelms any protection provided by this or other mechanisms. Later stages of the dystrophy may not be hypoxia-induced.

BACKGROUND

Neovascularization induced by basic fibroblast growth factor (basic FGF) or FGF-like cytokines is thought to play a substantial role in the pathogenesis of human immunodeficiency virus (HIV)-associated Kaposi's sarcoma. Pentosan polysulfate has been shown to inhibit basic FGF and FGF-like dependent tumor growth both in vitro and in vivo. Moreover, it has been found to inhibit the growth of Kaposi's sarcoma-derived spindle cells in vitro. These observations suggested that pentosan polysulfate might be worth exploring as a potential agent for the treatment of Kaposi's sarcoma.

OBJECTIVE

The purpose of this phase 1 clinical trial was to determine the maximum tolerated dose of pentosan polysulfate in patients with HIV-associated Kaposi's sarcoma and whether or not this compound had activity against this neoplasm.

METHODS

Sixteen HIV-seropositive patients with Kaposi's sarcoma received pentosan polysulfate via continuous venous infusion for 3-6 weeks and then received a subcutaneous dose three times per week. Three different doses of pentosan polysulfate were administered: 2 mg/kg per day by infusion followed by 2 mg/kg per dose given subcutaneously (six patients), 3 mg/kg per day by infusion followed by 3 mg/kg per dose given subcutaneously (five patients), and 4 mg/kg per day by infusion followed by 4 mg/kg per dose given subcutaneously (five patients). Five of the 16 patients in the study also received injections of 1 mg of pentosan polysulfate into two different lesions two times a week for 3 weeks, followed by intralesional therapy once weekly. After receiving pentosan polysulfate for 6 weeks, patients were administered 100 mg zidovudine (AZT) orally every 4 hours in conjunction with pentosan polysulfate.

RESULTS

The maximally tolerated dose of pentosan polysulfate given by continuous venous infusion was found to be 3 mg/kg per day. No patient had an objective clinical antitumor response to either systemic or intralesional pentosan polysulfate administration; however, three patients had stable Kaposi's sarcoma for 3-27 weeks. No statistically significant effect on CD4 cells or serum HIV p24 antigen was noted during pentosan polysulfate administration. Dose-limiting toxic effects were characterized by anticoagulation and thrombocytopenia and were reversible.

CONCLUSIONS

Pentosan polysulfate was well tolerated in this patient population. However, no objective tumor response or evidence of anti-HIV activity was noted; therefore, no claim of activity can be made in this trial.

CONCLUSIONS

Continued investigation into the use of angiogenesis inhibitors with improved activity and toxicity profiles or different mechanisms of action is warranted.

Cell therapy using multipotent mesenchymal stromal cells (MSCs) is of high interest in various indications. As the pleiotropic effects mediated by MSCs rely mostly on their unique secretory profile, long-term persistence of ex-vivo-expanded cells in the recipient may not always be desirable. Irradiation is a routine procedure in transfusion medicine to prevent long-term persistence of nucleated cells and could therefore also be applied to MSCs. We have exposed human bone-marrow-derived MSCs to 30 or 60 Gy of γ-irradiation and assessed cell proliferation, clonogenicity, differentiation, cytokine levels in media supernatants, surface receptor profile, as well as expression of proto-oncogenes/cell cycle markers, self-renewal/stemness markers, and DNA damage/irradiation markers. Irradiated MSCs show a significant decrease in proliferation and colony-forming unit-<em>fibroblasts</em>. However, a subpopulation of surviving cells is able to differentiate, but is unable to form colonies after irradiation. Irradiated MSCs showed stable expression of CD73 and CD90 and absence of CD3, CD34, and CD45 during a <em>16</em>-week follow-up period. We found increased vascular endothelial <em>growth</em> <em>factor</em> (VEGF) levels and a decrease of platelet-derived <em>growth</em> <em>factor</em> (PDGF)-AA and PDGF-AB/BB in culture media of nonirradiated cells. Irradiated MSCs showed an inverse pattern, that is, no increase of VEGF, and less consumption of PDGF-AA and PDGF-AB/BB. Interestingly, interleukin-6 (IL-6) levels increased during culture regardless of irradiation. Cells with lower sensitivity toward γ-irradiation showed positive β-galactosidase activity 10 days after irradiation. Gene expression of both irradiated and nonirradiated MSCs 13-<em>16</em> weeks after irradiation with 60 Gy predominantly followed the same pattern; cell cycle regulators CDKN1A (p21) and CDKN2A (p<em>16</em>) were upregulated, indicating cell cycle arrest, whereas classical proto-oncogenes, respectively, and self-renewal/stemness markers MYC, TP53 (p53), and KLF4 were downregulated. In addition, DNA damage/irradiation markers ATM, ATR, BRCA1, CHEK1, CHEK2, MDC1, and TP53BP1 also mostly showed the same pattern of gene expression as high-dose γ-irradiation. In conclusion, we demonstrated the existence of an MSC subpopulation with remarkable resistance to high-dose γ-irradiation. Cells surviving irradiation retained their trilineage differentiation capacity and surface marker profile but changed their cytokine secretion profile and became prematurely senescent.

BACKGROUND

Evidence suggests that keloid scar formation may be mediated, in part, by deranged growth factor activity, including that of transforming growth factor (TGF) beta1. Tamoxifen citrate has shown promise in the treatment of keloids.

OBJECTIVE

To evaluate the effect of tamoxifen on autocrine growth factor expression in keloid and fetal dermal fibroblasts, which exhibit scar-free healing.

METHODS

Serum-free cell lines of keloid and fetal dermal fibroblasts were established. Cell cultures were exposed to different concentrations of tamoxifen solution (8 and 12 or 16 micromol/L). Cell counts were performed and supernatants collected at 24, 48, and 96 hours. Cell-free supernatants were quantitatively assayed for TGF-beta1 expression.

RESULTS

Keloid fibroblasts show increased per-cell TGF-beta1 production compared with fetal fibroblasts. Tamoxifen appeared to decrease per-cell TGF-beta1 production at each of the time points evaluated.

CONCLUSIONS

Keloids likely arise due to locally insufficient or excessive concentrations of specific growth factors. The higher level of TGF-beta1 produced by keloid cells compared with fetal fibroblasts could be related to the aberrant wound healing seen with keloids. The addition of tamoxifen may lead to improved wound healing in keloids by decreasing the expression of TGF-beta1.

Giant cell tumors (GCTs) of the bone are osteolytic neoplasms with variable degrees of aggressiveness. The aim of this study was the molecular characterization of GCT tissue. We established gene expression profiles and discovered a number of genes that have not been described in GCTs before. RNA was prepared from 7 cryopreserved GCTs (primary tumors n = 5, relapses n = 2) and was hybridized to Affymetrix HG U133A microarrays. Paraffin-embedded samples were used for immunohistochemical validation (primary tumors n = <em>16</em>, relapses n = 6). Gene ontology revealed that the majority of genes, found to be differentially expressed between primary and recurrent GCTs, were associated with receptor tyrosine kinase activity. We selected one upregulated gene (Claudin 7) and four downregulated genes (CD52, Ephrin A1 receptor, autocrine motility <em>factor</em> receptor [AMFR] and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 [FGFR3] for further analysis using immunohistochemistry. Immunohistochemical analysis of CD52, AMFR, and Ephrin A1 receptor revealed expression profiles concordant with the microarray data, also with regard to differences between primary tumors and relapses.

BACKGROUND

Tubulointerstitial fibrosis (fibrosis), a histologic feature associated with a failing kidney allograft, is diagnosed using the invasive allograft biopsy. A noninvasive diagnostic test for fibrosis may help improve allograft outcome.

METHODS

We obtained 114 urine specimens from 114 renal allograft recipients: 48 from 48 recipients with fibrosis in their biopsy results and 66 from 66 recipients with normal biopsy results. Levels of messenger RNAs (mRNAs) in urinary cells were measured using kinetic, quantitative polymerase chain reaction assays, and the levels were related to allograft diagnosis. A discovery set of 76 recipients (32 with allograft fibrosis and 44 with normal biopsy results) was used to develop a diagnostic signature, and an independent validation set of 38 recipients (<em>16</em> with allograft fibrosis and 22 with normal biopsy results) was used to validate the signature.

RESULTS

In the discovery set, urinary cell levels of the following mRNAs were significantly associated with the presence of allograft fibrosis: vimentin (P<0.0001, logistic regression model), hepatocyte growth factor (P<0.0001), α-smooth muscle actin (P<0.0001), fibronectin 1 (P<0.0001), perforin (P=0.0002), plasminogen activator inhibitor 1 (P=0.0002), transforming growth factor β1 (P=0.0004), tissue inhibitor of metalloproteinase 1 (P=0.0009), granzyme B (P=0.0009), fibroblast-specific protein 1 (P=0.006), CD103 (P=0.02), and collagen 1A1 (P=0.04). A four-gene model composed of the levels of mRNA for vimentin, NKCC2, and E-cadherin and of 18S ribosomal RNA provided the most accurate, parsimonious diagnostic model of allograft fibrosis with a sensitivity of 93.8% and a specificity of 84.1% (P<0.0001). In the independent validation set, this same model predicted the presence of allograft fibrosis with a sensitivity of 77.3% and a specificity of 87.5% (P<0.0001).

CONCLUSIONS

Measurement of mRNAs in urinary cells may offer a noninvasive means of diagnosing fibrosis in human renal allografts.

Enamel matrix derivative, obtained from developing porcine teeth, is composed mainly of amelogenin proteins and used topically in periodontal surgery for advanced periodontitis to regenerate lost connective tissues. The primary objective of this study was to investigate the effects of enamel matrix derivative on skin wound healing. Secondly, in vitro effects of enamel matrix derivative on dermal <em>fibroblasts</em> and microvascular endothelial cells were examined. Full-thickness, circular 2-cm skin wounds in white <em>16</em>-week-old rabbits were treated thrice weekly with enamel matrix derivative (30 mg/ml) in the vehicle propylene glycol alginate or with vehicle alone. Enamel matrix derivative treatment increased the amount of granulation tissue and accelerated time to complete epithelialization by 3 days (p < 0.001) compared to vehicle treatment. In cultured <em>fibroblasts</em>, vascular endothelial <em>growth</em> <em>factor</em> levels in conditioned media were increased more than fivefold (p < 0.001) with enamel matrix derivative treatment (0.1mg/ml) over control, measured by specific enzyme-linked immunosorbent assay. Enamel matrix derivative also increased release of matrix metalloproteinase-2 more than threefold from <em>fibroblasts</em> (p < 0.001) and from endothelial cells (p < 0.001). Thus, enamel matrix derivative significantly accelerated wound closure in rabbits, possibly by increasing levels of <em>growth</em> <em>factors</em> and proteinases important for granulation tissue formation and remodeling.

Collagen overproduction characteristic for dilated cardiomyopathy (DCM) is coregulated by endothelin (ET)-1, transforming <em>growth</em> <em>factor</em> (TGF)-beta1, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and matrix metalloproteases (MMPs). Whether these molecules affect grafts transplanted to heart failure patients is unknown. In 67 idiopathic DCM patients, 31 patients with ischemic cardiomyopathy (ICM) and <em>16</em> controls, the myocardial bFGF, TGF-beta1, pro-collagen (PrCol) type 1 (PrCol1-alpha1, -alpha2) and MMP expressions were examined using real-time RT-PCR or Western blotting. mRNA expression was measured in grafts for 1 year. TGF-beta1/bFGF stimulation or gene silencing was used to examine their effect on collagen synthesis in cardiac tissue cultures. TGF-beta1 and PrCol1 were upregulated in DCM only, while bFGF was upregulated in both groups versus controls. TGF-beta1 downregulated MMP-1 and upregulated collagen 1, whereas bFGF upregulated MMP-13 in DCM tissue. Post-transplant PrCol1-alpha1, -alpha2 and ET-1 mRNA increased over time in grafts of DCM patients only, while other <em>factors</em> returned to control baseline levels in DCM and ICM. These data indicate that cardiac transplantation corrects the dysregulated TGF/bFGF/MMP-1/MMP-13, but not the excess collagen and ET-1 synthesis in cardiac grafts transplanted to DCM patients. ET-1 might be a major pathologic trigger for graft fibrosis in DCM.

A number of angiogenic <em>growth</em> <em>factors</em> have been shown to accelerate wound healing. Previous work has demonstrated that topical application of epidermal <em>growth</em> <em>factor</em> is effective in healing chronic tympanic membrane perforations in an animal model. Theoretically, <em>fibroblast</em> <em>growth</em> <em>factor</em> may result in a superior healed membrane through preferential stimulation of the <em>fibroblasts</em> within the middle layer of the tympanic membrane. To test this hypothesis, the effects of exogenously applied <em>fibroblast</em> <em>growth</em> <em>factor</em> on the chronically perforated tympanic membrane were evaluated. A buffered solution of <em>fibroblast</em> <em>growth</em> <em>factor</em> (25 microliters of <em>fibroblast</em> <em>growth</em> <em>factor</em>, 0.2 mg/ml) was administered to a Gelfoam pledget placed over chronic tympanic membrane perforations in chinchillas. Control ears were treated with Gelfoam and the buffer solution only. Complete closure of the tympanic membrane perforation was observed in 81% (13 of <em>16</em>) of the <em>fibroblast</em> <em>growth</em> <em>factor</em>-treated ears, but in only 41% (7 of 17) of the controls (p = 0.05). Heading took place gradually, requiring an average of 4 weeks for the <em>fibroblast</em> <em>growth</em> <em>factor</em>-treated and 6.5 weeks for the control ears that healed. The relatively high healing rate for the control group does not imply that the pretreatment perforations were not chronic, rather there appears to be some efficacy to the control protocol of repeated applications of Gelfoam and buffer. A histologic analysis of the <em>fibroblast</em> <em>growth</em> <em>factor</em>-healed eardrums immediately after closure demonstrated hypertrophy of the squamous and fibrous layers of the tympanic membrane. Over time, the eardrum thinned to reach proportions similar to those of the normal tympanic membrane, including the presence of a substantial middle fibrous layer. A screening ototoxicity study revealed no structural damage to the organ of Corti after <em>growth</em> <em>factor</em> treatment. To assess the potential for systemic toxicity, blood and peripheral tissues were analyzed for radioactivity at time points during a 48-hour period after application of 25 microliters of 125I-<em>fibroblast</em> <em>growth</em> <em>factor</em> to the perforated tympanic membrane. More than 78% of the radioactivity remained at the application site. Given the tiny original dosage, the small fraction absorbed systemically is minuscule and highly unlikely to induce adverse effects in light of published toxicity data. On the basis of these promising safety and efficacy data in the chinchilla model, clinical trials of <em>fibroblast</em> <em>growth</em> <em>factor</em> in repair of chronic tympanic membrane perforations in human beings are being initiated.

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. <em>Fibroblast</em> <em>growth</em> <em>factor</em> 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/<em>16</em>/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.

Here we report the successful generation and long-term expansion of SOX9-expressing CD271(+)PDGFRα(+)CD73(+) chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1(-)CD271(hi)PDGFRα(lo)CD73(-) neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/transforming <em>growth</em> <em>factor</em>β (TGFβ) inhibitor and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). When "primed" with TGFβ, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. The ectomesenchymal cells were expandable without loss of chondrogenic potential for at least <em>16</em> passages. They maintained normal karyotype for at least 10 passages and expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B), cranial mesenchyme (ALX1/3/4), and chondroprogenitors (SOX9, COL2A1) of neural crest origin (SOX8/9, NGFR, NES). Ectomesenchyme is a source of many craniofacial bone and cartilage structures. The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.

The E5 oncoprotein of the human papillomavirus type <em>16</em> (HPV<em>16</em> E5) cooperates in cervical carcinogenesis and in epithelial transformation deregulating cell <em>growth</em>, survival and differentiation through the modulation of <em>growth</em> <em>factor</em> receptors. Among the epithelial receptor tyrosine kinases, the keratinocyte <em>growth</em> <em>factor</em> receptor/<em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2b (KGFR/FGFR2b) is a major paracrine mediator of epithelial homeostasis and appears to have an unique and unusual role in epithelial tissues, exerting a tumor-suppressive function in vitro and in vivo. With the aim to better elucidate the molecular events involved in the pathological activity of <em>16</em>E5, we investigated if the viral protein would be able to affect the KGFR expression, signaling and turnover by interference with its degradative and recycling endocytic pathways. Quantitative reverse transcriptase-PCR and biochemical approaches on human keratinocytes transfected with <em>16</em>E5-HA showed that E5 protein is able to induce KGFR down-modulation at both transcript and protein levels. Immunofluorescence microscopy in double-transfected cells expressing both E5 and KGFR revealed that the viral protein alters the receptor endocytic trafficking and triggers its endosomal sorting to the indirect juxtanuclear recycling pathway. The shift from lysosomal degradation to recycling at the plasma membrane correlates with a reduced phosphorylation of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor substrate-2α tyrosine 196, the major docking site for Grb2-Cbl complexes responsible for receptor ubiquitination and degradation. 5'-Bromo-deoxyuridine incorporation assay demonstrated that expression of <em>16</em>E5 induces a decrease in the <em>growth</em> response to the receptor ligands as a consequence of KGFR down-modulation, suggesting that <em>16</em>E5 might have a role on HPV infection in perturbing the KGFR-mediated physiological behavior of confluent keratinocytes committed to differentiation.

Stimulation of <em>growth</em> of endothelial cells from preexisting blood vessels, i.e., angiogenesis, is one of the essential elements necessary to create a permissive environment in which a tumor can grow. During angiogenesis, the matrix metalloproteinase (MMP) family of tissue enzymes contributes to normal (embriogenesis or wound repair) and pathologic tissue remodeling (chronic inflammation and tumor genesis). The proposed pathogenic roles of MMPs in cancer are tissue breakdown and remodeling during invasive tumor <em>growth</em> and tumor angiogenesis. Tissue inhibitors of metalloproteinases (TIMPs) form a complex with MMPs, which in turn inhibits active MMPs. Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) are unique among mediators of angiogenesis with synergistic effect, and both can also be secreted by thyroid cancer cells. The goal of the study was to evaluate the plasma blood concentration of VEGF, bFGF, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, TIMP-1, and TIMP-2 in patients with cancer and in normal subjects. Twenty-two patients with thyroid cancers (papillary cancer, 11; partly papillary and partly follicular cancer, 3; anaplastic cancer, 5; medullary cancer, 3) and <em>16</em> healthy subjects (controls) were included in the study. VEGF, bFGF MMPs, and TIMPs were evaluated by enzyme-linked immunosorbent assay (ELISA). In patients with thyroid cancer, normal VEGF concentrations (74.29 +/- 13.38 vs. 84.85 +/- 21.71 pg/mL; p>> 0.05) and increased bFGF (29.52 +/- 4.99 vs. 6.05 +/- 1.43 pg/mL; p < 0.001), MMP-2 (605.95 +/- 81.83 vs. 148.75 +/- 43.53 ng/mL; p < 0.001), TIMP-2 (114.19 +/- 6.62 vs. 60.75 +/- 9.18 ng/mL; p < 0.001), as well as lower MMP-1 (0.70 +/- 0.42 vs. 3.87 +/- 0.53; p < 0.001) levels have been noted. Increased plasma levels of MMP-3 and MMP-9 were also found in patients with medullary carcinoma. In conclusion, predominance of MMP-2 over TIMP-2 and TIMP-1 over MMP-1 as well as increased concentration of bFGF in peripheral blood are common features in patients with thyroid cancer.

The ability of oocytes to resume meiosis, become fertilized, and generate viable pregnancies is controlled during folliculogenesis by several endocrine and paracrine <em>factors</em>. The aim of this work is to determine whether <em>fibroblast</em> <em>growth</em> <em>factor</em> 10 (FGF10) is an oocyte competent <em>factor</em>. Transcripts for each of the four FGF receptor types (FGFR) were present in cumulus and oocytes after their extraction from the follicles. FGFR1 transcripts predominated in cumulus cells whereas FGFR2 was most abundant in oocytes. Exposing the cumulus-oocyte complexes to FGF10 during in vitro maturation did not affect cleavage rates, but increases (P<0.05) in the percentage of embryos at the 8-<em>16</em>-cell stage on day 3 and at the blastocyst stage on day 7, which were evident in FGF10-supplemented oocytes. The progression of oocytes through meiosis and cumulus expansion was increased (P<0.05) by FGF10. The importance of the endogenous sources of FGFs was examined by adding anti-FGF10 IgG during oocyte maturation. Blocking endogenous FGF10 activity decreased (P<0.05) the percentage of oocytes developing into blastocysts and limited (P<0.05) cumulus expansion. Expression profiles of putative cumulus and oocyte competency markers were examined for their involvement in FGF10-mediated responses. FGF10 influenced the expression of CTSB and SPRY2 in cumulus cells and BMP15 in oocytes. In summary, this work provides new insight into the importance of FGFRs and locally derived FGF10 during oocyte maturation in cattle. Its subsequent impact on in vitro embryo development implicates it as a noteworthy oocyte competent <em>factor</em>.

The purpose of these experiments was to identify <em>growth</em> <em>factors</em> produced during the formation of a peritoneal wound in relation to tumour cell seeding and stimulated <em>growth</em> in granulation tissue. Gelfoam(c) gelatin sponge was implanted in the mesenteric fan of nude mice to initiate the granulation process. Human HT29 colon carcinoma cells were inoculated intraperitoneally at various times after sponge implantation and tumour <em>growth</em> in granulation tissue was determined. RNA isolated from granulation tissue was used for polymerase chain reaction analysis of the expression of specific <em>growth</em> <em>factors</em> and receptors [vascular endothelial <em>growth</em> <em>factor</em> (VEGF), transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) and lysophosphatic acid (LPA)], and for microarray analysis of differentially expressed genes in early vs. late granulation tissue. Inflammatory cells infiltrated the sponge within 1 day, followed by <em>fibroblasts</em> and the formation of an extracellular matrix. Tumour cell inoculation at 8 h to 3 days after sponge implantation resulted in extensive tumour formation in all cases. Inoculation at 10-28 days resulted in focal tumour <em>growth</em> in only <em>16</em>-33% of the sponges. Low amounts of VEGF, TGF-beta(1-3), TGF-beta RIII and LPA receptors 1,2 were detected in early granulation tissue, with increased expression from day 10. Microarray analysis identified additional differentially expressed genes that may stimulate tumour take and <em>growth</em> in early granulation tissue.

BACKGROUND

Vitamin D transcriptional effects were linked to tumor growth control, however, the hormone targets were determined in cell cultures exposed to supra physiological concentrations of 1,25(OH)(2)D(3) (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)(2)D(3) in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)(2)D(3) at concentrations that can be attained in vivo.

METHODS

Tumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)(2)D(3) 0.5nM or 100nM. Gene expression was analyzed by microarray (SAM paired analysis, FDR≤0.1) or RT-qPCR (p≤0.05, Friedman/Wilcoxon test). Expression of candidate genes was then evaluated in mammary epithelial/breast cancer lineages and cancer associated fibroblasts (CAFs), exposed or not to 1,25(OH)(2)D(3) 0.5nM, using RT-qPCR, western blot or immunocytochemistry.

RESULTS

1,25(OH)(2)D(3) 0.5nM or 100nM effects were evaluated in five tumor samples by microarray and seven and 136 genes, respectively, were up-regulated. There was an enrichment of genes containing transcription factor binding sites for the vitamin D receptor (VDR) in samples exposed to 1,25(OH)(2)D(3) near physiological concentration. Genes up-modulated by both 1,25(OH)(2)D(3) concentrations were CYP24A1, DPP4, CA2, EFTUD1, TKTL1, KCNK3. Expression of candidate genes was subsequently evaluated in another 16 samples by RT-qPCR and up-regulation of CYP24A1, DPP4 and CA2 by 1,25(OH)(2)D(3) was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)(2)D(3) 0.5nM were restricted to the epithelial or stromal compartments, gene expression was examined in HB4A, C5.4, SKBR3, MDA-MB231, MCF-7 lineages and CAFs, using RT-qPCR. In epithelial cells, there was a clear induction of CYP24A1, CA2, CD14 and IL1RL1. In fibroblasts, in addition to CYP24A1 induction, there was a trend towards up-regulation of CA2, IL1RL1, and DPP4. A higher protein expression of CD14 in epithelial cells and CA2 and DPP4 in CAFs exposed to 1,25(OH)(2)D(3) 0.5nM was detected.

CONCLUSIONS

In breast cancer specimens a short period of 1,25(OH)(2)D(3) exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)(2)D(3) effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment.

FGFRL1 is a novel member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) family. To investigate its expression during mammalian embryonic development, we have used the mouse system. Expression of Fgfrl1 is very low in mouse embryos of day 6 but steadily increases until birth. As demonstrated by in situ hybridization of <em>16</em>-day-old embryos, the Fgfrl1 mRNA occurs in cartilaginous structures such as the primordia of bones and the permanent cartilage of the trachea, the ribs and the nose. In addition, some muscle types, including the muscles of the tongue and the diaphragm, express Fgfrl1 at relatively high level. In contrast, the heart and the skeletal muscles of the limbs, as well as many other organs (brain, lung, liver, kidney, gut) express Fgfrl1 only at basal level. It is conceivable that Fgfrl1 interacts with other Fgfrs, which are expressed in cartilage and muscle, to modulate FGF signaling.

The localization of <em>fibroblast</em> <em>growth</em> <em>factor</em>-1 (FGF-1) and FGF-2 in human oral squamous cell carcinoma (SCC) was examined by immunohistochemical techniques using anti-FGF-1 and anti-FGF-2 monoclonal antibodies. Immunofluorescence staining of two oral SCC cell lines revealed that <em>growing</em> cancer cells were intensely positive for both FGF-1 and FGF-2, but confluent cells showed a faint immunostaining. In addition, two molecular mass species of FGF-1 (<em>16</em> and 18 kDa) and one of FGF-2 (18 kDa) were identified by Western blot in cell extracts derived from <em>growing</em> SCC cells, but not from confluent SCC cells. The <em>growing</em> cell extracts significantly stimulated the proliferation of human umbilical vein endothelial cells. Immunoperoxidase staining of 13 oral SCC cases showed that both well-differentiated and poorly-differentiated cancer cells were positive for FGF-1 and FGF-2 with high frequency and intensity as compared to normal oral epithelium. These results indicate that SCC cells express high levels of endogenous FGF-1 and FGF-2, and suggest that these <em>growth</em> <em>factors</em> may contribute to cancer cell <em>growth</em>.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are secreted polypeptide <em>growth</em> <em>factors</em>. The FGF signaling system plays crucial roles in multiple developmental processes in vertebrates. The human FGF family comprises 22 members. Although <em>16</em> zebrafish fgfs have been reported, the zebrafish fgf family has not been well elucidated. We have identified 11 additional zebrafish fgfs by conducting a homology-based search in the zebrafish genome and cDNA databases. The zebrafish fgf family now comprises at least 27 members. By conducting phylogenetic and gene location analyses, we examined relationships of zebrafish fgf genes with human FGF genes. All the zebrafish orthologs of human FGFs except for FGF9 have been identified. Zebrafish fgf9 might have been lost from the genome during evolution. In addition, six paralogs of zebrafish fgf genes have been identified. The phylogenetic analysis suggests that the zebrafish fgf gene family can be divided into seven subfamilies. The zebrafish fgf subfamilies are essentially consistent with the human FGF subfamilies, although some include potential paralogs. As the zebrafish system has proved useful for studying gene functions and genetic diseases, the present findings will be useful for elucidation of roles of FGFs in zebrafish and humans.

The effects of various cytokines on survival and differentiation of an astrocyte progenitor cell line (AP-<em>16</em>) were examined. Epidermal <em>growth</em> <em>factor</em> (EGF) deprivation caused death of AP-<em>16</em> cells by apoptosis. Transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) prevented the apoptosis occurring in the absence of EGF. Leukemia inhibitory <em>factor</em> (LIF) and ciliary neurotrophic <em>factor</em> (CNTF) induced glial fibrillary acidic protein (GFAP) and decreased A2B5 antigen in AP-<em>16</em> cells, indicating that these cytokines induced AP-<em>16</em> cells to differentiate into astrocytes.

A cDNA clone encoding rat basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was used as a hybridization probe to determine the level of bFGF mRNA during rat brain development as well as in different adult rat brain regions. In the rat brain, a 3.7kb bFGF mRNA was detected together with lower levels of two minor bFGF mRNA species of 1.8kb and 1.5kb, respectively. The 3.7kb bFGF mRNA was detected in the rat brain already at embryonic day <em>16</em>, the earliest time point tested. The embryonic brain contained 1.5 to 2 times higher levels of the 3.7kb bFGF mRNA than the adult brain. The amount of the 3.7kb bFGF mRNA in the adult rat brain was approximately 50 times higher than the level of beta-nerve <em>growth</em> <em>factor</em> mRNA in the rat brain. bFGF mRNA was found in all 12 brain regions tested in the adult rat brain with the highest level in colliculi, cerebral cortex, thalamus, and ol<em>factor</em>y bulb. The lowest levels were found in pons and medulla oblongata. All three bFGF mRNA species showed the same regional distribution in the brain. In contrast to nerve <em>growth</em> <em>factor</em> mRNA, the level of bFGF mRNA in the neonatal hippocampus was slightly decreased 10 days after a cholinergic denervation by transection of the fimbria-fornix.

The distribution of the beta-subunit of platelet-derived <em>growth</em> <em>factor</em> receptor (PDGFR-beta) was assessed by a sensitive immunoalkaline phosphatase technique using the monoclonal antibody PR7212. Frozen tissue sections of several nonneoplastic human tissues were stained along with 42 soft tissue sarcomas, <em>16</em> benign soft tissue proliferations, and 7 epithelial tumors. In all nonneoplastic tissue, there was intense labeling of cell processes of perivascular <em>fibroblasts</em> or pericytes in and about the walls of muscular blood vessels and of <em>fibroblast</em> cell processes around some glandular and ductal epithelia. No PDGFR-beta was found in the endothelial cells of muscular arteries and veins, but cells of uncertain identity within some capillaries were immunoreactive and the possibility that endothelial cells of some small capillaries express PDGFR-beta could not be excluded. In kidney there was strong labeling of glomerular mesangial cells and interstitial <em>fibroblasts</em>. Some histological types of soft tissue sarcomas were uniformly and strongly labeled with anti-PDGFR-beta, but other types were infrequently labeled or unreactive. The order of decreasing frequency and strength of labeling of the various types of benign and malignant soft tissue proliferations was as follows: benign fibromatosis and neurofibroma greater than malignant fibrous histiocytoma greater than liposarcoma greater than leiomyosarcoma greater than rhabdomyosarcoma. No tumor cell labeling was detected in epithelioid, synovial or clear cell sarcomas, leiomyomas, or carcinomas, but there was usually strong labeling of <em>fibroblast</em> and/or pericyte cell processes within tumor, especially around blood vessels. We conclude that PDGFR-beta is strongly expressed by vascular and stromal tissues of most tumors and normal organs and by tumor cells of several types of soft tissue tumors and proliferations, most notably those of <em>fibroblast</em>ic origin.

The mechanisms involved in normal cranial suture development and fusion as well as in the pathophysiology of craniosyostosis are not well understood. The purpose of this study was to investigate the expression of several cytokines--transforming <em>growth</em> <em>factor</em>-beta-1 (TGF-beta1), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and interleukin-6 (IL-6)--during cranial suture fusion. TGF-beta exists in three mammalian isoforms that are abundant in bone and stimulate calvarial bone formation when delivered locally. Other bone <em>growth</em> <em>factors</em> including basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and the interleukins regulate bone <em>growth</em> and are mitogenic for bone marrow cells and osteoblasts. The involvement of <em>growth</em> <em>factors</em> in the pathophysiology of craniosynostosis is supported by recent genetics data linking <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor mutations to syndromal craniosynostoses. In this experimental study, in situ hybridization was used to localize and quantify the gene expression of TGF-beta1, bFGF, and IL-6 during cranial suture fusion. In the Sprague-Dawley rat, the posterior frontal cranial suture normally undergoes fusion between 12 and 22 days of age, whereas all other cranial sutures remain patent. All in situ analyses of fusing posterior frontal sutures were compared with the patent, control, sagittal sutures. Posterior frontal and sagittal sutures, together with underlying dura, were harvested from rats at 8, 12, <em>16</em>, and 35 days of postnatal life to analyze posterior frontal suture activity before, during, and after fusion. In situ hybridization was performed on frozen sections of these specimens using DNA probes specific for TGF-beta1, bFGF, and IL-6 mRNA. A negative control probe to IL-6 in the sense orientation was also used to validate the procedure. Cells expressing cytokine-specific mRNA were quantified (in cells positive per 10(-1) mm2) and analyzed using the unpaired Student's t test. Areas encompassing the fibrous suture and the surrounding bone plates were analyzed for cellular mRNA activity. IL-6 mRNA expression showed a minimal rise in the posterior frontal suture at days 12 and <em>16</em>, with an average count of 10 and 6 cells per 10(-1) mm2, respectively. The sagittal suture remained negative for IL-6 mRNA at all time points. TGF-beta1 and bFGF analyses were most interesting, showing marked increases specifically in the posterior frontal suture during the time of active suture fusion. On postnatal day 8, a 1.5-fold increase in posterior frontal suture TGF-beta1 mRNA was found compared with sagittal sutures (p = 0.1890, unpaired Student's t test). This difference was increased 26-fold on day 12 in posterior frontal suture TGF-beta1 expression (p = 0.0005). By day 35, posterior frontal suture TGF-beta1 mRNA had nearly returned to prefusion levels, whereas TGF-beta1 mRNA levels in the sagittal suture remained low. A similar upregulation of bFGF mRNA, peaking at day 12, was observed in posterior frontal but not sagittal sutures (p = 0.0003). Furthermore, both TGF-beta1 and bFGF mRNA samples with intact dura showed an intense dural mRNA expression in the time preceding and during active posterior frontal suture fusion but not in sagittal tissues. Our data demonstrate that TGF-beta1 and bFGF mRNA are up-regulated in cranial suture fusion, possibly signaling in a paracrine fashion from dura to suture. TGF-beta1 and bFGF gene expression were dramatically increased both in and surrounding the actively fusing suture and followed the direction of fusion from endocranial to epicranial. These experimental data on bone <em>growth</em> <em>factors</em> support the recent human genetics data linking <em>growth</em> <em>factor</em>/<em>fibroblast</em> <em>growth</em> <em>factor</em> receptor deletions to syndromal craniosynostoses. The ultimate aim of these studies is to understand the underlying mechanisms regulating suture <em>growth</em>, development, and fusion so surgeons may one day manipulate the biology of premature cranial suture fusion.