This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by <em>16</em> d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived <em>growth</em> <em>factors</em>, a deleted variant of hepatocyte <em>growth</em> <em>factor</em> (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.

During wound repair, <em>fibroblasts</em> accumulate in the injured area until any defect is filled with stratified layers of cells and matrix. Such fibroplasia also occurs in many fibrotic disorders. Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), a promotor of granulation tissue in vivo and extracellular matrix production in vitro, is expressed during the active fibroplasia of wound healing and fibroproliferative diseases. Under usual tissue culture conditions, normal <em>fibroblasts</em> grow to confluence and then cease proliferation. In this study, culture conditions with TGF-beta 1 have been delineated that promote human <em>fibroblasts</em> to grow in stratified layers mimicking in vivo fibroplasia. When medium supplemented with serum, ascorbate, proline, and TGF-beta was added thrice weekly to normal human dermal <em>fibroblasts</em>, the cells proliferated and stratified up to <em>16</em> cell layers thick within the culture dish, producing a tissue-like fibroplasia. TGF-beta stimulated both DNA synthesis as measured by 3H-thymidine uptake and cell proliferation as measured by a Hoechst dye DNA assay in these postconfluent cultures. The stratification was dependent on fibronectin assembly, as demonstrated by anti-fibronectin antibodies which inhibited both basal and TGF-beta-stimulated cell proliferation and stratification. Suppression of collagen matrix assembly in cell layers with beta-amino-proprionitrile (BAPN) did not inhibit basal or TGF-beta stimulated in vitro fibroplasia. BAPN did not interfere with fibronectin matrix assembly as judged by immunofluorescence microscopy. Thus, in concert with serum <em>factors</em>, TGF-beta stimulates postconfluent, fibronectin matrix-dependent, <em>fibroblast</em> <em>growth</em> creating a fibroplasia-like tissue in vitro.

Conversion of <em>fibroblasts</em> into myo<em>fibroblasts</em> as mediated by transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) is the most prominent stromal reaction to a number of epithelial lesions including breast cancer. To identify genes which are regulated during this process, the mRNA profiles from primary breast <em>fibroblasts</em> treated with or without TGF-beta1 were analyzed by differential display. Ninety-five differentially expressed transcripts were PCR-cloned and sequenced, and 28 clones were selected for verification in a hybridization array. By use of gene-specific sequence tags, nine differentially expressed genes were identified. One of the clones, identified as CLIC4, a member of the CLIC family of chloride channels, was up-regulated more than <em>16</em> times in myo<em>fibroblasts</em> and was therefore chosen for further analysis. Using RT-PCR, comparison with CLIC1, CLIC2, CLIC3, and CLIC5 demonstrated that CLIC4 was unique by being up-regulated by TGF-beta1 in myo<em>fibroblasts</em>. Immunohistochemistry showed a hitherto unknown, distinctive pattern of CLIC4 expression in breast stroma. Whereas normal breast <em>fibroblasts</em> were devoid of CLIC4 protein expression, myo<em>fibroblasts</em> of breast carcinomas were strongly CLIC4-positive. The functional significance of CLIC4 was analyzed in MEF/3T3 <em>fibroblasts</em> by conditional expression using the tetracycline-repressive gene regulation system. In a migration assay, we found that CLIC4 inhibited cell motility by 27%. These results suggest that CLIC4 is differentially regulated in <em>fibroblasts</em> and that its expression contributes to a collective stationary myofibroblast phenotype.

The adenovirus E1A, SV40 large T and papillomavirus E7 proteins immortalize primary cells by virtue of their ability to bind the retinoblastoma gene product (pRB) and other cellular proteins, including cyclin A and the prRB-related protein, p107. It has been demonstrated that these viral oncogene products will prevent the inhibition of positive <em>growth</em> regulators by pRB, one of them being the E2F transcription <em>factor</em>. Here we show that the interactions of pRB and cyclin A with E2F are present also in normal keratinocytes and in primary human <em>fibroblasts</em>. In human keratinocytes immortalized by human papillomavirus <em>16</em> (HPV-<em>16</em>), expressing high levels of HPV-<em>16</em> E7 protein, complexes between E2F and pRB are disrupted. In this cell line, as well as in HeLa cells which express HPV-18 E7, complexes containing E2F and cyclin A are maintained, indicating that this interaction is not sensitive to the viral oncoprotein and that cyclin A can associate with E2F independently of pRB. In vitro binding experiments suggest that the E7 gene product is able to preferentially abolish the interaction of pRB with E2F, leaving the cyclin A complexes intact. Our findings suggest that E7-dependent immortalization of human cells is associated with modifications of E2F multiprotein complexes.

Insulin-like <em>growth</em> <em>factor</em>-binding protein-3 (IGFBP-3) is a multifunctional protein that induces apoptosis utilizing both insulin-like <em>growth</em> <em>factor</em> receptor (IGF)-dependent and -independent mechanisms. We investigated the effects of IGFBP-3 on tumor <em>growth</em> and angiogenesis utilizing a human CaP xenograft model in severe-combined immunodeficiency mice. A <em>16</em>-day course of IGFBP-3 injections reduced tumor size and increased apoptosis and also led to a reduction in the number of vessels stained with CD31. In vitro, IGFBP-3 inhibited both vascular endothelial <em>growth</em> <em>factor</em>- and IGF-stimulated human umbilical vein endothelial cells vascular network formation in a matrigel assay. This action is primarily IGF independent as shown by studies utilizing the non-IGFBP-binding IGF-1 analog Long-R3. Additionally, we used a <em>fibroblast</em> <em>growth</em> <em>factor</em>-enriched matrigel-plug assay and chick allantoic membrane assays to show that IGFBP-3 has potent antiangiogenic actions in vivo. Finally, overexpression of IGFBP-3 or the non-IGF-binding GGG-IGFBP-3 mutant in Zebrafish embryos confirmed that both IGFBP-3 and the non-IGF-binding mutant inhibited vessel formation in vivo, indicating that the antiangiogenic effect of IGFBP-3 is an IGF-independent phenomenon. Together, these studies provide the first evidence that IGFBP-3 has direct, IGF-independent inhibitory effects on angiogenesis providing an additional mechanism by which it exerts its tumor suppressive effects and further supporting its development for clinical use in the therapy of patients with prostate cancer.

BACKGROUND

The clinical management of leprosy Type 1 (T1R) and Type 2 (T2R) reactions pose challenges mainly because they can cause severe nerve injury and disability. No laboratory test or marker is available for the diagnosis or prognosis of leprosy reactions. This study simultaneously screened plasma factors to identify circulating biomarkers associated with leprosy T1R and T2R among patients recruited in Goiania, Central Brazil.

METHODS

A nested case-control study evaluated T1R (n = 10) and TR2 (n = 10) compared to leprosy patients without reactions (n = 29), matched by sex and age-group (+/- 5 years) and histopathological classification. Multiplex bead based technique provided profiles of 27 plasma factors including 16 pro inflammatory cytokines: tumor necrosis factor-alpha (TNF-alpha), Interferon-gamma (IFN-gamma), interleukin (IL)- IL12p70, IL2, IL17, IL1 beta, IL6, IL15, IL5, IL8, macrophage inflammatory protein (MIP)-1 alpha (MIP1alpha), 1 beta (MIP1beta), regulated upon activation normal T-cell expressed and secreted (RANTES), monocyte chemoattractrant protein 1 (MCP1), CC-chemokine 11 (CCL11/Eotaxin), CXC-chemokine 10 (CXCL10/IP10); 4 anti inflammatory interleukins: IL4, IL10, IL13, IL1Ralpha and 7 growth factors: IL7, IL9, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), platelet-derived growth factor BB (PDGF BB), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF).

RESULTS

Elevations of plasma CXCL10 (P = 0.004) and IL6 (p = 0.013) were observed in T1R patients compared to controls without reaction. IL6 (p = 0.05), IL7 (p = 0.039), and PDGF-BB (p = 0.041) were elevated in T2R. RANTES and GMCSF were excluded due to values above and below detection limit respectively in all samples.

CONCLUSIONS

Potential biomarkers of T1R identified were CXCL10 and IL6 whereas IL7, PDGF-BB and IL6, may be laboratory markers of TR2. Additional studies on these biomarkers may help understand the immunopathologic mechanisms of leprosy reactions and indicate their usefulness for the diagnosis and for the clinical management of these events.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) has been implicated in the progression of human tumours via both autocrine and paracrine (angiogenic) activities. We investigated the expression of FGF-2 and FGF receptors (FGFR-1 to -4) in NSCLC cell lines (N = <em>16</em>), NSCLC surgical specimens (N = 21) and 2 control cell lines. Our data show that almost all NSCLC cells produce elevated levels of FGF-2 and FGFR in vitro and in vivo. FGF-2 expression did correlate with a short doubling time as well as with potent anchorage-independent <em>growth</em> of NSCLC cell lines. In contrast with control cells, NSCLC cells did not secrete considerable amounts of FGF-2 into the extracellular space. Expression levels of FGFR-1 and -2 in NSCLC cell lines correlated with FGF-2 production. FGFR were located at the plasma membranes in some low FGF-2-producing NSCLC and control cell lines. These cells were sensitive to the proliferative effect of recombinant FGF-2 (rFGF-2). In NSCLC cell lines with an enhanced FGF-2 production, representing the majority studied, FGFR localisation was predominantly intracellular. These cells were insensitive to both the proliferative effect of rFGF-2 and <em>growth</em> inhibition by FGF-2-neutralising antibodies. In contrast, several agents antagonised FGF-2 intracellularly impaired <em>growth</em> of almost all NSCLC cell lines. Our data suggest a role of FGF-2 and FGFR in the <em>growth</em> stimulation of NSCLC cells possibly via an intracrine mechanism.

Increased mortality of adult chronic hemodialysis (HD) patients is associated with coronary calcifications (CC), increased serum phosphorus (P), use of calcium (Ca)-containing P-binders, and vitamin D deficiency. Serum concentration of <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF 23) is markedly elevated in adults receiving dialysis and is independently associated with increased mortality. Although coronary calcifications have been described in pediatric and adult HD patients, no significant association between serum FGF 23 and CC has been reported. In our study, 5/<em>16</em> patients had CC. Patients with CC were older, had longer dialysis vintage and higher serum P. Serum Ca, total PTH, elemental Ca intake, and calcitriol doses were not different for CC patients. Serum FGF 23 levels were markedly elevated in all patients (mean 4,024, range 874-8,253), but significantly higher in patients with CC (4,247 ± 10,35 vs 2,427 ± 11,92, p = 0.01) and positively correlated with Agatston calcification score (r = 0.69, p = 0.003) and serum P (r = 0.49, p = 0.05). Using multivariate analysis, serum FGF 23 and serum P remained the most significant <em>factors</em> associated with Agatston score. This study confirms the occurrence of CC in pediatric HD patients and is the first to show a significant association between CC and elevated serum FGF 23 in children.

OBJECTIVE

To determine the role of the microvascular endothelium in the regulation of regenerating liver mass after partial hepatectomy.

BACKGROUND

Angiogenesis is critical for both pathologic and physiologic processes. The ability of certain tissues, such as the liver, kidney, and spleen, to regenerate after injury is poorly understood. The liver will regenerate to its normal mass within 8 days of surgical excision. Because the authors have previously shown that the endothelial cell regulates tumor mass, we hypothesized that normal adult organ mass is also controlled by the endothelial cell.

METHODS

Two-thirds partial hepatectomy was performed in 7- to 8-week-old C57 BL/6 mice, followed by systemic treatment with either the angiogenesis stimulator basic fibroblast growth factor (bFGF) (1 microg/g/d intraperitoneal) or the angiogenesis inhibitor TNP-470 (30 mg/kg/qod subcutaneous). Groups of three mice were then euthanized on postoperative days 2, 4, 6, and 8, and the livers were weighed and analyzed by immunohistochemistry.

RESULTS

bFGF accelerated hepatic regeneration by 42%, 19%, 16%, and 16% on postoperative days 2, 4, 6, and 8, respectively. TNP-470 inhibited hepatic regeneration by 46%, 74%, 67%, and 64% on postoperative days 2, 4, 6, and 8, respectively. Immunohistochemistry revealed that bFGF and TNP-470 primarily affected the endothelial compartment. Specifically, bFGF increased endothelial proliferation and decreased endothelial apoptosis. TNP-470, in contrast, inhibited endothelial cell proliferation. The cessation of the regenerative process correlated with a decrease in endothelial proliferation and an increase in endothelial apoptosis.

CONCLUSIONS

The systemic administration of angiogenesis agents modulates the regeneration of hepatic mass primarily by affecting endothelial cell proliferation or apoptosis. Endothelial cell apoptosis is associated with the cessation of the regenerative process in control mice. These results suggest that the endothelial cell is one of the key mediators of regenerating adult tissue mass in this partial hepatectomy model.

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenemia that is amplified by insulin in the presence of resistance to insulin's action to stimulate glucose uptake in muscle and fat. To explore the mechanisms for this paradox, we examined the metabolic and mitogenic actions of insulin and insulin-like <em>growth</em> <em>factor</em> I (IGF-I) in cultured skin <em>fibroblasts</em> from PCOS (n = <em>16</em>) and control (n = 11) women. There were no significant decreases in the number or affinity of insulin- or IGF-I-binding sites in PCOS compared to control <em>fibroblasts</em>. Basal rates were similar, but there were significant decreases in insulin-stimulated (control, 51.8 +/- 7.0; PCOS, 29.5 +/- 2.9 nmol/10(6) cells x 2 h at 1,000,000 pmol/L; P < 0.005) and IGF-I-stimulated (control, 48.9 +/- 6.7; PCOS, 33.0 +/- 3.2 PCOS nmol/10(6) cells x 2 h at 100,000 pmol/L IGF-I; P < 0.05) glucose incorporation into glycogen in PCOS <em>fibroblasts</em>, a metabolic action of insulin. Stimulation of thymidine incorporation, a mitogenic action of insulin, was similar in PCOS and control <em>fibroblasts</em> in response to both insulin and IGF-I. There were also no significant differences in insulin- or IGF-I-stimulated insulin receptor substrate-1-associated phosphatidylinositol-3-kinase activity in PCOS compared to control <em>fibroblast</em> cells. We conclude that 1) there is a selective defect in insulin action in PCOS <em>fibroblasts</em> that affects metabolic, but not mitogenic, signaling pathways; 2) there is a similar defect in IGF-I action, suggesting that insulin and IGF-I stimulate glycogen synthesis by the same postreceptor pathways; and 3) insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activation by insulin and IGF-I is similar to the control value, suggesting that the metabolic signaling defect is in another pathway or downstream of this signaling step in PCOS <em>fibroblasts</em>.

Heparanase activity correlates with the metastatic potential of lymphoma, melanoma and mammary adenocarcinoma cell lines. We investigated the ability of various modified species of heparin and size-homogeneous oligosaccharides derived from depolymerized heparin to inhibit (1) heparanase-mediated degradation of heparan sulfate in a naturally produced subendothelial extracellular matrix (ECM), and (2) lung colonization of B<em>16</em>-BL6 melanoma cells in C57BL mice. Inhibition of heparanase was best achieved by heparin species containing <em>16</em> or more sugar units and having sulfate groups at both the N and O positions. Low-sulfate oligosaccharides were less effective heparanase inhibitors than medium- and high-sulfate fractions of the same-size saccharide. While O-desulfation abolished the heparanase-inhibiting effect of heparin, O-sulfated, N-substituted (e.g. N-acetyl or N-hexanoyl) species of heparin retained high inhibitory activity. Potent inhibitors of heparanase activity were also efficient inhibitors of tumor invasion and lung colonization. Heparin fractions with high and low anticoagulant activity expressed similar high antiheparanase and antimetastatic activities. Structural requirement for the inhibition of melanoma cell heparanase and lung colonization by species of heparin were different from those identified for (1) release of ECM-bound basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (b-FGF) and (2) stimulation of b-FGF receptor binding and mitogenic activity. These results indicate that various nonanticoagulant species of heparin and other polyanionic molecules differing in size, sulfation and substituted groups can be designed to elicit specific effects resulting in the inhibition of cell invasion in tumor metastasis and autoimmunity, or stimulation of neovascularization and wound healing.

The anaplastic lymphoma kinase (ALK), tyrosine kinase oncogene is implicated in a wide variety of cancers. In this study we used conditional onco-ALK (NPM-ALK and TPM3-ALK) mouse MEF cell lines (ALK+ <em>fibroblasts</em>) and transgenic models (ALK+ B-lymphoma) to investigate the involvement and regulation of angiogenesis in ALK tumor development. First, we observed that ALK expression leads to downregulation of miR-<em>16</em> and increased Vascular Endothelial <em>Growth</em> <em>Factor</em> (VEGF) levels. Second, we found that modification of miR-<em>16</em> levels in TPM3-ALK MEF cells greatly affected VEGF levels. Third, we demonstrated that miR-<em>16</em> directly interacts with VEGF mRNA at the 3'-untranslated region and that the regulation of VEGF by miR-<em>16</em> occurs at the translational level. Fourth, we showed that expression of both the ALK oncogene and hypoxia-induced <em>factor</em> 1α (HIF1α) is a prerequisite for miR-<em>16</em> downregulation. Fifth, in vivo, miR-<em>16</em> gain resulted in reduced angiogenesis and tumor <em>growth</em>. Finally, we highlighted an inverse correlation between the levels of miR-<em>16</em> and VEGF in human NPM-ALK+ Anaplastic Large Cell Lymphomas (ALCL). Altogether, our results demonstrate, for the first time, the involvement of angiogenesis in ALK+ ALCL and strongly suggest an important role for hypoxia-miR-<em>16</em> in regulating VEGF translation.

OBJECTIVE

In the first months after successful kidney transplantation, hypophosphatemia and renal phosphorus wasting are common and related to inappropriately high parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF-23) levels. Little is known about the long-term natural history of renal phosphorus homeostasis in renal transplant recipients.

METHODS

We prospectively followed parameters of mineral metabolism (including full-length PTH and FGF-23) in 50 renal transplant recipients at the time of transplantation (Tx), at month 3 (M3) and at month 12 (M12). Transplant recipients were (1:1) matched for estimated GFR with chronic kidney disease (CKD) patients.

RESULTS

FGF-23 levels (Tx: 2816 [641 to 10665] versus M3: 73 [43 to 111] versus M12: 56 [34 to 78] ng/L, median [interquartile range]) and fractional phosphorus excretion (FE(phos); M3: 45 +/- 19% versus M12: 37 +/- 13%) significantly declined over time after renal transplantation. Levels 1 yr after transplantation were similar to those in CKD patients (FGF-23: 47 [34 to 77] ng/L; FE(phos) 35 +/- 16%). Calcium (9.1 +/- 0.5 versus 8.9 +/- 0.3 mg/dl) and PTH (27.2 [17.0 to 46.0] versus 17.5 [11.7 to 24.4] ng/L) levels were significantly higher, whereas phosphorus (3.0 +/- 0.6 versus 3.3 +/- 0.6 mg/dl) levels were significantly lower 1 yr after renal transplantation as compared with CKD patients.

CONCLUSIONS

Data indicate that hyperphosphatoninism and renal phosphorus wasting regress by 1 yr after successful renal transplantation.

A <em>16</em>-kilodalton N-terminal fragment of PRL (<em>16</em>K PRL) is formed by enzymatic cleavage of intact 23-kilodalton PRL (23K PRL) in the pituitary gland and in target tissues for PRL. <em>16</em>K PRL inhibits the <em>growth</em> of capillary endothelial cells, while intact PRL was inactive suggesting that <em>16</em>K PRL acted via a receptor other than the PRL receptor. To analyze whether this inhibitory effect could be mediated through an specific <em>16</em>K PRL receptor, we characterized the binding of <em>16</em>K PRL to membrane preparations of bovine brain capillary endothelial (BBE) cells. <em>16</em>K PRL was generated by the proteolysis of rat 23K PRL with a particulate fraction from rat mammary gland homogenates and purified by gel filtration. The specific binding of [125I]<em>16</em>K PRL to BBE cell membranes was high affinity (Kd = 9.9 nM), saturable (Bmax = 4.8 pmol/mg protein), and reversible. In competition studies for [125I]<em>16</em>K PRL binding, <em>16</em>K PRL was most potent, while little displacement was observed with high concentrations of 23K PRLs, <em>growth</em> hormones, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Blockade of reformation of disulfide bonds by carbamidomethylation of <em>16</em>K PRL, a procedure which increases the biological activity of the molecule, increased its binding affinity (Kd = 0.9 nM). Cross-linking experiments identified a 52,000 and a 32,000 mol wt protein as the major <em>16</em>K PRL binding species. These data demonstrate the presence of specific, high affinity, saturable binding sites for <em>16</em>K PRL on BBE cell membranes and support biological findings that <em>16</em>K PRL inhibits capillary endothelial cell proliferation, through a novel, high affinity receptor.

Chondrocyte is a unique cell type in articular cartilage tissue and is essential for cartilage formation and functionality. It arises from mesenchymal stem cells (MSCs) and is regulated by a series of cytokine and transcription <em>factor</em> interactions, including the transforming <em>growth</em> <em>factor</em>-beta super family, <em>fibroblast</em> <em>growth</em> <em>factors</em>, and insulin-like <em>growth</em> <em>factor</em>-1. To understand the biomechanisms of the chondrocyte differentiation process, various cellular model systems have been employed, such as primary chondrocyte culture, clonal normal cell lines (HCS-2/8, Ch-1, ATDC5, CFK-2, and RCJ3.1C5.18), and transformed clonal cell lines (T/C-28a2, T/C-28a4, C-28/I2, tsT/AC62, and HPV-<em>16</em> E6/E7). Additionally, cell culture methods, including conventional monolayer culture, three-dimensional scaffold culture, bioreactor culture, pellet culture, and organ culture, have been established to create stable environments for the expansion, phenotypic maintenance, and subsequent biological study of chondrocytes for clinical application. Knowledge gained through these study systems has allowed for the use of chondrocytes in orthopedics for the treatment of cartilage injury and epiphyseal <em>growth</em> plate defects using tissue-engineering approaches. Furthermore, the potential of chondrocyte implantation for facial reconstruction, the treatment of long segmental tracheal defects, and urinary incontinence and vesicoureteral reflux are being investigated. This review summarizes the present study of chondrocyte biology and the potential uses of this cell in orthopedics and other disciplines.

BACKGROUND

Biliary tract cancers (BTCs) typically present at an advanced stage, and systemic chemotherapy is often of limited benefit.

METHODS

Hybrid capture-based comprehensive genomic profiling (CGP) was performed for 412 intrahepatic cholangiocarcinomas (IHCCAs), 57 extrahepatic cholangiocarcinomas (EHCCAs), and 85 gallbladder carcinomas (GBCAs). The mutational profile was correlated with the clinical outcome of standard and experimental therapies for 321 patients. Clinical variables, detected mutations, and administered therapies were correlated with overall survival (OS) in a Cox regression model.

RESULTS

The most frequent genetic aberrations (GAs) observed were tumor protein 53 (TP53; 27%), cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B; 27%), KRAS (22%), AT-rich interactive domain-containing protein 1A (ARID1A; 18%), and isocitrate dehydrogenase 1 (IDH1; <em>16</em>%) in IHCCA; KRAS (42%), TP53 (40%), CDKN2A/B (17%), and SMAD4 (21%) in EHCCA; and TP53 (59%), CDKN2A/B (19%), ARID1A (13%), and ERBB2 (<em>16</em>%) in GBCA. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR; 11%) and IDH mutations (20%) were mostly limited to IHCCA but appeared to be mutually exclusive. In the IHCCA group, TP53 and KRAS mutations were associated significantly with poor OS, whereas FGFR2 mutations were associated with improved OS (P = .001), a younger age at onset, and female sex. IDH1/2 mutations were not prognostic. In a multivariate model, the effects of TP53 and FGFR GAs remained significant (P < .05). Patients with FGFR GAs had superior OS with FGFR-targeted therapy versus standard regimens (P = .006). Targeted therapy in IHCCA was associated with a numerical OS improvement (P = .07).

CONCLUSIONS

This is the largest clinically annotated data set of BTC cases with CGP and indicates the potential of CGP for improving outcomes. CGP should be strongly considered in the management of BTC patients. Cancer 20<em>16</em>;122:3838-3847. © 20<em>16</em> American Cancer Society.

Intact 23 kilodalton (kDa), rat PRL is enzymatically cleaved in many target tissues to a <em>16</em> kDa (<em>16</em>K PRL) and an 8 kDa fragment. After reduction of an internal disulfide bond the fragments are released. <em>16</em>K PRL was shown to be a potent mitogen on mammary epithelial cells via PRL receptors. Since estradiol-induced prolactinomas develop a new blood supply we tested the action of intact PRL and <em>16</em>K PRL on <em>growth</em> of new vessels (angiogenesis). The angiogenic action of intact PRL and <em>16</em>K PRL was tested in cultured bovine brain and adrenal cortex endothelial cells. Basal (b) or b-<em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) stimulated <em>growth</em> was estimated by counting cells or measuring the level of incorporation of 3H-thymidine into DNA. Paradoxically, <em>16</em>K PRL inhibited the basal and FGF-stimulated <em>growth</em> of cultured endothelial cells in a dose-dependent fashion. Intact PRL or the cleaved but not reduced PRL were inactive even at a 100-fold higher concentration. When reformation of disulfide bonds was inhibited by carbamidomethylation of <em>16</em>K PRL the preparations were more potent. <em>16</em>K PRL had no effect on the mitogenic action of bFGF on baby hamster kidney cells which are known to have FGF receptors. These data demonstrate that in vitro <em>16</em>K PRL is a potent and specific angiolytic <em>factor</em>, i.e. it inhibits angiogenesis. Furthermore, the action of <em>16</em>K PRL does not appear to be via the known PRL or FGF receptors. Since angiogenesis is an essential component of tumor <em>growth</em> <em>16</em>K PRL has potential as a therapeutic agent for the treatment of cancer.

An Epstein-Barr virus (EBV) recombinant (MS231) that expresses the first 231 amino acids (aa) of LMP1 and is truncated 155 aa before the carboxyl terminus transformed resting B lymphocytes into lymphoblastoid cell lines (LCLs) only when the infected cells were grown on <em>fibroblast</em> feeder cells (K. M. Kaye et al., J. Virol. 69:675-683, 1995). Higher-titer MS231 virus has now been compared to wild-type (WT) EBV recombinants for the ability to cause resting primary B-lymphocyte transformation. Unexpectedly, MS231 is as potent as WT EBV recombinants in causing infected B lymphocytes to proliferate in culture for up to 5 weeks. When more than one transforming event is initiated in a microwell, the MS231 recombinant supports efficient long-term LCL out<em>growth</em> and <em>fibroblast</em> feeder cells are not required. However, with limited virus input, MS231-infected cells differed in their <em>growth</em> from WT virus-infected cells as early as 6 weeks after infection. In contrast to WT virus-infected cells, most MS231-infected cells could not be grown into long-term LCLs. Thus, the LMP1 amino-terminal 231 aa are sufficient for initial <em>growth</em> transformation but the carboxyl-terminal 155 aa are necessary for efficient long-term out<em>growth</em>. Despite the absence of the carboxyl-terminal 155 aa, MS231- and WT-transformed LCLs are similar in latent EBV gene expression, in ICAM-1 and CD23 expression, and in NF-kappaB and c-jun N-terminal kinase activation. MS231 recombinant-infected LCLs, however, require <em>16</em>- to 64-fold higher cell density than WT-infected LCLs for re<em>growth</em> after limiting dilution. These data indicate that the LMP1 carboxyl-terminal 155 aa are important for <em>growth</em> at lower cell density and appear to reduce dependence on paracrine <em>growth</em> <em>factors</em>.

OBJECTIVE

Proliferative vitreoretinopathy (PVR) is a disorder characterized by the formation of cellular membranes on both surfaces of the retina and within the vitreous cavity. It occurs in 5% to 10% of patients who undergo retinal reattachment surgery. In the rabbit model of the disease, the platelet-derived growth factor alpha receptor (PDGFRalpha) is dramatically more capable of promoting PVR than is closely related PDGFRbeta. To test the ligand hypothesis (i.e., that this phenomenon can be explained by a predominance of PDGFRalpha-specific ligands) this study was conducted to determine the profile of PDGF ligands expressed by cells that induce PVR and in the vitreous of rabbits that have PVR. In addition, we examined which PDGF isoforms were present in the vitreous of patients with PVR, to assess the relevance of the rabbit model to the clinical setting.

METHODS

PDGF isoforms were detected and quantified by Western blot analysis and ELISA. An assay was performed of conditioned medium from mouse embryo fibroblasts expressing the PDGFRalpha (Falpha) and rabbit conjunctival fibroblasts (RCFs), both of which cause PVR in the experimental model, and from human retinal pigment epithelial cells (ARPE-19). Because PDGF-C is secreted in a latent form and must be proteolytically processed to become biologically active, a PDGF-C processing assay was established, and conditioned medium was tested from these cells lines, for processing activity. Vitreous specimens, from control and PVR rabbits and from patients undergoing vitrectomy surgery, either to repair retinal detachment or for other reasons, were also tested for PDGF isoforms and for PDGF-C processing activity.

RESULTS

PDGF isoforms that activate PDGFRbeta (PDGF-B and -D) were either undetectable or were present at very low levels in all the samples tested. Relatively low levels of PDGF-A and -AB were detected, whereas PDGF-C was the predominant isoform. Falpha, RCFs, and ARPE-19 cells accumulated PDGF-C in the conditioned medium at an average rate of 2.0 +/- 0.2, 2.9 +/- 0.3, and 71.3 +/- 6.0 ng/mL per day, respectively. Although there was no detectable PDGF-C in the vitreous of control rabbits (n = 8), there was an average of 1784 +/- 1150 ng/mL latent PDGF-C in the vitreous from rabbits with PVR (n= 14). Of the patients with PVR, eight of nine contained PDGF-C (range, 50-1000 ng/mL). In contrast, PDGF-C was detected in only 1 of 16 of the patients without PVR. In both conditioned medium and vitreous samples, the latent (instead of the active) form of PDGF-C was detected, even though processing activity was present in all the samples tested.

CONCLUSIONS

The predominance of PDGF isoforms that activate PDGFRalpha support the ligand hypothesis as an explanation of why PDGFRalpha is more capable of inducing PVR than is PDGFRbeta. Furthermore, the profile of PDGF isoforms observed in the rabbit model accurately reflected the clinical specimens from patients with PVR. Finally, these findings implicate one of the new PDGF family members as an important contributor to experimental and clinical PVR.

The binding of human acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) to heparin has been analyzed by a variety of different approaches to better elucidate the nature of this protein/sulfated polysaccharide interaction. Static and dynamic light scattering as well as analytical ultracentrifugation analyses indicates that 14-15 molecules of a FGF can bind to a <em>16</em>-kDa heparin chain, with approximately 10 of these bound relatively uniformly to high-affinity sites. The dissociation constants of these latter sites are estimated to be approximately 50-140 nM on the basis of surface plasmon resonance experiments in which the association and dissociation rates of aFGF interaction with immobilized heparin were measured. The size of the binding site of a FGF on heparin was also determined by heparin lyase digestion of a FGF/heparin complexes followed by isolation and characterization of protected oligosaccharides. The smallest aFGF-protected oligosaccharide comigrated with delta UA2S(1-->4)-alpha-D-GlcNp2S6S(1-->4)-alpha-L-IdoAp-2S( 1-->4)-alpha-D-GlcNp2S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid and S is sulfate). Thus, aFGF appears to bind at high density (one molecule every 4-5 polysaccharide units) and with high affinity to heparin. This potentially provides a concentrated, stabilized storage form of the <em>growth</em> <em>factor</em> that can be released for receptor-mediated cellular activation in response to the proper stimuli. It is also possible that close proximity of aFGF molecules on the highly sulfated regions of heparan chains may be involved in the induction of receptor aggregation as suggested by Ornitz et al. [Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E., & Leder, P. (1992) Mol. Cell. Biol. 12, 240-247].

OBJECTIVE

Signaling through the fibroblast growth factor (FGF) pathway may account for tumor resistance to antiangiogenic therapies targeting the VEGF pathway. Here, dovitinib (TKI258), a potent oral inhibitor of FGF receptor, VEGF receptor (VEGFR), and platelet-derived growth factor receptor tyrosine kinases, is studied in a dose escalation trial.

METHODS

Patients with advanced or metastatic renal cell carcinoma (RCC) with predominant clear cell histology were treated with oral dovitinib 500 or 600 mg/day (5-days-on/2-days-off schedule).

RESULTS

Twenty heavily pretreated patients (median 3 prior regimens) were enrolled, with 16, 11, and 12 patients having previously received at least 1: VEGFR inhibitor, mTOR inhibitor, and immunotherapy, respectively. Fifteen and 5 patients were treated in 500- and 600-mg cohorts, respectively. Three patients experienced dose-limiting toxicities: grade 2 bradycardia (500 mg), grade 4 hypertensive crisis (600 mg), and grade 3 asthenia with grade 2 nausea and vomiting (600 mg). The most common adverse events related to dovitinib were nausea (75%), diarrhea (70%), vomiting (70%), and asthenia (50%), the majority of which were mild (grade 1 or 2), with grade 3 events 5% or less (except asthenia, 15%) and only one grade 4 event (hypertensive crisis). Two patients achieved a partial response (500 mg), and 12 patients had stable disease, including 2 patients with long lasting disease stabilizations (>1 year) in the 500-mg cohort.

CONCLUSIONS

Dovitinib was tolerable and showed antitumor activity at a maximum tolerated dose of 500 mg on a 5-days-on/2-days-off schedule in heavily pretreated RCC patients.

Farnesoid X receptor (FXR) is the master regulator of bile acid (BA) homeostasis because it controls BA synthesis, influx, efflux, and detoxification in the gut/liver axis. Deregulation of BA homeostasis has been linked to hepatocellular carcinoma (HCC), and spontaneous hepatocarcinogenesis has been observed in FXR-null mice. This dreaded liver neoplasm has been associated with both FXR gene deletion and BA-mediated metabolic abnormalities after inactivation of FXR transcriptional activity. In the present study, we addressed the hypothesis that intestinal selective FXR reactivation would be sufficient to restore the <em>fibroblast</em> <em>growth</em> <em>factor</em> 15 (FGF15)/cholesterol-7alpha-hydroxylase (Cyp7a1) enterohepatic axis and eventually provide protection against HCC. To this end, we generated FXR-null mice with re-expression of constitutively active FXR in enterocytes (FXR(-/-)iVP<em>16</em>FXR) and corresponding control mice (FXR(-/-)iVP<em>16</em>). In FXR-null mice, intestinal selective FXR reactivation normalized BA enterohepatic circulation along with up-regulation of intestinal FXR transcriptome and reduction of hepatic BA synthesis. At <em>16</em> months of age, intestinal FXR reactivation protected FXR-null mice from spontaneous HCC development that occurred in otherwise FXR-null mice. Activation of intestinal FXR conferred hepatoprotection by restoring hepatic homeostasis, limiting cellular proliferation through reduced cyclinD1 expression, decreasing hepatic inflammation and fibrosis (decreased signal transducer and activator of transcription 3 activation and curtailed collagen deposition).

CONCLUSIONS

Intestinal FXR is sufficient to restore BA homeostasis through the FGF15 axis and prevent progression of liver damage to HCC even in the absence of hepatic FXR. Intestinal-selective FXR modulators could stand as potential therapeutic intervention to prevent this devastating hepatic malignancy, even if carrying a somatic FXR mutation.

Fgf-8 is a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family that was initially identified as an androgen-inducible <em>growth</em> <em>factor</em> in a mammary carcinoma cell line. Alternative splicing of the primary Fgf-8 transcript results in three messenger RNAs which code for secreted FGF-8 protein isoforms that differ only in their mature amino termini. Fgf-8 RNA is present from day 10 through 12 of murine gestation when analyzed by northern blot analysis, suggesting that Fgf-8 normally functions during post-gastrulation development. To characterize the temporal, spatial and isoform-specific aspects of Fgf-8 expression during mouse development, we performed in situ hybridization and ribonuclease protection assays between the days 8 and <em>16</em> of gestation. Fgf-8 expression is first detected at day 9 of gestation in the surface ectoderm of the first branchial arches, the frontonasal process, the forebrain and the midbrain-hindbrain junction. At days 10-12 of gestation, Fgf-8 expression is detected in the surface ectoderm of the forelimb and hindlimb buds, in the nasal pits and nasopharynx, in the infundibulum and in the telencephalon, diencephalon and metencephalon. Fgf-8 expression continues in the developing hindlimbs through day 13 of gestation but is undetectable thereafter. Ribonuclease protection assays reveal that RNAs coding for all three FGF-8 isoforms are present at days 10-12 of gestation. These results reveal a unique temporal and spatial pattern of Fgf-8 expression in the developing mouse and suggest a role for this FGF in multiple regions of ectodermal differentiation in the post-gastrulation mouse embryo.

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of benign <em>growths</em> in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome <em>16</em>. The TSC2 gene on chromosome <em>16</em> encodes a 1784-amino acid tumor suppressor protein, tuberin, that functions as a GTPase-activating protein for Rap1, a member of the superfamily of Ras-related proteins. By immunoblot analyses, we found TSC2 expression to be high in G0 as well as in early small G1 cells. Analyses after different cell synchronization procedures revealed that TSC2 mRNA and protein expression do not fluctuate throughout the cell cycle. Using inducible expression systems we further demonstrated that TSC2 expression is not affected by overexpression of the mitogenic transcription <em>factor</em> E2F-1 or c-Myc. Nevertheless, antisense inhibition of tuberin expression in logarithmically <em>growing</em> cells markedly decreased the percentage of cells in G1. Furthermore, we found that cells exposed to TSC2 antisense oligonucleotides did not undergo G0 arrest after serum withdrawal. Antisense inhibition of TSC2 expression also induced quiescent G0-arrested <em>fibroblasts</em> to reenter the cell cycle. Our data show for the first time that the absence of tuberin can both induce cells to pass through the G1/S transition of the eukaryotic cell cycle and prevent them from entering a quiescent state. These results have clear implications for the tumor suppressor function of TSC2. We further found that reentry into the cell cycle upon loss of TSC2 is dependent on the activity of the G1 cyclin-dependent kinases (CDKs), Cdk2 or Cdk4. Taken together with our finding that antisense inhibition of TSC2 causes up-regulation of cyclin D1 expression, these results provide the first evidence for a connection between tuberin/Rap1 and the G1 CDK-dependent regulation of the transition from G0/G1 to S phase.