In the preceding report (Kelvin, D.J., G. Simard, H.H. Tai, T.P. Yamaguchi, and J.A. Connolly. 1989. J. Cell Biol. 108:<em>15</em>9-167) we demonstrated that pertussis toxin (PT) blocked proliferation and induced differentiation in BC3H1 muscle cells. In the present study, we have used PT to examine specific <em>growth</em> <em>factor</em> signaling pathways that may regulate these processes. Inhibition of [3H]thymidine by PT in 20% FBS was reversed in a dose-dependent fashion by purified <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). In 0.5% FBS, the normally induced increase in creatine kinase (CK) activity was blocked by FGF in both the presence and absence of PT. Similar results were obtained with purified epidermal <em>growth</em> <em>factor</em> (EGF). We subsequently examined the effect of a family of <em>growth</em> <em>factors</em> linked to inositol lipid hydrolysis and found that thrombin, like FGF, would increase [3H]thymidine incorporation and block CK synthesis. However, PT blocked thymidine incorporation induced by thrombin, and blocked the inhibition of CK turn-on in 0.5% FBS by thrombin. The ras oncogene, a G protein homologue, has previously been shown to block muscle cell differentiation in C2 muscle cells (Olson, E.N., G. Spizz, and M.A. Tainsky. 1987. Mol. Cell. Biol. 7:2104-2111); we have characterized a BC3H1 cell line, BCT31, which we transfected with the val12 oncogenic Harvey ras gene. This cell line did not express CK in response to serum deprivation. Whereas [3H]thymidine incorporation was inhibited by 70-80% by increasing doses of PT in control cells, BCT31 cells were only inhibited by <em>15</em>-20%. ADP ribosylation studies indicate this PT-insensitivity is not because of the lack of a PT substrate in this cell line. Furthermore, PT could not induce CK expression in BCT31 cells as it did in parental cells. We conclude that there are at least two distinct <em>growth</em> <em>factor</em> pathways that play a key role in regulating proliferation and differentiation in BC3H1 muscle cells, one of which is PT sensitive, and postulate that a G protein is involved in transducing signals from the thrombin receptor. We believe that ras functions in the transduction of <em>growth</em> <em>factor</em> signals in the nonPT-sensitive pathway or downstream from the PT substrate in the second pathway.

Human osteoclasts are well characterized multinucleated cells whose function is the directed resorption of normal bone (NB). Osteoclastic bone destruction accompanies lytic solid tumors and myeloma as well as Paget's disease (PD) of bone and giant cell tumors of bone (GCTB). The mechanism of this stimulation of osteoclastic bone resorption is unknown. This study was designed to detect cytokines present in the multinucleated cells of PD and GCTB in order to determine whether cytokine abnormalities exist to account for bone lysis. Nine cytokines, representing the functions of bone resorption, angiogenesis, tumor necrosis, bone cell proliferation, and osteoblast-osteoclast coupling, were examined by immunohistochemistry using tissue samples from <em>15</em> NB, 17 PD, and 19 GCTB patients. Standard nonparametric statistical analysis showed a significant increase (P < 0.01 to 0.05) in immunostaining between osteoclasts of PD and NB for interleukin-6 (Il-6), tumor necrosis <em>factor</em> beta (TNFbeta), epidermal <em>growth</em> <em>factor</em> (EGF), platelet derived <em>growth</em> <em>factor</em> (PDGF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). There was a statistically significant decrease in immunostaining of giant cells of GCTB as compared with NB for transforming <em>growth</em> <em>factor</em> beta (TGFbeta), but no other differences from normal osteoclasts. The increase in staining of PD osteoclasts over the giant cells of GCTB was significant (P < 0.01) for Il-6, TNFbeta, PDGF, bFGF and insulin <em>growth</em> <em>factor</em>-1 (IGF-1), and (P < 0. 05) for Il-1 and EGF. It was concluded that marked cytokine differences exist in vivo between osteoclasts of NB and PD lesions consistent with stimulated resorption. Alternatively, "osteoclastoma" cells in the center of the tumor did not overexpress the cytokines associated with bone lysis, suggesting some other mechanism for stimulated resorption.

Two kidney-derived mitogens have been isolated by ion exchange, heparin-Sepharose and reverse-phase high-performance liquid chromatography on the basis of their capacity to stimulate the proliferation of bovine vascular endothelial cells in vitro. Gas phase sequence analysis identified the amino terminal sequences His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-X-Lys-Asn-Gly-Gly-Phe-Phe-Leu and His-Phe-Lys-Asp-Pro-Lys-Arg-Leu, respectively. The sequences are identical to residues 16-32 and 16-23 of bovine basic pituitary <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> (FGF). The possibility that these kidney-derived mitogens are related, if not identical, to pituitary basic FGF is supported by the observations that they have similar molecular weights (<em>15</em>-16 kDa), similar retention behavior on all steps of chromatography and similar amino acid compositions, and they share at least some structural homology. Moreover, the kidney-derived <em>growth</em> <em>factors</em>, like basic FGF, are potent stimulators of capillary endothelial cells, granulosa cells, adrenocortical cells and vascular smooth-muscle cells (ED50 = 50 pg). The results demonstrate the existence of a kidney-derived FGF and suggest that at least some of the mitogenic, angiogenic and neovascularising activities described to be present in the kidney are due to the presence of an FGF-like molecule in this tissue.

Oligomerization of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) induced on binding to heparin or heparan sulfate proteoglycan is considered to be crucial for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the receptor by FGFs, in the present study we investigate the effect(s) of interaction of a heparin analog, sucrose octasulfate (SOS), on the structure, stability, and biological activities of a recombinant acidic FGF from Notophthalmus viridescens (nFGF-1). SOS is found to bind to nFGF-1 and significantly increase the thermodynamic stability of the protein. Using a variety of techniques such as size-exclusion chromatography, sedimentation velocity, and multidimensional nuclear magnetic resonance (NMR) spectroscopy, it is shown that binding of SOS to nFGF-1 retains the protein in its monomeric state. In its monomeric state (complexed to SOS), n-FGF-1 shows significant cell proliferation activity. (<em>15</em>)N and (1)H chemical shift perturbation and the intermolecular nuclear Overhauser effects (NOEs) between SOS and nFGF-1 reveal that the ligand binds to the dense, positively charged cluster located in the groove enclosed by beta-strands 10 and 11. In addition, molecular modeling based on the NOEs observed for the SOS-nFGF-1 complex, indicates that SOS and heparin share a common binding site on the protein. In conclusion, the results of the present study clearly show that heparin-induced oligomerization of nFGF-1 is not mandatory for its cell proliferation activity.

This study demonstrates the synthesis and release of prolactin (PRL) from dermal <em>fibroblasts</em> (>98%) in vitro, suggesting a potential local source of PRL in skin. PRL release was first detected in confluent cultures (0.25 x 10(6) plated cells) on or before day 18 and increased to a maximal level of 2 ng/72 h by day 30. Medroxyprogesterone acetate and estradiol (E2) had no effect on PRL release, but prostaglandin E2 (PGE2) reduced the time required for PRL induction to 6-9 days. The steroids and PGE2 together were synergistic, reaching maximal values of approximately 10 ng/72 h after 2 or more weeks of treatment. Dibutyryl-cyclic AMP, a second messenger in prostaglandin signal transduction, was also synergistic with medroxyprogesterone acetate and E2, but induced significant PRL expression in the absence of the steroids (28 and 12 ng/72 h, respectively). The increase in PRL release was not a result of increased cell proliferation, because the PRL-secreting cultures had 32.2 +/- 8.8% less DNA (N = 3 individuals, 93% confidence limit) than control cultures after 3 weeks of treatment with dibutyryl-cyclic AMP, medroxyprogesterone acetate, and E2. Dermal <em>fibroblast</em> PRL was immunologically and electrophoretically identical to decidual and pituitary PR-Ls, and Northern blot analysis demonstrated a PRL mRNA size of 1.<em>15</em> kb. Maximal PRL release from <em>fibroblast</em> cells was 32.0 +/- 6.1 ng/72 h (mean +/- SD at 95% confidence limit) for a donor population representing both males (n = <em>15</em>) and females (n = 7) between the ages of 20-week gestation to 52 years. In contrast to term decidual <em>fibroblast</em> cells that also express PRL, dermal <em>fibroblasts</em> did not co-express insulin-like <em>growth</em> <em>factor</em>-binding protein-1.

The success of embryonic neural transplants as a treatment for patients with Parkinson's disease has been limited by poor survival of transplanted dopamine neurons. To see if a new partially intact tissue preparation method improves survival, we have developed a technique for extruding embryonic tissue into strands. We expected this method to reduce cell damage and improve transplant survival as well as provide improved tissue delivery. We have compared transplants of tissue strands with mechanically dispersed suspensions of embryonic day <em>15</em> rat ventral mesencephalon. Tissue from ventral mesencephalon was transplanted into a single site in dopamine denervated striatum of unilateral 6-hydroxydopamine (6-OHDA) lesioned rats. To evaluate the effects of striatal cografts and <em>growth</em> <em>factors</em> on dopamine cell survival, dispersed mesencephalic cells were cotransplanted with dispersed striatal cells. Another group had dispersed mesencephalic cells cotransplanted with striatal cells incubated in the cold for 2 h with glial cell line-derived neurotrophic <em>factor</em> (GDNF, 100 ng/ml), insulin-like <em>growth</em> <em>factor</em>-I (IGF-I, <em>15</em>00 ng/ml), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, <em>15</em>0 ng/ml). Behavioral improvement was assessed monthly by changes in methamphetamine-induced rotational behavior. Animals were sacrificed after 3 months, and dopamine neurons were identified by tyrosine hydroxylase (TH) immunohistochemistry. Transplants of tissue strands produced better dopamine neuron survival and led to more robust behavioral restoration than did cell suspensions even when suspensions were supported with cografts of striatal cells or pretreatment with <em>growth</em> <em>factors</em>.

The purpose of this study was to dramatically enhance the solubility >> 400 fold) and stability of a therapeutic protein (<em>Fibroblast</em> <em>Growth</em> <em>Factor</em> 20) and to perform detailed biophysical characterization for the optimization of its formulation. The solubility of FGF-20 strongly depends on pH, arginine concentration and anions present in a buffer system. In the absence and presence of arginine, solubility was higher at lower pH (5 < or = pH < or = 6) and then decreased steadily with a minimum solubility at around pH 6.3 and plateaus at around pH 7.5 respectively. For a given pH, the protein was most soluble in arginine-sulfate. The solubility of FGF-20 increases with an increase in arginine-sulfate concentration for a given pH. However, a salting out effect was observed at higher arginine-sulfate concentration. Polysorbate-80 did not have any striking effect on solubility and no effect on thermal stability, but it significantly prevented the loss of protein under agitated conditions. Thermal stability of FGF-20 measured by DSC was increased with an increase in arginine-sulfate concentration (at least up to 0.5M). A sturdy dependence of thermal stability on pH was observed with about a <em>15</em> degrees C increase in T(m) (melting temperature) at pH 7.0 in comparison to pH 5.0. From the DSC data, approximate stability curves were generated and cold denaturation temperatures were predicted. Denaturant induced unfolding studies provided better insight of FGF-20 in different solution conditions in terms of structure and stability than the DSC data. An inverse relationship of solubility and thermal stability was observed in the pH range of 5.0 to 8.5 at a fixed arginine concentration and is consistent with Linderstrom-Lange's smeared model. A direct correlation between solubility and thermal stability was observed at different arginine concentrations for a fixed pH. The effect of arginine on the solubility and stability of FGF-20 was dominated by the preferential binding interaction.

In mice, two pluripotent cell lines, embryonic stem (ES) cells and embryonic germ (EG) cells, have been identified. We present here results indicating that porcine EG cell lines can be isolated, genetically transformed, and utilized to make transgenic chimeras. Briefly, primordial germ cells (PGCs) were isolated from Day 25-27 fetuses and plated on STO feeder cells in Dulbecco's modified Eagle's medium:Ham's F-10 medium supplemented with 0.01 mM nonessential amino acids, 2 mM glutamine, <em>15</em>% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 40 ng/ml human stem cell <em>factor</em>, 20 ng/ml human basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and 20 ng/ml human leukemia inhibitory <em>factor</em>. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein under control of the cytomegalovirus promoter. After electroporation, cells were plated and later examined under fluorescein isothiocyanate excitation. Fluorescent colonies were selected for chimera generation. Blastocysts collected from gilts on Day 5 were injected with 10-<em>15</em> transgenic PGC-derived cells and transferred into recipient gilts. Gilts were hysterectomized on Day 25, and fetal tissues were analyzed by Southern blotting. Three chimeras out of 20 fetuses analyzed were transgenic. Additionally, when one recipient gilt was allowed to go to term, one piglet with transgenic contribution was identified.

Treatment of quiescent rat aortic smooth muscle cells with either alpha-thrombin or a thrombin receptor-derived agonist peptide (SFLLRNP) resulted in pronounced increases in [3H]thymidine incorporation that were concentration dependent and reached a maximum of approximately <em>15</em>-fold above serum-starved controls. However, in contrast to FBS, PDGF-BB, or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), that initiated DNA synthesis promptly after 16-19 h, thymidine incorporation in response to thrombin was delayed by an additional 3-6 h. Delayed mitogenesis correlated with the appearance of a potent mitogenic activity in conditioned media samples obtained from thrombin-stimulated rat aortic smooth muscle cells, as assayed using Swiss 3T3 <em>fibroblasts</em>. This activity was not inhibited by neutralizing antibodies directed against PDGF or bFGF. Furthermore, in the Swiss 3T3 cells, simple addition of either alpha-thrombin or SFLLRNP failed to elicit a significant mitogenic response. In signal transduction studies, both thrombin and SFLLRNP treatment led to rapid tyrosine phosphorylation of proteins with apparent molecular masses of 42, 44, 75, 120, and 190 kD, respectively, as assessed by antiphosphotyrosine immunoblotting. The overall pattern of protein tyrosine phosphorylation was distinct from that observed after PDGF-BB addition. Activation of Raf-1 and the mitogen-activated protein (MAP) kinases p44mapk and p42mapk was also observed. However, the time course and duration of Raf-1/MAP kinase activation after thrombin stimulation were similar to those elicited by PDGF-BB. Taken together, our results indicate that thrombin-stimulated vascular smooth muscle proliferation is delayed and requires the de novo expression of one or more autocrine mitogens. In addition, the rapid induction of discrete intracellular signaling mechanisms by thrombin, including the Raf-1/MAP kinase pathway, appears to be insufficient alone to promote vascular smooth muscle cell mitogenesis.

The study was designed 1) to determine whether treatment with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and PTH is more efficacious than treatment with PTH alone for increasing bone mass and strength and improving trabecular microarchitecture in osteopenic ovariectomized rats, and 2) to assess whether prior and concurrent administration of the antiresorptive agents estrogen and risedronate suppresses the bone anabolic response to treatment with bFGF alone and sequential treatment with bFGF and PTH. Three-month-old female Sprague Dawley rats were ovariectomized (OVX) or sham-operated (sham) and maintained untreated for 1 yr. Baseline sham and OVX rats were killed at this time (<em>15</em> months of age). Groups of rats were injected sc with estrogen (10 microg/kg, 4 d/wk), risedronate (5 microg/kg, 2 d/wk), or vehicle. At the end of the second week of antiresorptive treatment, catheters were inserted into the jugular veins of all rats, and vehicle or bFGF at a dose of 250 microg/kg was injected daily for 14 d. Three groups of rats were killed at the end of bFGF treatment. The remaining rats were continued on their respective antiresorptive therapy and injected sc with vehicle or synthetic human PTH-(1-34) at a dose of 80 microg/kg, 5 d/wk, for 8 wk. Lumbar vertebrae were processed for cancellous bone histomorphometry and biomechanical testing. Ovariectomy resulted in a decrease in vertebral bone mass and strength. Treatment of OVX rats for 14 d with bFGF markedly increased osteoblast surface, osteoid surface, and osteoid volume compared with vehicle treatment of sham and OVX rats. Furthermore, osteoid bridges were observed extending between preexisting trabeculae in bFGF-treated OVX rats. Prior and concurrent administration of estrogen and risedronate did not suppress these bone anabolic effects of bFGF. Treatment of OVX rats with PTH alone increased vertebral cancellous bone mass and strength to the level of vehicle-treated sham rats. Sequential treatment of OVX rats with bFGF and PTH further augmented vertebral bone mass and strength to a level above that observed in OVX rats treated with PTH alone. The improvements in bone mass and strength were associated with an increase in trabecular thickness in OVX rats treated with PTH alone and with an increase in trabecular thickness and node to terminus ratio, an index of trabecular connectivity, in OVX rats treated sequentially with bFGF and PTH. Cotreatment with estrogen and risedronate did not suppress the anabolic response of bone to bFGF and PTH. In fact, a trend for an even greater increase in cancellous bone mass and node to terminus ratio was observed in OVX rats treated with risedronate, bFGF, and PTH. These findings indicate that sequential treatment with bFGF and PTH is more efficacious than treatment with PTH alone for increasing bone mass and strength and improving trabecular microarchitecture in osteopenic OVX rats.

The D-group cyclins play a key role in the progression of cells through the G(1) phase of the cell cycle. Treatment of MCF-7 breast cancer cells with the cyclopentenone prostaglandin <em>15</em>-deoxy-Delta(12,14)-PGJ(2) (<em>15</em>d-PGJ(2)) results in rapid down-regulation of cyclin D1 protein expression and <em>growth</em> arrest in the G(0)/G(1) phase of the cell cycle. <em>15</em>d-PGJ(2) also down-regulates the expression of cyclin D1 mRNA; however, this effect is delayed relative to the effect on cyclin D1 protein levels, suggesting that the regulation of cyclin D1 occurs at least partly at the level of translation or protein turnover. Treatment of MCF-7 cells with <em>15</em>d-PGJ(2) leads to a rapid increase in the phosphorylation of protein synthesis initiation <em>factor</em> eukaryotic initiation <em>factor</em> 2alpha (eIF-2alpha) and a shift of cyclin D1 mRNA from the polysome-associated to free mRNA fraction, indicating that <em>15</em>d-PGJ(2) inhibits the initiation of cyclin D1 mRNA translation. The selective rapid decrease in cyclin D1 protein accumulation is facilitated by its rapid turnover (t(1/2) = 34 min) after inhibition of cyclin D1 protein synthesis. The half-life of cyclin D1 protein is not significantly altered in cells treated with <em>15</em>d-PGJ(2). Treatment of cells with <em>15</em>d-PGJ(2) results in strong induction of heat shock protein 70 (HSP70) gene expression, suggesting that <em>15</em>d-PGJ(2) might activate protein kinase R (PKR), an eIF-2alpha kinase shown previously to be responsive to agents that induce stress. <em>15</em>d-PGJ(2) strongly stimulates eIF-2alpha phosphorylation and down-regulates cyclin D1 expression in a cell line derived from wild-type mouse embryo <em>fibroblasts</em> but has an attenuated effect in PKR-null cells, providing evidence that PKR is involved in mediating the effect of <em>15</em>d-PGJ(2) on eIF-2alpha phosphorylation and cyclin D1 expression. In summary, treatment of MCF-7 cells with <em>15</em>d-PGJ(2) results in increased phosphorylation of eIF-2alpha and inhibition of cyclin D1 mRNA translation initiation. At later time points, repression of cyclin D1 mRNA expression may also contribute to the decrease in cyclin D1 protein.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a potent endothelial cell mitogen whose actions are mediated by binding to specific cell surface receptors on a variety of cell types. However, the amino acid sequence of bFGF does not contain a classical signal peptide sequence and the extent to which cellular stores of this mitogen are released is still a matter of some controversy. In the present study we examined the release of immunoreactive bFGF into serum-free conditioned medium of bovine corneal endothelial cells (BCE) and a human astrocytoma cell line, U87-MG. Western blotting analysis of BCE conditioned medium using N-terminal specific anti-bFGF serum revealed a single immunoreactive band of 32 kilodaltons, which was reduced to 18 kilodaltons in the presence of 8 M urea. Using a sensitive two-site immunoradiometric assay we were able to quantify the release of immunoreactive bFGF into the culture medium by BCE cells and by the human astrocytoma cell line U87-MG. In each case the release of bFGF was cell density dependent, but under all conditions the level of bFGF released was significantly greater in the transformed astrocytoma line, ranging from <em>15</em>- to 50-fold higher than in the BCE cultures under various conditions. At 30% confluence the concentration of immunoreactive bFGF in the medium was maintained at a constant level for up to 24 h. However, the level of immunoreactive bFGF declined rapidly in confluent cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand stimulation of the platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptor results in its association with phosphoinositide 3-kinase activity and a corresponding synthesis of 3'-phosphorylated lipids. Early studies that examined this interaction in vivo employed anti-phosphotyrosine antiserum or antiserum against the PDGF receptor. The recent identification of multiple isoforms of both the regulatory and the catalytic subunit of the enzyme have led us to utilize antisera against p85alpha and p110alpha to characterize the association of this particular phosphoinositide 3-kinase complex with the PDGF receptor following ligand stimulation of murine <em>fibroblasts</em>. Both the p85alpha and p110alpha subunits rapidly associated with the ligand-activated receptor resulting in a transient, 2-fold increase in the total pool of p110alpha lipid kinase activity. This association was stable for <em>15</em> min after initial stimulation. Subsequently, both subunits began to dissociate from the receptor with similar kinetics. By 60 min this process was complete, demonstrating that p85alpha and p110alpha both associate with the receptor and dissociate from the receptor as a dimeric complex. At this time, marked PDGF receptor down-regulation was observed. Immunoprecipitation from metabolically labeled cells revealed that p85alpha is constitutively phosphorylated on serine residues in quiescent cultures. Upon PDGF stimulation, this phosphorylation upon serine residues was maintained in addition to tyrosine phosphorylation of this subunit. No phosphorylation of the p110alpha subunit was detected in either quiescent or PDGF-stimulated cells. Quantitation of Western blot analysis demonstrated that only 5% of the total pool of p85alpha associated with the PDGF receptor upon ligand stimulation. The 2-fold increase in the lipid kinase activity measured in immunoprecipitates using either anti-p85alpha or anti-p110alpha antiserum therefore reflects a far greater increase in the specific activity of the enzyme upon its association with the PDGF receptor.

The expression of the major protein kinase C (PKC) substrate, originally called "80K" for acidic SDS/PAGE-observed 80-kDa PKC substrate and now called "MARCKS" for myristoylated alanine-rich C kinase substrate, in Swiss 3T3 <em>fibroblasts</em> changes strikingly (<em>15</em>- to 22-fold) during transitions of cell <em>growth</em>. Quiescent cells in G0 express high levels of MARCKS mRNA and protein. However, plating these cells in fresh medium at low density to stimulate multiple rounds of cell division caused a striking down-regulation of MARCKS expression. The mRNA level declined to a minimum of 4.5% compared with quiescent control cells 6 hr after plating, and protein levels declined during the same period to 6.5% of the control value. This rapid down-regulation was independent of PKC activation and length of exposure to trypsin (1-10 min) but required plating in medium containing fresh serum. MARCKS mRNA and protein levels remained down-regulated for 3 days, during which time the cells were actively progressing through the cell cycle as judged by fluorescence-activated cell sorting analysis. However, on reaching quiescence, the expression of MARCKS mRNA and protein increased markedly. Furthermore, the rate of recovery of MARCKS mRNA and protein levels was shown to be dependent on the supply of serum-derived <em>growth</em> <em>factors</em> in the medium. Addition of hydroxyurea to arrest the cells in S phase or at the G1/S boundary rather than G0 completely prevented the recovery of MARCKS protein. The down-regulation of MARCKS following plating and its serum-dependent recovery was also demonstrated in tertiary cultures of mouse embryo <em>fibroblasts</em>. The results suggest that MARCKS may play a role in the regulation of entry and exit of cells from G0.

Several <em>growth</em> <em>factors</em> implicated in the process of cellular transformation were tested for their ability to induce anchorage-independent (AI) <em>growth</em> of primary rat embryo (RE) cells. Our results show that in the presence of 10% calf serum, platelet-derived <em>growth</em> <em>factor</em> (PDGF), 1-30 ng/ml, has the strongest effect of all <em>growth</em> <em>factors</em> tested on AI <em>growth</em>. Type-beta transforming <em>growth</em> <em>factor</em> (TGF-beta), by itself, does not stimulate AI <em>growth</em>, and it inhibits the PDGF-induced colony formation in a dose-dependent manner (ED50 approximately 0.03 ng/ml). Qualitatively similar responses are obtained by using an established line of <em>fibroblasts</em>, NIH 3T3 cells; the principal difference between the response of the primary cells and the established cell line is in colony-forming efficiency in soft agar culture (<em>15</em>% and 90%, respectively, for <em>growth</em> of colonies greater than 1,500 micron2 diameter in the presence of 10 ng/ml PDGF). Since AI <em>growth</em> has been shown to correlate well with tumorigenicity in vivo, our results suggest that the transforming potential of PDGF in an appropriate responsive cell can be controlled not only through its interaction with its own receptor, but also by the presence of inhibitory <em>factors</em> such as TGF-beta.

Controlled scaffold degradation is a critical design criterion for the clinical success of tissue-engineered constructs. Here, we exploited a biomimetic poly(ethylene glycol) diacrylate (PEGDA) hydrogel system immobilized with tethered YRGDS as the cell adhesion ligand and with either single (SSite) or multiple (MSite) collagenase-sensitive domains between crosslinks, to systematically study the effect of proteolytic cleavage site presentation on hydrogel degradation rate and three-dimensional (3-D) <em>fibroblast</em> invasion in vitro. Through the incorporation of multiple collagenase-sensitive domains between cross-links, hydrogel degradation rate was controlled and enhanced independent of alterations in compressive modulus. As compared to SSite hydrogels, MSite hydrogels resulted in increased 3-D <em>fibroblast</em> invasion in vitro, which occurred over a wider range of compressive moduli. Furthermore, encapsulated soluble acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-1), a potent mitogen during processes such as vascularization and wound healing, was incorporated into SSite and MSite PEGDA scaffolds to determine its in vitro potential on <em>fibroblast</em> cell invasion. Hydrogels containing soluble FGF-1 significantly enhanced 3-D <em>fibroblast</em> invasion in a dose-dependent manner within the different types of PEG matrices investigated over a period of <em>15</em> days. The methodology presented provides flexibility in designing PEG scaffolds with desired mechanical properties, but with increased susceptibility to proteolytically mediated degradation. These results indicate that effective tuning of initial matrix stiffness and hydrogel degradation kinetics plays a critical role in effectively designing PEG scaffolds that promote controlled 3-D cellular behavior and in situ tissue regeneration.

OBJECTIVE

The aim of this study was to investigate plasma and synovial fluid basic fibroblast growth factor (bFGF) levels in patients with primary knee osteoarthritis (OA) and to evaluate the correlation between bFGF levels and disease severity.

METHODS

Thirty-five patients with knee OA and 15 healthy individuals were recruited into this study. Knee OA grading was performed according to the Kellgren-Lawrence classification. bFGF concentrations in both plasma and synovial fluid were determined using enzyme-linked immunosorbent assay.

RESULTS

Plasma and synovial fluid bFGF levels in knee OA patients were significantly higher than in controls (P < 0.001). Moreover, plasma and synovial fluid bFGF concentrations were positively correlated with radiographic severity (r = 0.535, P < 0.001 and r = 0.570, P < 0.001, respectively). Further analysis revealed that there was a positive correlation between plasma and synovial fluid bFGF levels (r = 0.674, P < 0.001).

CONCLUSIONS

Plasma and synovial fluid bFGF levels were significantly increased in OA patients, and these elevated levels were positively correlated with radiographic severity. These findings indicate that bFGF levels may be a monitor of disease severity and could play an essential part in the pathophysiology of degenerative process in OA.

The fibroproliferative changes in pulmonary artery (PA) remodeling are partially prevented by antifibrotic agents. Relaxin (Rlx), a hormone involved in loosening collagen bundles in ligaments during parturition, has antifibrotic and vasodilator properties that may prevent pulmonary vascular remodeling. In the hypoxia model of pulmonary hypertension, two doses of recombinant human relaxin (rhRlx 24 [high] or 5 [low] mg X 10(-2)/kg d(-1)) were administered subcutaneously continuously for 10d to hypoxic (10% O2) rats. At day 11, right ventricular pressure (Pa X 10(2)) was reduced by rhRlx in a dose-dependent manner (<em>15</em> +/- 1* control; 28 +/- 1 hypoxia; 23 +/- 1* low; 20+/-1* high; n = 10-14/group, *P < 0.05 vs. hypoxia). High rhRlx ameliorated increased collagen accumulation (mug hydroxyproline/vessel) in main PAs (87 +/- 6) vs. untreated hypoxia (102 +/- 2) (n=5/group, P < 0.05). Infusion of rhRlx had no effect on air-breathing rats, and acute administration did not alter blood pressure in hypoxic rats. <em>Fibroblasts</em> cultured from rat PAs spontaneously expressed collagen and fibronectin, and treatment with TGF-beta increased secretion 26- and 25 X 10(-1)-fold, respectively. Addition of rhRlx to transforming <em>growth</em> <em>factor</em>-beta-stimulated <em>fibroblasts</em> inhibited collagen (37%) and fibronectin (38%) secretion vs. vehicle (n = 4 per group, both P < 0.05). We conclude that rhRlx inhibits the early fibroproliferative response in hypoxic pulmonary hypertension and the mechanism may be due in part to suppression of collagen synthesis.

Secreted proteins of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGF-ligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1, -2, -22, FgfR-2c, -3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-<em>15</em> (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-<em>15</em>, FGF-17a) were functionally characterized by plasmid-based over-expression in dissociated E11.5 VM cell cultures, however, such a continuous exposure had no influence on the yield of dopaminergic neurons in vitro.

OBJECTIVE

We analyzed the actions of zoledronic acid (10-250 microM) and doxorubicin (10-250 nM) on PC-3 prostate cancer cells using both continuous (48-96 hr) and pulsatile exposures (<em>15</em> min/day for up to three consecutive days).

RESULTS

The proliferation of PC-3 cells was inhibited by either continuous or pulsatile exposures of zoledronic acid in a dose-dependent manner. In contrast, pulsatile exposures of doxorubicin failed to inhibit the growth of PC-3 cells. In addition, the inhibition of PC-3 cells by zoledronic acid was partially neutralized by exogenous administration of geranylgeranyl pyrophosphate (GGPP), however, not by farnesyl pyrophosphate (FPP). Furthermore, exogenous administration of transforming growth factor beta 1 (TGF-beta1), interleukin 6 (IL-6), basic fibroblast growth factor (bFGF), and more potently, insulin-like growth factor 1 (IGF-1) inhibited the doxorubicin-induced apoptosis of PC-3 cells. Under identical experimental conditions, these growth factors failed to alter the cytotoxicity of PC-3 cells induced by zoledronic acid.

CONCLUSIONS

These data suggest that (i) repetitive and pulsatile (<em>15</em> min/day) exposure to zoledronic acid inhibited the growth of PC-3 cells, (ii) this anticancer action of zoledronic acid was partially mediated by the attenuation of GGPP production, and (iii) bone microenvironment-related growth factors do not alter the anticancer actions of zoledronic acid on PC-3 cells.

Nuclear <em>factor</em> kappa B (NF-kappa B) modulates the expression of numerous genes via interaction with a specific DNA sequence termed the kappa B site. Its activity is modulated by a cytosolic inhibitor protein termed I kappa B, and its activation occurs in response to a variety of agents in a variety of cell types, most notably B and T lymphocytes. Data presented here show that an activity (designated complex I) that binds specifically to the kappa B site is induced in density-arrested Balb/c-3T3 mouse <em>fibroblasts</em> by platelet-derived <em>growth</em> <em>factor</em> (PDGF), a potent mitogen for these cells. Increased levels of complex I, as evaluated by electrophoretic mobility shift assays of nuclear extracts, were observed in cells treated for 1-4 h (but not <em>15</em> min) with the BB isoform of PDGF. 12-O-tetradecanoylphorbol 13-acetate (TPA) and the AA isoform of PDGF also stimulated this response and both isoforms, but not TPA, were effective in cells depleted of protein kinase C. Complex I most likely is authentic NF-kappa B, a p50-p65 heterodimer, or a closely related <em>factor</em> because it exhibited properties characteristic of those previously described for NF-kappa B including inducibility by deoxycholate and cycloheximide and sensitivity to I kappa B. A second kappa B binding activity (complex II), which apparently contained p50 homodimers, displayed limited induction by PDGF, whereas a third complex (complex III) migrated faster than but behaved similarly to complex I. These studies suggest that NF-kappa B or an NF-kappa B-like <em>factor</em> may participate in the expression of PDGF-inducible genes.

Targeted delivery of anticancer drugs using antibodies specific for tumor-associated antigens represents one of the most important approaches in current immuno-oncology research. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) has been demonstrated to be a high-frequency targetable oncogene specific for smoking-associated lung cancers, present in over 20% of lung squamous cell carcinoma cases. This report describes the generation of a potent, fully human antibody fragment in scFv-Fc format efficiently targeting FGFR1. Antibody phage display was used to select high-affinity scFv antibody fragments against the extracellular domain of FGFR1(IIIc). Enzyme immunoassay (ELISA) and surface plasmon resonance (SPR) analysis were used for antibody screening and characterization. The best binder (named D2) was cloned to diabody and Fc fusion formats. All D2 antibodies demonstrated high affinity for FGFR1 with dissociation constants of 18 nmol/L (scFvD2), 0.82 nmol/L (scFvD2 diabody), and 0.59 nmol/L (scFvD2-Fc). scFvD2 was found to be exquisitely selective for FGFR1 versus other FGFR family members and bound FGFR1 even in the presence of its natural ligand FGF2, as shown by competitive analysis. Confocal microscopy revealed that scFvD2-Fc was specifically and rapidly internalized by a panel of cell lines overexpressing FGFR1. Finally, it was demonstrated that scFvD2-Fc mediated specific delivery of a cytotoxic payload into lung cancer cells harboring oncogenic FGFR1 gene amplifications.Implications: This study reports a highly specific internalizing antibody fragment that can serve as a therapeutic targeting agent for efficient delivery of cytotoxic drugs into FGFR1-positive lung cancer cells. Mol Cancer Res; <em>15</em>(8); 1040-50. ©2017 AACR.

The <em>growth</em> of human cerebral meningiomas depends on various <em>growth</em> <em>factors</em>, including epidermal <em>growth</em> <em>factor</em> (EGF), transforming <em>growth</em> <em>factor</em> (TGF)-alpha and TGF-beta, platelet-derived <em>growth</em> <em>factor</em> (PDGF)-BB, insulin-like <em>growth</em> <em>factor</em> (IGF)-I and IGF-II, and acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em>. The latter three have been shown to form autocrine loops that are thought to be a major component of uncontrolled <em>growth</em> in meningioma tissue. Suramin is known to prevent binding of a variety of <em>growth</em> <em>factors</em> to their receptors in mammalian tissue, thus abolishing para- and/or autocrine-mediated cell <em>growth</em>. The authors therefore tested the effect of suramin on the proliferation of cultured human meningioma cells. Suramin (10(-5) to 10(-4) M) significantly inhibited the <em>growth</em> of meningioma cells in culture. The maximum effect observed was with the higher dose (10(-4) M), which resulted in a 40% to 70% reduction in cellular proliferation. This effect was observed in all <em>15</em> tumor samples studied and was confirmed by [3H]thymidine uptake. In studies using DNA flow cytometry, suramin inhibited meningioma cell proliferation in five tumor samples by arresting cells in the S and G2/M phases of the cell cycle. <em>Growth</em> <em>factor</em> (EGF, IGF-I, and PDGF-BB)-induced cell proliferation was completely abolished in five tumor samples when 10(-4) M suramin was applied to meningioma cells. Western blot analysis of three tumor samples showed that the intracellular PDGF-BB content of meningioma cells was significantly reduced after treating the cells with 10(-4) M suramin. Binding of iodinated <em>growth</em> <em>factors</em> (that is, [125I]EGF, [125I]IGF-I, and [125I]PDGF-BB) to their receptor sites was prevented by suramin in a dose-dependent manner in 10 meningioma membrane fractions. Lowering of the intracellular PDGF content and prevention of extracellular <em>growth</em> <em>factor</em> receptor binding demonstrates that suramin disrupts autocrine loops and paracrine <em>growth</em> stimulation in meningioma tissue. These data provide evidence that <em>growth</em> of cerebral meningiomas in culture is strongly inhibited by suramin at a concentration of 10(-4) M. Suramin acts as a scavenger neutralizing exogenous <em>growth</em> <em>factors</em>; thus it can interrupt autocrine loops and paracrine stimulation of human meningioma cell <em>growth</em>. The evidence favors suramin as a therapeutic option for controlling meningioma proliferation in patients with inoperable and recurrent high-grade meningiomas.

The Baculoviridae is a large family of pathogens that are infectious for arthropods, particularly insects of the Lepidoptera. Nucleopolyhedroviruses (NPVs), a genus of Baculoviridae, have a large circular, supercoiled, and double-stranded DNA genome packaged into rod-shaped virions. The Bombyx mori NPV (BmNPV), an NPV pathogenic for B. mori, is known to potentially encode 136 proteins. Using the B. mori genome information, we found that <em>15</em> of 136 BmNPV proteins (11%) show significant similarity to the B. mori proteins. Among them, genes encoding nine proteins can be deleted in B. mori cultured cell line BmN by homologous recombination, indicating that these genes are dispensable for normal virus production. Interestingly, most of non-essential auxiliary genes encode proteins controlling host physiology at cellular and/or organismal levels: ecdysteroid UDP-glucosyltransferase inactivates an insect molting hormone ecdysone, protein tyrosine phosphatase is involved in wandering behavior at the late stage of infection, <em>fibroblast</em> <em>growth</em> <em>factor</em> induces host cell chemotaxis, and chitinase and cathepsin are required for postmortem host liquefaction. Deletion analysis of other non-essential genes also showed that three of them are viral pathogenicity <em>factors</em> for B. mori. These findings suggest that the modern lepidopteran baculovirus may have acquired auxiliary genes from an ancestral host insect to control host physiology and to increase the efficiency of virus transmission in nature.