Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.

Differential tubulogenic and branching morphogenetic activities of growth factors: implications for epithelial tissue development.

At least two kidney epithelial cell lines, the Madin-Darby canine kidney (MDCK) and the murine inner medullary collecting duct line mIMCD-3, can be induced to form branching tubular structures when cultured with hepatocyte growth factor (HGF) plus serum in collagen I gels. In our studies, whereas MDCK cells remained unable to form tubules in the presence of serum alone, mIMCD-3 cells formed impressive branching tubular structures with apparent lumens, suggesting the existence of specific factors in serum that are tubulogenic for mIMCD-3 cells but not for MDCK cells. Since normal serum does not contain enough HGF to induce tubulogenesis, these factors appeared to be substances other than HGF. This was also suggested by another observation: when MDCK cells or mIMCD-3 cells were cocultured under serum-free conditions with the embryonic kidney, both cell types formed branching tubular structures similar to those induced by HGF; however, only in the case of MDCK cells could this be inhibited by neutralizing antibodies against HGF. Thus, the embryonic kidney produces growth factors other than HGF capable of inducing tubule formation in the mIMCD-3 cells. Of a number of growth factors examined, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to be tubulogenic for mIMCD-3 cells. Whereas only HGF was a potent tubulogenic factor for MDCK cells, HGF, TGF-alpha, and EGF were potent tubulogenic factors for mIMCD-3 cells. Nevertheless, there were marked differences in the capacity of these tubulogenic factors to induce tubulation as well as branching events in those tubules that did form (HGF >> TGF-alpha > EGF). Thus, at least three different growth factors can induce tubulogenesis and branching in a specific epithelial cell in vitro (though to different degrees), and different epithelial cells that are capable of forming branching tubular structures demonstrate vastly different responses to tubulogenic growth factors. The results are discussed in the context of branching morphogenesis during epithelial tissue development.

Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid.

mac25, the subject of this report, was selected by the differential display of mRNA method in a search for genes overexpressed in senescent human mammary epithelial cells. mac25 had previously been cloned as a discrete gene, preferentially expressed in normal, leptomeningial cells compared with meningioma tumors. mac25 is another member of the insulin growth factor-binding protein (IGFBP) family. Insulin-like growth factors are potent mitogens for mammary epithelial cells, and the IGFBPs have been shown to modulate this mitogenic activity. We report here that mac25, unlike most IGFBPs, is down-regulated at the transcription level in mammary carcinoma cell lines, suggesting a tumor-suppressor role. The gene was mapped to chromosome 4q12. We found that mac25 accumulates in senescent cells and is up-regulated in normal, growing mammary epithelial cells by all-trans-retinoic acid or the synthetic retinoid fenretinide. These findings suggest that mac25 may be a downstream effector of retinoid chemoprevention in breast epithelial cells and that its tumor-suppressive role may involve a senescence pathway.

Interferons alpha and beta down-regulate the expression of basic fibroblast growth factor in human carcinomas.

We investigated the influence of interferons alpha, beta, and gamma (IFN-alpha, -beta, and -gamma) on the production of basic fibroblast growth factor (bFGF) by human renal carcinoma cells. The human renal carcinoma cell metastatic line SN12PM6 was established in culture from a lung metastasis and SN12PM6-resistant cells were selected in vitro for resistance to the antiproliferative effects of IFN-alpha or IFN-beta. IFN-alpha and IFN-beta, but not IFN-gamma, down-regulated the expression of bFGF at the mRNA and protein levels by a mechanism independent of their antiproliferative effects. Down-regulation of bFGF required a long exposure (> 4 days) of cells to low concentrations (> 10 units/ml) of IFN-alpha or IFN-beta. The withdrawal of IFN-alpha or IFN-beta from the medium permitted SN12PM6-resistant cells to resume production of bFGF. The incubation of human bladder, prostate, colon, and breast carcinoma cells with noncytostatic concentrations of IFN-alpha or IFN-beta also produced down-regulation of bFGF production.

Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells.

Wound repair and tumor vascularization depend upon blood vessel growth into hypoxic tissue. Although hypoxia slows endothelial cell (EC) proliferation and suppresses EC basic fibroblast growth factor (bFGF) expression, we report that macrophages (MPs) exposed to PO2 approximately 12-14 torr (1 torr = 133.3 Pa) synthesize and release in a time-dependent manner platelet-derived growth factor (PDGF) and acidic/basic FGFs (a/bFGFs), which stimulate the growth of hypoxic ECs. Chromatography of hypoxic MP-conditioned medium on immobilized heparin with an ascending NaCl gradient resolved three peaks of mitogenic activity: activity of the first peak was neutralized by antibody to PDGF; activity of the second peak was neutralized by antibody to aFGF; and activity of the third peak was neutralized by antibody to bFGF. Metabolically labeled lysates and supernatants from MPs exposed to hypoxia showed increased synthesis and release of immunoprecipitable PDGF and a/bFGF in the absence of changes in cell viability. Possible involvement of a heme-containing oxygen sensor in MP elaboration of growth factors was suggested by the induction of bFGF and PDGF by normoxic MPs exposed to nickel or cobalt, although metabolic inhibitors such as sodium azide were without effect. These results suggest a paracrine model in which hypoxia stimulates MP release of PDGF and a/bFGF, inducing EC proliferation and potentially promoting angiogenesis in hypoxic environments.

Bridging grafts and transient nerve growth factor infusions promote long-term central nervous system neuronal rescue and partial functional recovery.

Grafts of favorable axonal growth substrates were combined with transient nerve growth factor (NGF) infusions to promote morphological and functional recovery in the adult rat brain after lesions of the septohippocampal projection. Long-term septal cholinergic neuronal rescue and partial hippocampal reinnervation were achieved, resulting in partial functional recovery on a simple task assessing habituation but not on a more complex task assessing spatial reference memory. Control animals that received transient NGF infusions without axonal-growth-promoting grafts lacked behavioral recovery but also showed long-term septal neuronal rescue. These findings indicate that (i) partial recovery from central nervous system injury can be induced by both preventing host neuronal loss and promoting host axonal regrowth and (ii) long-term neuronal loss can be prevented with transient NGF infusions.

Basic fibroblast growth factor can mediate the early inductive events in renal development.

The earliest characterized events during induction of tubulogenesis in renal anlage include the condensation or compaction of metanephrogenic mesenchyme with the concurrent upregulation of WT1, the gene encoding the Wilms tumor transcriptional activator/suppressor. We report that basic fibroblast growth factor (FGF2) can mimic the early effects of an inductor tissue by promoting the condensation of mesenchyme and inhibiting the tissue degeneration associated with the absence of an inductor tissue. By in situ hybridization, FGF2 was also found to mediate the transcriptional activation of WT1 and of the hepatocyte growth factor receptor gene, c-met. Although FGF2 can induce these early events of renal tubulogenesis, it cannot promote the epithelial conversion associated with tubule formation in metanephrogenic mesenchyme. For this, an undefined factor(s) from pituitary extract in combination with FGF2 can cause tubule formation in uninduced mesenchyme. These findings support the concept that induction in kidney is a multiphasic process that is mediated by more than a single comprehensive inductive factor and that soluble molecules can mimic these inductive activities in isolated uninduced metanephrogenic mesenchyme.

Functional interactions between the retinoblastoma (Rb) protein and Sp-family members: superactivation by Rb requires amino acids necessary for growth suppression.

The transient expression of the retinoblastoma protein (Rb) regulates the transcription of a variety of growth-control genes, including c-fos, c-myc, and the gene for transforming growth factor beta 1 via discrete promoter sequences termed retinoblastoma control elements (RCE). Previous analyses have shown that Sp1 is one of three RCE-binding proteins identified in nuclear extracts and that Rb functionally interacts with Sp1 in vivo, resulting in the "superactivation" of Sp1-mediated transcription. By immunochemical and biochemical criteria, we report that an Sp1-related transcription factor, Sp3, is a second RCE-binding protein. Furthermore, in transient cotransfection assays, we report that Rb "superactivates" Sp3-mediated RCE-dependent transcription in vivo and that levels of superactivation are dependent on the trans-activator (Sp1 or Sp3) studied. Using expression vectors carrying mutated Rb cDNAs, we have identified two portions of Rb required for superactivation: (i) a portion of the Rb "pocket" (amino acids 614-839) previously determined to be required for physical interactions between Rb and transcription factors such as E2F-1 and (ii) a novel amino-terminal region (amino acids 140-202). Since both of these regions of Rb are targets of mutation in human tumors, our data suggest that superactivation of Sp1/Sp3 may play a role in Rb-mediated growth suppression and/or the induction of differentiation.

Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol.

Antisense oligodeoxyribonucleotides targeted to the epidermal growth factor (EGF) receptor were encapsulated into liposomes linked to folate via a polyethylene glycol spacer (folate-PEG-liposomes) and efficiently delivered into cultured KB cells via folate receptor-mediated endocytosis. The oligonucleotides were a phosphodiester 15-mer antisense to the EGF receptor (EGFR) gene stop codon (AEGFR2), the same sequence with three phosphorothioate linkages at each terminus (AEGFR2S), a randomized 15-mer control of similar base composition to AEGFR2 (RC15), a 14-mer control derived from a symmetrized Escherichia coli lac operator (LACM), and the 5'-fluorescein-labeled homologs of several of the above. Cellular uptake of AEGFR2 encapsulated in folate-PEG-liposomes was nine times higher than AEGFR2 encapsulated in nontargeted liposomes and 16 times higher than unencapsulated AEGFR2. Treatment of KB cells with AEGFR2 in folate-PEG-liposomes resulted in growth inhibition and significant morphological changes. Curiously, AEGFR2 and AEGFR2S encapsulated in folate-PEG-liposomes exhibited virtually identical growth inhibitory effects, reducing KB cell proliferation by > 90% 48 hr after the cells were treated for 4 hr with 3 microM oligonucleotide. Free AEGFR2 caused almost no growth inhibition, whereas free AEGFR2S was only one-fifth as potent as the folate-PEG-liposome-encapsulated oligonucleotide. Growth inhibition of the oligonucleotide-treated cells was probably due to reduced EGFR expression because indirect immunofluorescence staining of the cells with a monoclonal antibody against the EGFR showed an almost quantitative reduction of the EGFR in cells treated with folate-PEG-liposome-entrapped AEGFR2. These results suggest that antisense oligonucleotide encapsulation in folate-PEG-liposomes promise efficient and tumor-specific delivery and that phosphorothioate oligonucleotides appear to offer no major advantage over native phosphodiester DNA when delivered by this route.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.