A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.

Comparison of fusion phage libraries displaying VH or single-chain Fv antibody fragments derived from the antibody repertoire of a vaccinated melanoma patient as a source of melanoma-specific targeting molecules

A single-chain Fv (scFv) fusion phage library derived from random combinations of VH and VL (variable heavy and light chains) domains in the antibody repertoire of a vaccinated melanoma patient was previously used to isolate clones that bind specifically to melanoma cells. An unexpected finding was that one of the clones encoded a truncated scFv molecule with most of the VL domain deleted, indicating that a VH domain alone can exhibit tumor-specific binding. In this report a VH fusion phage library containing VH domains unassociated with VL domains was compared with a scFv fusion phage library as a source of melanoma-specific clones; both libraries contained the same VH domains from the vaccinated melanoma patient. The results demonstrate that the clones can be isolated from both libraries, and that both libraries should be used to optimize the chance of isolating clones binding to different epitopes. Although this strategy has been tested only for melanoma, it is also applicable to other cancers. Because of their small size, human origin and specificity for cell surface tumor antigens, the VH and scFv molecules have significant advantages as tumor-targeting molecules for diagnostic and therapeutic procedures and can also serve as probes for identifying the cognate tumor antigens.

ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins

The adenylate uridylate-rich elements (AREs) mediate the rapid turnover of mRNAs encoding proteins that regulate cellular growth and body response to exogenous agents such as microbes, inflammatory and environmental stimuli. However, the full repertoire of ARE-containing mRNAs is unknown. Here, we explore the distribution of AREs in human mRNA sequences. Computational derivation of a 13-bp ARE pattern was performed using multiple expectation maximization for motif elicitations (MEME) and consensus analyses. This pattern was statistically validated for the specificity towards the 3′-untranslated region and not coding region. The computationally derived ARE pattern is the basis of a database which contains non-redundant full-length ARE-mRNAs. The ARE-mRNA database (ARED; http://rc.kfshrc.edu.sa/ared) reveals that ARE-mRNAs encode a wide repertoire of functionally diverse proteins that belong to different biological processes and are important in several disease states. Cluster analysis was performed using the ARE sequences to demonstrate potential relationships between the type and number of ARE motifs, and the functional characteristics of the proteins.

Localization of vasopressin mRNA and immunoreactivity in pituicytes of pituitary stalk-transected rats after osmotic stimulation.

The presence of [arginine] vasopressin (AVP) mRNA and AVP immunoreactivity in pituicytes of the neural lobe (NL) of intact and pituitary stalk-transected rats, with and without osmotic stimulation, was examined. AVP mRNA was analyzed by Northern blotting, as well as by in situ hybridization in combination with immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) as a marker for pituicytes. In intact rats, a poly(A) tail-truncated 0.62-kb AVP mRNA was detected in the NL and was found to increase 10-fold with 7 days of continuous salt loading. Morphological analysis of the NL of 7-day salt-loaded rats revealed the presence of AVP mRNA in a significant number of GFAP-positive pituicytes in the NL and in areas most probably containing nerve fibers. Eight days after pituitary stalk transection the NL AVP mRNA diminished in animals given water to drink, whereas in those given 2% saline for 18 h followed by 6 h of water, a treatment repeated on 6 successive days beginning 2 days after surgery, the 0.62-kb AVP mRNA was present. The AVP mRNA in the pituitary stalk-transected, salt-loaded rats showed an exclusive cellular distribution in the NL, indicative of localization in pituicytes. Immunoelectron microscopy showed the presence of AVP immunoreactivity in a subpopulation of pituicytes 7 and 10 days after pituitary stalk transection in salt-loaded animals, when almost all AVP fibers had disappeared from the NL. These data show that a subset of pituicytes in the NL is activated to synthesize AVP mRNA and AVP in response to osmotic stimulation.

Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation

Electrical stimulation of neonatal cardiac myocytes produces hypertrophy and cellular maturation with increased mitochondrial content and activity. To investigate the patterns of gene expression associated with these processes, cardiac myocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation [c-fos, c-jun, JunB, nuclear respiratory factor 1 (NRF-1)], mitochondrial proliferation [cytochrome c (Cyt c), cytochrome oxidase], and mitochondrial differentiation [carnitine palmitoyltransferase I (CPT-I) isoforms] were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25–3 hr) and followed sequentially by c-jun (0.5–3 hr), JunB (0.5–6 hr), NRF-1 (1–12 hr), Cyt c (12–72 hr), and muscle-specific CPT-I (48–72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA, thus supporting the developmental fidelity of this pattern of gene regulation. Consistent with a transcriptional mechanism, electrical stimulation increased c-fos, β-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element, and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the NRF-1 and CRE sites inhibited the induction by electrical stimulation (5-fold and 2-fold, respectively) whereas mutation of the Sp-1 site maintained or increased the fold induction. This finding is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c and suggests that induction of these transcription factors is a prerequisite for the transcriptional activation of Cyt c expression. These results support a regulatory role for NRF-1 and possibly AP-1 in the initiation of mitochondrial proliferation.

Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca2+ influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca2+ entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca2+ influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.

Autoregulation at the level of mRNA 3′ end formation of the suppressor of forked gene of Drosophila melanogaster is conserved in Drosophila virilis

The Drosophila melanogaster Suppressor of forked [Su(f)] protein shares homology with the yeast RNA14 protein and the 77-kDa subunit of human cleavage stimulation factor, which are proteins involved in mRNA 3′ end formation. This suggests a role for Su(f) in mRNA 3′ end formation in Drosophila. The su(f) gene produces three transcripts; two of them are polyadenylated at the end of the transcription unit, and one is a truncated transcript, polyadenylated in intron 4. Using temperature-sensitive su(f) mutants, we show that accumulation of the truncated transcript requires wild-type Su(f) protein. This suggests that the Su(f) protein autoregulates negatively its accumulation by stimulating 3′ end formation of the truncated su(f) RNA. Cloning of su(f) from Drosophila virilis and analysis of its RNA profile suggest that su(f) autoregulation is conserved in this species. Sequence comparison between su(f) from both species allows us to point out three conserved regions in intron 4 downstream of the truncated RNA poly(A) site. These conserved regions include the GU-rich downstream sequence involved in poly(A) site definition. Using transgenes truncated within intron 4, we show that sequence up to the conserved GU-rich domain is sufficient for production of the truncated RNA and for regulation of this production by su(f). Our results indicate a role of su(f) in the regulation of poly(A) site utilization and an important role of the GU-rich sequence for this regulation to occur.

Models of immune memory: On the role of cross-reactive stimulation, competition, and homeostasis in maintaining immune memory

There has been much debate on the contribution of processes such as the persistence of antigens, cross-reactive stimulation, homeostasis, competition between different lineages of lymphocytes, and the rate of cell turnover on the duration of immune memory and the maintenance of the immune repertoire. We use simple mathematical models to investigate the contributions of these various processes to the longevity of immune memory (defined as the rate of decline of the population of antigen-specific memory cells). The models we develop incorporate a large repertoire of immune cells, each lineage having distinct antigenic specificities, and describe the dynamics of the individual lineages and total population of cells. Our results suggest that, if homeostatic control regulates the total population of memory cells, then, for a wide range of parameters, immune memory will be long-lived in the absence of persistent antigen (T1/2 > 1 year). We also show that the longevity of memory in this situation will be insensitive to the relative rates of cross-reactive stimulation, the rate of turnover of immune cells, and the functional form of the term for the maintenance of homeostasis.

Karyopherin β2 mediates nuclear import of a mRNA binding protein

We have cloned and sequenced cDNA for human karyopherin β2, also known as transportin. In a solution binding assay, recombinant β2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1’s previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin β1, karyopherin β2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β1 and β2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β2 inhibited karyopherin α/β1-mediated import of a classical NLS containing substrate and, vice versa, β1 inhibited β2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β1 and β2 to repeat nucleoporins.

Stimulation of amyloid precursor protein synthesis by adrenergic receptors coupled to cAMP formation

Amyloid plaques in Alzheimer disease are primarily aggregates of Aβ peptides that are derived from the amyloid precursor protein (APP). Neurotransmitter agonists that activate phosphatidylinositol hydrolysis and protein kinase C stimulate APP processing and generate soluble, non-amyloidogenic APP (APPs). Elevations in cAMP oppose this stimulatory effect and lead to the accumulation of cell-associated APP holoprotein containing amyloidogenic Aβ peptides. We now report that cAMP signaling can also increase cellular levels of APP holoprotein by stimulating APP gene expression in astrocytes. Treatment of astrocytes with norepinephrine or isoproterenol for 24 h increased both APP mRNA and holoprotein levels, and these increases were blocked by the β-adrenergic antagonist propranolol. Treatment with 8-bromo-adenosine 3′,5′-cyclic monophosphate or forskolin for 24 h similarly increased APP holoprotein levels; astrocytes were also transformed into process-bearing cells expressing increased amounts of glial fibrillary acidic protein, suggesting that these cells resemble reactive astrocytes. The increases in APP mRNA and holoprotein in astrocytes caused by cAMP stimulation were inhibited by the immunosuppressant cyclosporin A. Our study suggests that APP overexpression by reactive astrocytes during neuronal injury may contribute to Alzheimer disease neuropathology, and that immunosuppressants can inhibit cAMP activation of APP gene transcription.

Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.

Inhibition of Rho Is Required for cAMP-induced Melanoma Cell Differentiation

Up-regulation of the cAMP pathway by forskolin or α-melanocyte stimulating hormone induces melanocyte and melanoma cell differentiation characterized by stimulation of melanin synthesis and dendrite development. Here we show that forskolin-induced dendricity is associated to a disassembly of actin stress fibers. Since Rho controls actin organization, we studied the role of this guanosine triphosphate (GTP)-binding protein in cAMP-induced dendrite formation. Clostridium botulinum C3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia coli toxin cytotoxic necrotizing factor-1 (CNF-1), which activates Rho and the expression of a constitutively active Rho mutant, blocked forskolin-induced dendrite outgrowth. In addition, overexpression of a constitutively active form of the Rho target p160 Rho-kinase (P160ROCK) prevented the dendritogenic effects of cAMP. Our results suggest that inhibition of Rho and of its target p160ROCK are required events for cAMP-induced dendrite outgrowth in B16 cells. Furthermore, we present evidence that Rho is involved in the regulation of melanogenesis. Indeed, Rho inactivation enhanced the cAMP stimulation of tyrosinase gene transcription and protein expression, while Rho constitutive activation impaired these cAMP-induced effects. This reveals that, in addition to controlling dendricity, Rho also participates in the regulation of melanin synthesis by cAMP.

Subcellular Localization of Myosin-V in the B16 Melanoma Cells, a Wild-type Cell Line for the dilute Gene

The discovery that the dilute gene encodes a class V myosin led to the hypothesis that this molecular motor is involved in melanosome transport and/or dendrite outgrowth in mammalian melanocytes. The present studies were undertaken to gain insight into the subcellular distribution of myosin-V in the melanoma cell line B16-F10, which is wild-type for the dilute gene. Immunofluorescence studies showed some degree of superimposed labeling of myosin-V with melanosomes that predominated at the cell periphery. A subcellular fraction highly enriched in melanosomes was also enriched in myosin-V based on Western blot analysis. Immunoelectron microscopy showed myosin-V labeling associated with melanosomes and other organelles. The stimulation of B16 cells with the α-melanocyte-stimulating hormone led to a significant increase in myosin-V expression. This is the first evidence that a cAMP signaling pathway might regulate the dilute gene expression. Immunofluorescence also showed an intense labeling of myosin-V independent of melanosomes that was observed within the dendrites and at the perinuclear region. Although the results presented herein are consistent with the hypothesis that myosin-V might act as a motor for melanosome translocation, they also suggest a broader cytoplasmic function for myosin-V, acting on other types of organelles or in cytoskeletal dynamics.

Melanoma angiogenesis and metastasis modulated by ribozyme targeting of the secreted growth factor pleiotrophin

Clinical and experimental evidence suggests that spreading of malignant cells from a localized tumor (metastasis) is directly related to the number of microvessels in the primary tumor. This tumor angiogenesis is thought to be mediated by tumor-cell-derived growth factors. However, most tumor cells express a multitude of candidate angiogenesis factors and it is difficult to decipher which of these are rate-limiting factors in vivo. Herein we use ribozyme targeting of pleiotrophin (PTN) in metastatic human melanoma cells to assess the significance of this secreted growth factor for angiogenesis and metastasis. As a model we used human melanoma cells (1205LU) that express high levels of PTN and metastasize from subcutaneous tumors to the lungs of experimental animals. In these melanoma cells, we reduced PTN mRNA and growth factor activity by transfection with PTN-targeted ribozymes and generated cell lines expressing different levels of PTN. We found that the reduction of PTN does not affect growth of the melanoma cells in vitro. In nude mice, however, tumor growth and angiogenesis were decreased in parallel with the reduced PTN levels and apoptosis in the tumors was increased. Concomitantly, the metastatic spread of the tumors from the subcutaneous site to the lungs was prevented. These studies support a direct link between tumor angiogenesis and metastasis through a secreted growth factor and identify PTN as a candidate factor that may be rate-limiting for human melanoma metastasis.

Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G1/S checkpoint and maintain high levels of p27KIP1 mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27KIP1 is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin α2β1, which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.