Adenoviral gene transfer of beta3 integrin subunit induces conversion from radial to vertical growth phase in primary human melanoma

Expression of the beta3 subunit of the alphavbeta3 vitronectin receptor on melanoma cells is associated with tumor thickness and the ability to invade and metastasize. To address the role of alphavbeta3 in the complex process of progression from the nontumorigenic radial to the tumorigenic vertical growth phase of primary melanoma, we examined the biological consequences of overexpressing alphavbeta3 in early-stage melanoma cells using an adenoviral vector for gene transfer. Overexpression of functional alphavbeta3 in radial growth phase primary melanoma cells 1) promotes both anchorage-dependent and -independent growth, 2) initiates invasive growth from the epidermis into the dermis in three-dimensional skin reconstructs, 3) prevents apoptosis of invading cells, and 4) increases tumor growth in vivo. Thus, alphavbeta3 serves diverse biological functions during the progression from the nontumorigenic radial growth phase to the tumorigenic and-invasive vertical growth phase primary melanoma.     

Transforming growth factor beta 1 induces squamous carcinoma cell variants with increased metastatic abilities and a disorganized cytoskeleton

Previous studies indicated that mouse transformed keratinocytes undergo an epithelial-fibroblastic conversion when cultured in the presence of TGF-beta1. This conversion is associated in vivo with a squamous-spindle carcinoma transition. We derived epithelioid (A6, FPA6) and spindle (B5) clonal cell variants from a squamous carcinoma cell line (PDV) after treatment with TGF-beta1. FPA6 cells were isolated from the ascites fluid of an A6-tumor-bearing mouse. FPA6 and A6 cell lines produced in nude mice mixed carcinomas with a squamous and poorly differentiated component. Both cell lines coexpressed keratins and vimentin and synthesized E-cadherin protein, although FPA6 cells cultured at early passages (FPA6-ep) had reduced levels of E-cadherin mRNA and increased synthesis of keratin K8, a marker of malignant progression. Immunofluorescence analysis revealed that FPA6-ep cells exhibited a disorganized cytoskeleton with keratins forming focal juxtanuclear aggregates and loss of F-actin stress fibers and cortical bundles, and E-cadherin was localized in the cytoplasm out of cell-cell contact areas. Sporadic cells in A6 and PDV cultures also presented those anomalous keratin structures, suggesting that FPA6 cells originated from a subpopulation of A6 tumor cells that metastasized into the peritoneal cavity. The analysis of the spontaneous and experimental metastatic potentials of the cell lines showed that epithelioid and fibroblastic cell variants had acquired metastatic abilities compared to PDV which was nonmetastatic. The FPA6-ep cell line exhibited a highly aggressive behavior, killing the animals at about 17 days after intravenous injection of the cells into athymic mice. The phenotype of FPA6-ep cells was unstable and reverted at later passages in which the normal organization of keratin and F-actin in filaments and the localization of E-cadherin at cell-cell contacts were restored. This phenotypic reversion occurred concomitantly with a reduction of the experimental metastatic potential of FPA6 cells.     

The C825T polymorphism in the human G-protein beta3 subunit gene is not associated with diabetic nephropathy in Type I diabetes mellitus

In Type I (insulin-dependent) diabetes mellitus a genetic predisposition exists to nephropathy and is related to parental hypertension. Enhanced G-protein activation, a cellular phenotype observed in cultured cells from patients with essential hypertension, was recently documented in Type I diabetic subjects with nephropathy. This enhanced G-protein activation has been associated with a genetic variant in the G-protein beta3 subunit, GNB3. A C-->T polymorphism at position 825 in exon 10 is associated with G-protein activation, the T allele associated with enhanced activity. Furthermore the T allele was observed more frequently in a group with essential hypertension. In this report we have analysed the role of the C825T polymorphism in the predisposition to diabetic nephropathy in Type I diabetes. We have investigated the frequency of this polymorphism in a large case-control study and found no association of the T allele with diabetic nephropathy. Specifically carriage of the T allele as CT or TT was observed in 49% of 200 Type I diabetic control subjects with normoalbuminuria (diabetes duration 24 years) compared with 53% of 216 Type I diabetic subjects with nephropathy (overt proteinuria or end-stage renal failure). Within this group we have also examined the inheritance of C825T alleles in a family study and found no evidence for excess transmission of the T allele to Type I diabetic offspring with nephropathy (T allele transmitted to 51% of nephropathy offspring, C allele transmitted to 49% of nephropathy offspring, p = 0.79). In none of the Type I diabetic datasets examined was there any effect of genotype on variation in systolic or diastolic blood pressure. In conclusion we can find no evidence for the C825T polymorphism of the beta3 G-protein subunit as a major gene in the susceptibility to diabetic nephropathy in Type I diabetes.     

A peptidomimetic antagonist of the integrin alpha(v)beta3 inhibits Leydig cell tumor growth and the development of hypercalcemia of malignancy

The integrin alpha(v)beta3 interacts with the arginine-glycine-aspartic acid (RGD) tripeptide recognition sequence of a variety of extracellular matrix proteins. Recent studies show that alpha(v)beta3 plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha(v)beta3 inhibit angiogenic processes that include endothelial cell adhesion and migration. Consequently, we reasoned that an RGD-based peptidomimetic antagonist of alpha(v)beta3 might inhibit tumor angiogenesis and tumor growth in vivo. An RGD-peptidomimetic library was screened to identify antagonists of vitronectin binding to alpha(v)beta3, and the compounds chosen were modified to produce selective and potent inhibitors of alpha(v)beta3. One of these compounds, beta-[[2-2-[[[3-[(aminoiminomethyl)amino]-phenyl]carbonyl]amino]ac etyl]amino]-3,5-dichlorobenzenepropanoic acid (SC-68448), inhibited vitronectin binding to both alpha(v)beta3 and the closely related platelet receptor, alpha(IIb)beta3, in a dose-responsive manner. SC-68448 inhibited vitronectin binding to alpha(v)beta3 (IC50, 1 nM) and fibrinogen binding to the platelet receptor alpha(IIb)beta3 (IC50, >100 nM), demonstrating that SC-68448 was 100-fold more potent as an inhibitor of alpha(v)beta3 versus alpha(IIb)beta3. In cell-based studies, SC-68448 inhibited alpha(v)beta3-mediated endothelial cell proliferation in a dose-dependent manner but did not inhibit tumor cell proliferation, suggesting that effects on endothelial cell proliferation were not due to SC-68448-induced cytotoxicity. In accord with these results, SC-68448 inhibited angiogenesis in vivo in a basic fibroblast growth factor-induced rat corneal neovascularization model. A xenogeneic severe combined immune deficiency mouse/rat Leydig cell tumor model was developed for testing SC-68448 as an inhibitor of tumor growth in vivo. Rat Leydig cell tumors grew rapidly in severe combined immune deficiency mice and produced humoral hypercalcemia of malignancy. SC-68448 inhibited the growth of the tumors in mice by up to 80% and completely blocked the development of hypercalcemia. Together, these results demonstrate the feasibility of antitumor therapies based upon the development of nontoxic small molecule pharmacological antagonists of integrin alpha(v)beta3.     

Calretinin- and parvalbumin-immunoreactive neurons in the rat main olfactory bulb do not express NADPH-diaphorase activity

The presence of nitric oxide synthase (NOS) in neuronal elements expressing the calcium-binding proteins calretinin (CR) and parvalbumin (PV) was studied in the rat main olfactory bulb. CR and PV were detected by using immunocytochemistry and the nitric oxide (NO) -synthesizing cells were identified by means of the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) direct histochemical method. The possible coexistence of NADPH-diaphorase and each calcium-binding protein marker was determined by sequential histochemical-immunohistochemical double-labeling of the same sections. Specific neuronal populations were positive for these three markers. A subpopulation of olfactory fibers and olfactory glomeruli were positive for either NADPH-diaphorase or CR. In the most superficial layers, groups of juxtaglomerular cells, superficial short-axon cells and Van Gehuchten cells demonstrated staining for all three markers. In the deep regions, abundant granule cells were NADPH-diaphorase- and CR-positive and a few were PV-immunoreactive. Scarce deep short-axon cells demonstrated either CR-, PV-, or NADPH-diaphorase staining. Among all these labeled elements, no neuron expressing CR or PV colocalized NADPH-diaphorase staining. The present data contribute to a more detailed classification of the chemically- and morphologically-defined neuronal types in the rodent olfactory bulb. The neurochemical differences support the existence of physiologically distinct groups within morphologically homogeneous populations. Each of these groups would be involved in different modulatory mechanisms of the olfactory information. In addition, the absence of CR and PV in neuronal groups displaying NADPH-diaphorase, which moreover are calmodulin-negative, indicate that the regulation of NOS activity in calmodulin-negative neurons of the rat olfactory bulb is not mediated by CR or PV.     

The reactivity of o-quinones which do not isomerize to quinone methides correlates with alkylcatechol-induced toxicity in human melanoma cells

Catechols are widespread in the environment, especially as constituents of edible plants. A number of these catechols may undergo oxidative metabolism to electrophilic o-quinones (3,5-cyclohexadien-1,2-dione) by oxidative enzymes such as cytochrome P450 and peroxidases. Alkylation of cellular nucleophiles by these intermediates and the formation of reactive oxygen species, especially through redox cycling of o-quinones, could contribute to the cytotoxic properties of the parent catechols. In contrast, isomerization of the o-quinones to electrophilic quinone methides (4-methylene-2,5-cyclohexadien-1-one, QM) could cause cellular damage primarily through alkylation. In this investigation, we treated human melanoma cells with two groups of catechols. These cells have high levels of tyrosinase required to oxidize catechols to quinoids. For catechols which are oxidized to o-quinones that cannot isomerize to quinone methides or form unstable quinone methides, plots of the cytotoxicity data (ED50) versus the reactivity of the o-quinones gave an excellent linear correlation; decreasing o-quinone reactivity led to a decrease in the cytotoxic potency of the catechol. In contrast, catechols which are metabolized by the o-quinone/p-quinone methide bioactivation pathway were equally cytotoxic but showed no correlation between the reactivity of the o-quinones and the cytotoxic potency of the catechols. The most likely explanation for this effect is a change in cytotoxic mechanism from o-quinone-mediated inhibition of cell growth to a bioactivation pathway based on both o-quinone and p-QM formation. These results substantiate the conclusion that the involvement of the o-quinone/ QM pathway in catechol toxicity depends on a combination between the rate of enzymatic formation of the o-quinone, the rate of isomerization to the more electrophilic QM, and the chemical reactivity of the quinoids.     

Activation status and function of the VLA-4 (alpha4beta1) integrin expressed on human melanoma cell lines

A highly sensitive microspectrophotometer was developed to measure spectral changes of oxyhemoglobin (oxy Hb) in single red blood cells (RBC) incubated with stimulated macrophages as a model of nitric oxide (NO) dependent cytotoxicity. Our microspectrophotometer, using a modified acousto-optic tunable filter (AOTF) and a 2-dimensional CCD array, allows fast spectrophotometric data acquisition. Human RBC treated with various concentrations of NO showed spectral changes due to the conversion of oxy Hb to methemoglobin (met Hb), in which the change in absorption differences at alpha (557 590 nm) and beta (542-525 nm) bands showed a linear relationship with the concentration of NO up to 100 microM. In contrast to highly diffusible NO, nitrite ions (NO2-) seem to enter RBC very slowly, resulting in negligible formation of met Hb in the presence of 5 mM glucose even during a prolonged incubation period. RBC were incubated with murine macrophages with and without lipopolysaccharide (LPS) in the presence of glucose for 24 and 40 h and subjected to the microspectrophotometric assay. The RBC incubated with LPS-stimulated macrophages showed significant changes in the spectrum due to NO-dependent conversion of oxy Hb to met Hb, which corresponded to the spectral changes of RBC treated with a several times higher concentration of NO than that in the culture medium. The trapping efficiency was calculated from the amounts of the NO released from macrophages and of the met Hb formed in the RBC, which gave a high efficiency (43%). The results suggest that RBC trap NO directly by cell cell interaction with macrophages. This spectrophotometric system is available for use with just a few drops of samples to study NO-specific cytotoxicity as a model of RBC without the use of any chemical reagent, in parallel with microscopic observations on changes of the cellular morphology under physiological conditions, such as membrane damage leading to hemolysis, adherence, and phagocytosis.     

A conserved sequence motif in the integrin beta3 cytoplasmic domain is required for its specific interaction with beta3-endonexin

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed beta3-endonexin, which interacts selectively with the integrin beta3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin beta3 cytoplasmic domain and beta3-endonexin to map sites required for interaction. The interaction of the full-length beta3 integrin subunit with beta3-endonexin in vitro required the beta3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the beta3 cytoplasmic domain were necessary for interaction with beta3-endonexin. In particular, the membrane-distal NITY motif at beta3 756-759 was critical for the interaction. Exchange of beta3 residues 756-759 (NITY) for the corresponding residues in beta1 (NPKY) endowed the beta1 cytoplasmic domain with the ability to interact with beta3-endonexin. Conversely, exchange of the NPKY motif at beta1 772-775 for the NITY motif in beta3 abolished interaction of this chimeric cytoplasmic domain with beta3-endonexin. Because the NITY motif is present in the beta3 but not the beta1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with beta3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of beta3-endonexin is sufficient for specific binding to the beta3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.     

Clinical significance of alpha(v)beta3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions

Several lines of experimental evidence in in vitro and animal model systems suggest that the integrin alpha(v)beta3 plays a role in the tumorigenicity of human melanoma cells and that the blocking of alpha(v)beta3 ligand binding can inhibit tumor progression. However, there is only scanty information about the role of alpha(v)beta3 in malignant melanoma in a clinical setting. Therefore, in the present study, we have analyzed the distribution in lesions of melanocyte origin and in normal tissues of the alpha(v) integrin subunit and of the alpha(v)beta3 complex and their association with histopathological and clinical parameters of malignant melanoma. We have used as probes the monoclonal antibodies (mAbs) TP36.1 and VF27.263.15, which we have shown with a combination of serological and immunochemical assays to be specific for the alpha(v) subunit and for the alpha(v)beta3 complex, respectively. In immunohistochemical assays, mAb TP36.1 stained both benign and malignant lesions of melanocyte origin. In contrast, the reactivity of mAb VF27.263.15 was restricted to malignant lesions. Both mAbs displayed differential reactivity with primary melanoma lesions of different histotypes because they stained about 50% of acral lentiginous melanoma and superficial spreading melanoma lesions, at least 80% of nodular melanoma lesions, and none of the uveal melanoma lesions tested. Both mAbs TP36.1 and VF27.263.15 stained about 60% of lymph node metastases and 80% of cutaneous metastases. Expression of the alpha(v)beta3 complex in melanocytic lesions resembles that of intercellular adhesion molecule-1 (ICAM-1) in several respects: (a) both are expressed in a significantly (P < 0.004) larger proportion of malignant than of benign lesions; (b) expression of both molecules in primary melanoma lesions is significantly (P < 0.05) associated with lesion thickness; and (c) expression of both molecules in primary lesions from patients with stage I melanoma is significantly (P < 0.05) associated with an increased probability of disease recurrence following surgical excision. alpha(v)beta3 and ICAM-1 in primary melanoma lesions complement each other in predicting the outcome of the disease, because the association with prognosis was enhanced when primary lesions were stained by both anti-alpha(v)beta3 mAb VF27.263.15 and anti-ICAM-1 mAb CL203.4 or by neither mAb. Because alpha(v)beta3 has been suggested as a potential target of immunotherapy, its distribution in normal tissues was investigated. alpha(v)beta3 expression is restricted because it was only detected in ductal epithelium of parotid glands, thyrocytes, basal glands of the stomach, colonic and rectal epithelium glomeruli, Bowman's capsules and proximal and distal tubules of kidneys, and endometrial epithelium. These findings suggest that renal function will be a critical clinical parameter to monitor in therapies of malignant diseases relying on systemic administration of anti-alpha(v)beta3 mAb.     

In vivo tumor transfection with superantigen plus cytokine genes induces tumor regression and prolongs survival in dogs with malignant melanoma

In vivo transfection of established tumors with immunostimulatory genes can elicit antitumor immunity. Therefore, we evaluated the safety and efficacy of intratumoral injections of a bacterial superantigen with a cytokine gene in dogs with malignant melanoma, a spontaneous and highly malignant canine tumor. 26 dogs with melanoma were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin B and either GM-CSF or IL-2. Dogs were evaluated for treatment-associated toxicity, tumor responses, immunologic responses, and survival times. The overall response rate (complete or partial remissions) for all 26 dogs was 46% (12 of 26), and was highest in patients with smaller tumors. Toxicity was minimal or absent in all dogs. Injected tumors developed marked infiltrates of CD4+ and CD8+ T cells and macrophages, and tumor regression was associated with development of high levels of antitumor cytotoxic T lymphocyte activity in peripheral blood lymphocytes. Survival times for animals with stage III melanomas treated by intratumoral gene therapy were prolonged significantly compared with animals treated with surgical tumor excision only. Thus, local tumor transfection with superantigen and cytokine genes was capable of inducing both local and systemic antitumor immunity in an outbred animal with a spontaneously developing malignant tumor.     

Activation of alphav beta3 integrin on human osteoclast-like cells stimulates adhesion and migration in response to osteopontin

Integrins mediate cell adhesion and can induce different cellular responses, including changes in intracellular pH, changes and oscillation in intracellular free calcium, and protein phosphorylation on tyrosine. During bone resorption, the integrin alphav beta3 regulates adhesion of osteoclasts to bone extracellular matrix proteins, such us osteopontin (Opn). Adhesion via alphav beta3 is followed by osteoclast polarization onto the bone surface and by the onset of bone resorption. To characterize these events at the molecular level, we investigated the state of activation of alphav beta3 on the human osteoclast-like cell line GCT23 using the monoclonal antibody AP5 which binds to and can induce, under low calcium conditions, activated alphav beta3. By flow cytometry, approximately 50% of alphav beta3 on the surface of the osteoclast-like cell line GCT23 was reactive with AP5 and was therefore in the activated state. Incubation with AP5 in the presence of low calcium concentrations increased activated alphav beta3 to 90-100%. Activation of alphav beta3 increased the efficiency of GCT23 adhesion to Opn by 2-fold. Furthermore, haptotactic migration on Opn was also enhanced about 40% compared to control. We propose that changes in the activation state of alphav beta3 may be a regulation point for osteoclasts during bone resorption.     

Expression of transforming growth factors beta-1, beta 2 and beta 3 in human bladder carcinomas

We previously detected elevated transforming growth factor beta-1 (TGF-beta1) serum levels in patients with invasive bladder carcinomas. In this study, we therefore investigated whether elevated serum levels correlate with enhanced TGF-beta expression in human bladder tumours. mRNA levels of TGF-beta1, -beta2 and -beta3 were reduced in bladder tumour tissue to 86%, 68% and 56%, respectively, of the levels in normal urothelium. On the other hand, TGF-beta1 protein levels were found to be higher in superficial tumours (Ta-T1) (mean level of 0.153 ng mg(-1)) and in invasive T2/T3 tumours (mean level of 0.104 ng mg(-1)) compared with normal urothelium (mean level of 0.065 ng mg(-1)). Invasive T4 tumours, however, contained only low amounts of TGF-beta1 (mean level of 0.02 ng mg(-1)). Neither in mean nor in individual patients were serum and tissue TGF-beta levels correlated with each other. Cell culture experiments on primary bladder cells revealed a 57% decrease in TGF-beta1 mRNA levels in tumour compared with normal epithelial cells. Tumour epithelial cells contained about two times higher levels of TGF-beta2 and TGF-beta3 mRNA than normal epithelial cells. Fibroblasts expressed about the same amount of TGF-beta1 or TGF-beta2 as epithelial cells. Yet, fibroblasts released only 19% and 13% of the amount secreted by tumour epithelial cells into the supernatant. TGF-beta3, on the other hand, was expressed by fibroblasts with higher levels than by epithelial cells. TGF-beta1 was the predominent isoform in bladder tissue and cells at protein as well as on mRNA levels indicating that TGFs-beta2 and -beta3 are of minor importance in bladder cancer. In summary, there is a lack of correlation between TGF-beta serum levels and TGF-beta expression in tumour tissue in bladder cancer.     

Ligand-like effects induced by anti-c-erbB-2 antibodies do not correlate with and are not required for growth inhibition of human carcinoma cells

The c-erbB-2 gene encodes a M(r) 185,000 tyrosine kinase receptor (p185) with extensive homology to the epidermal growth factor receptor. We have conducted mechanistic studies with several anti-p185 monoclonal antibodies (TAb 250, -255, -257, -260, and -263) directed against the extracellular domain of p185 utilizing the SKBR-3, BT-474, and SKOV-3 cancer cell lines. Several of these antibodies exhibited ligand-mimicking properties: they induced tyrosine phosphorylation of p185; increased the catalytic activity of the receptor substrate phospholipase C-gamma 1; exhibited time- and pH-dependent internalization; induced receptor down-regulation; and increased the turnover of the p185 protein delta 3-fold. However, there was not a universal correlation between the antibody-mediated ligand-like effects and growth inhibition. TAb 250 inhibited BT-474 cells but did not alter p185 phosphotyrosine content or increase receptor turnover in these cells. TAb 260 increased p185 protein turnover but did not affect proliferation of the SKOV-3 cell line. Furthermore, blockade of TAb 250-induced receptor phosphorylation with the tyrosine kinase inhibitor tyrphostin 50864-2 did not abrogate TAb 250-mediated growth inhibition of SKBR-3 cells. These data suggest that ligand-like effects mediated by p185 antibodies are not critical for the growth inhibition of c-erbB-2-overexpressing carcinoma cells.     

Expression of platelet-derived growth factor (PDGF)-A, PDGF-B and the PDGF-alpha receptor, but not the PDGF-beta receptor, in human malignant melanoma in vivo

There has been considerable interest in the potential role of growth factors in the initiation and development of cutaneous malignant melanoma (CMM). Platelet-derived growth factor (PDGF) has been shown to be secreted by melanoma cell lines and by metastatic melanoma in vivo. PDGF also has been reported to stimulate the development of tumour stroma and new blood vessels. We studied the expression of PDGF and its receptors by both immunohistochemistry (IHC) and in situ hybridization (ISH) in primary and metastatic melanoma and in normal skin specimens. Cryostat sections were incubated with 35S-labelled riboprobes and antibodies for PDGF-AA, PDGF-alpha receptor, PDGF-BB and PDGF-beta receptor. Both primary and metastatic melanoma exhibited significant expression of PDGF-AA, PDGF-BB and PDGF-alpha receptor by both IHC and ISH, compared with only background expression in normal skin. We did not observe expression of PDGF-beta receptor in melanoma. Our results suggest that PDGF may function as an autocrine growth factor, as well as an angiogenesis factor, in CMM tumour development. This expression of the PDGF-alpha receptor rather than the beta receptor may be unique among solid tumours.     

Platelet integrin alpha IIb beta 3 (GPIIb-IIIa) is not implicated in the binding of LDL to intact resting platelets

The anti-tumor activity of irinotecan (CPT-11), a DNA-topoisomerase 1 inhibitor, was evaluated in 5 advanced stage subcutaneous medulloblastoma xenografts in nude mice, using different schedules of administration. With a 5-day schedule, the highest i.v. dose tested (40 mg kg-1 day-1) induced complete regressions in all xenografts but 1, and delays in tumor growth always exceeded 30 days. Two xenografts, IGRM11 and IGRM33, were highly sensitive, and animals survived tumor-free beyond 120 days after treatment. CPT-11 clearly retained its anti-tumor activity at a lower dosage (27 mg kg-1 day-1). CPT-11 was significantly more active than cyclophosphamide, thiotepa and etoposide against the 3 xenografts evaluated. To study the schedule dependency of its anti-tumor activity, CPT-11 was given i.v. at the same total doses over the same period (33 days) using either a protracted or a sequential schedule in IGRM34-bearing mice. With a dose of 10 mg kg-1 day-1 given on days 0-4, days 7-11, days 21-25 and days 28-32 (total dose, 200 mg kg-1), 3 of 6 animals were tumor free on day 378. The same total dose given with a sequential schedule, i.e., 20 mg kg-1 day-1 on days 0-4 and days 28-32, failed to induce complete regression. The plasma pharmacokinetics of CPT-11 and SN-38 were studied in IGRM34-bearing animals after a single i.v. dose of 10 and 40 mg kg-1. The plasma clearance rate of CPT-11 was dose dependent. The ratio between the SN-38 and CPT-11 area under the curve in plasma was 0.4-0.65, i.e., significantly higher than that observed in humans at the maximum tolerated dose (0.01-0.05). Conversely, this ratio was 10-fold lower in tumor than in plasma. Clinical development of irinotecan is warranted in pediatric malignancies.     

In vivo induction of the interferon-stimulated protein 2''5''-oligoadenylate synthetase in tumor and peripheral blood cells during IFN-alpha treatment of metastatic melanoma

Learning, making memories, and forgetting are thought to require changes in the strengths of connections between neurons. Such changes in synaptic strength occur in two phases: an early phase that is likely mediated by covalent modifications to existing proteins, and a delayed phase that depends on new gene expression and protein synthesis. However, the biochemical mechanisms by which neuronal activity leads to changes in synaptic strength are poorly understood. Recently, it has been shown that animals that lack Ras guanine nucleotide releasing factor (Ras-GRF), a Ca(2+)-dependent activator of the small GTP-binding protein, Ras, do not learn fear responses normally, although other types of learning appear normal. These animals show defects in the delayed phase of memory formation within the neuronal circuit that mediates fear conditioning. This paper suggests that Ras-GRF couples synaptic activity to the molecular mechanisms that consolidate changes in synaptic strength within specific neuronal circuits.     

Soluble ligands of the alpha v beta 3 integrin mediate enhanced tyrosine phosphorylation of multiple proteins in adherent bovine pulmonary artery endothelial cells

Binding of substrate-bound extracellular matrix proteins to cell surface integrins results in a variety of cellular responses including adhesion, cytoskeletal reorganization, and gene expression. We have previously shown that addition of soluble SC5b-9, the complement-vitronectin complex, resulted in an RGD-dependent increase in lung venular hydraulic conductivity (Ishikawa, S., Tsukada, H., and Bhattacharya, J. (1993) J. Clin. Invest. 91, 103-109). To identify specific integrin(s) and signal transduction pathways that are responsive to soluble vitronectin-containing ligands, we exposed confluent bovine pulmonary artery cells to purified soluble human mono- or multimeric vitronectin, or SC5b-9, and determined the extent of endothelial cell protein tyrosine phosphorylation. Monomeric vitronectin (Vn) did not induce enhanced protein tyrosine phosphorylation. However, multimeric Vn and SC5b-9 elicited time- and concentration-dependent increases in tyrosine phosphorylation of numerous proteins. Antiserum against vitronectin, RGD peptides, and monoclonal and polyclonal antibodies against the alpha v beta 3 integrin blocked the vitronectin- or SC5b-9-induced enhanced accumulation of tyrosine phosphoproteins, while antibodies against beta 1 integrins and the alpha v beta 5 integrin did not. Clustering of the alpha v beta 3 integrin using monoclonal antibody LM609 caused a pattern of enhanced tyrosine phosphorylation similar to that caused by multimeric Vn and SC5b-9, suggesting that aggregation of alpha v beta 3 was critical for signaling. Among the proteins that underwent enhanced tyrosine phosphorylation in response to vitronectin were the cytoskeletal proteins paxillin, cortactin, and ezrin, as well as the SH2 domain-containing protein Shc, and p125FAK. We conclude that ligation of the alpha v beta 3 integrin by soluble ligands promotes enhanced phosphorylation of several proteins implicated in tyrosine kinase signaling and suggest that this pathway may be important in inflammatory states which are accompanied by accumulation of SC5b-9.