The signaling network downstream of the ErbB family of receptors has been extensively targeted by cancer therapeutics; however, understanding the relative importance of the different components of the ErbB network is nontrivial. To explore the optimal way to therapeutically inhibit combinatorial, ligand-induced activation of the ErbB-phosphatidylinositol 3-kinase (PI3K) axis, we built a computational model of the ErbB signaling network that describes the most effective ErbB ligands, as well as known and previously unidentified ErbB inhibitors. Sensitivity analysis identified ErbB3 as the key node in response to ligands that can bind either ErbB3 or EGFR (epidermal growth factor receptor). We describe MM-121, a human monoclonal antibody that halts the growth of tumor xenografts in mice and, consistent with model-simulated inhibitor data, potently inhibits ErbB3 phosphorylation in a manner distinct from that of other ErbB-targeted therapies. MM-121, a previously unidentified anticancer therapeutic designed using a systems approach, promises to benefit patients with combinatorial, ligand-induced activation of the ErbB signaling network that are not effectively treated by current therapies targeting overexpressed or mutated oncogenes.

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Abnormalities in cellular di erentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant ®broblast interferon (IFN-b) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell di erentiation. Subtraction hybridization identi®ed melanoma di erentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful e ects occur when mda-7 is expressed in normal epithelial or ®broblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Humda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-b plus MEZ. Mda-7 expression is also induced during megakaryocyte di erentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-b+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-b+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in signi®cant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a di erentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers. Oncogene (2001) 20, 7051 ± 7063.

Glutamate transport is central to neurotransmitter functions in the brain. Impaired glutamate transport induces neurotoxicity associated with numerous pathological processes, including stroke͞ischemia, temporal lobe epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, HIV-1-associated dementia, and growth of malignant gliomas. Excitatory amino acid transporter-2 (EAAT2) is a major glutamate transporter in the brain expressed primarily in astrocytes. We presently describe the cloning and characterization of the human EAAT2 promoter, demonstrating elevated expression in astrocytes. Regulators of EAAT2 transport, both positive and negative, alter EAAT2 transcription, promoter activity, mRNA, and protein. These findings imply that transcriptional processes can regulate EAAT2 expression. Moreover, they raise the intriguing possibility that the EAAT2 promoter may be useful for targeting gene expression in the brain and for identifying molecules capable of modulating glutamate transport that could potentially inhibit, ameliorate, or prevent various neurodegenerative diseases.sequential progressive genomic scanning ͉ nuclear run-on assays ͉ promoter reporter assays ͉ mRNA and protein expression

Terminal differentiation and cellular senescence display common properties including irreversible growth arrest. To define the molecular and ultimately the biochemical basis of the complex physiological changes associated with terminal differentiation and senescence, an overlapping-pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes. This approach involved screening of a subtracted cDNA library prepared from human melanoma cells induced to terminally differentiate by treatment with fibroblast IFN and mezerein with mRNA derived from senescent human progeria cells. overlapping-pathway screen ͉ terminal cell differentiation ͉ senescent phenotype ͉ interferon-inducible gene ͉ evolutionary conserved gene P lasticity of the transformed phenotype is suggested by the ability of differentiation-inducing agents to revert the cancerous properties of specific tumors (1-3). This attribute of tumor cells provides the basis for a potentially less toxic form of therapy, ''differentiation therapy.'' In metastatic human melanoma, a combination of IFN-␤ and the protein kinase C activator mezerein (MEZ) produces irreversible growth arrest, a loss of tumorigenic competence, and terminal differentiation (1, 4). To define gene-expression changes associated with induction of terminal differentiation, a subtracted cDNA library enriched for genes associated with terminal differentiation was constructed (5). This construction was accomplished by subtracting control HO-1 human melanoma mRNAs from IFN-␤ ϩ MEZ-treated HO-1 mRNAs, which were temporally collected over a 24-h period (5). This subtracted cDNA library then was screened by random isolation of phage colonies and Northern blotting, high-density cDNA microarray analysis, and reverse Northern screening followed by Northern blotting (5-7). These approaches have identified both unknown and known genes associated with tumor and normal growth control, cell-cycle regulation, IFN response, differentiation, and apoptosis (5-12). Four classes of melanoma differentiation-associated (mda) genes have been identified (5, 10).Terminal cell differentiation and cellular senescence are characterized by changes in cell morphology, lack of responsiveness to mitogenic stimulation, and irreversible growth arrest (1, 4, 13-18).Normal cells cultured in vitro lose their proliferative potential after a finite number of doublings in a process described as cellular senescence (13). Experiments in human diploid fibroblasts and additional cell types document an inverse correlation between replicative senescence and donor age and a direct relationship between replicative senescence and donor-species life span (13,19,20). In agreement with this relationship, cells from patients with premature aging syndromes such as Werner's syndrome and progeria achieve a quiescent state more rapidly than normal human fibroblasts (21). Although senescence is a time-dependent process, terminal differentiation can be induced in a variety of cell types by appropriate treatme...

Terminal differentiation and senescence share several common properties, including irreversible cessation of growth and changes in gene expression profiles. To identify molecules that converge in both processes, an overlapping pathway screening was employed that identified old-35, which is human polynucleotide phosphorylase (hPNPase old-35 ), a 3,5-exoribonuclease. We previously demonstrated that hPNPase old-35 is a type I interferon-inducible gene that is also induced in senescent fibroblasts. In vitro RNA degradation assays confirmed its exoribonuclease properties, and overexpression of hPNPase There are two contrasting endpoints in the life of a replicating cell. One involves the normal physiological processes of differentiation or senescence. The other is the pathological process of neoplastic transformation characterized by uncontrolled proliferation and de-differentiation. Treatment of HO-1 metastatic human melanoma cells with fibroblast interferon (IFN-␤) 1 and the protein kinase C activator mezerein (MEZ) induces irreversible growth arrest and terminal differentiation characterized by changes in cell morphology, increase in melanin synthesis, modifications in gene expression, and alterations in surface antigen expression (1-5). Replicative or cellular senescence, a process leading to irreversible arrest of cell division, was first described in cultures of human fibroblasts that lost the ability to divide upon continuous subcultures (6). Replicative senescence can result from telomere shortening linked with a DNA end-replication problem, overexpression of certain oncogenes, or tumor suppressor genes, or it can be stress-induced premature senescence after exposure to a variety of oxidative stresses or DNA damaging agents (for a review, see Ref. 7).Terminal differentiation and cellular senescence share several common traits including irreversible growth arrest and changes in gene expression profiles. To understand the molecular and biochemical basis of the complex physiological changes associated with these phenomena, an overlapping pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes (8). A temporally spaced terminally differentiated human melanoma subtracted cDNA library was screened with cDNAs derived from senescent progeroid fibroblast cells. This led to the identification of old-35, which is human polynucleotide phosphorylase (hPNPase old-35 ), a 3Ј,5Ј exoribonuclease involved in RNA degradation (8). hPNPase old-35 is a highly evolutionary conserved gene in plants, prokaryotes and eukaryotes having similar domain structure and functional properties in all species. In vitro assays confirmed that hPNPase old-35 is involved in RNA degradation. Analysis of the expression profile of hPNPase old-35 revealed that it is predominantly a type I interferoninducible gene, and its expression is also induced in senescent fibroblasts in comparison with young fibroblasts. These findings indicate that hPNPase old-35 might play an essential role in

A cancer drug target is only truly validated by demonstrating that a given therapeutic agent is clinically effective and acts through the target against which it was designed. Nevertheless, it is desirable to declare an early-stage drug target as 'validated' before investing in a full-scale drug discovery programme dedicated to it. Although the outcome of validation studies can guide cancer research programmes, strictly defined universal validation criteria have not been established.

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