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Tumor metastasis comprises a series of coordinated events that culminate in dissemination of cancer cells to distant sites within the body representing the greatest challenge impeding effective therapy of cancer and the leading cause of cancer-associated morbidity. Cancer cells exploit multiple genes and pathways to colonize to distant organs. These pathways are integrated and regulated at different levels by cellular- and extracellular-associated factors. Defining the genes and pathways that govern metastasis can provide new targets for therapeutic intervention. Melanoma differentiation associated gene-9 (mda-9) (also known as Syntenin-1 and SDCBP (Syndecan binding protein)) was identified by subtraction hybridization as a novel gene displaying differential temporal expression during differentiation of melanoma. MDA-9/Syntenin is an established Syndecan binding protein that functions as an adaptor protein. Expression of MDA-9/Syntenin is elevated at an RNA and protein level in a wide-range of cancers including melanoma, glioblastoma, neuroblastoma, and prostate, breast and liver cancer. Expression is increased significantly in metastatic cancer cells as compared with non-metastatic cancer cells or normal cells, which make it an attractive target in treating cancer metastasis. In this review, we focus on the role and regulation of mda-9 in cancer progression and metastasis. [ABSTRACT FROM AUTHOR]

Molecular-genetic imaging is advancing from a valuable preclinical tool to a guide for patient management. The strategy involves pairing an imaging reporter gene with a complementary imaging agent in a system that can be used to measure gene expression or protein interaction or track gene-tagged cells in vivo. Tissue-specific promoters can be used to delineate gene expression in certain tissues, particularly when coupled with an appropriate amplification mechanism. Here we show that the progression elevated gene-3 (PEG-3) promoter, derived from a rodent gene mediating tumor progression and metastatic phenotypes, can be used to drive imaging reporters selectively to enable detection of micrometastatic disease in mouse models of human melanoma and breast cancer using bioluminescence and radionuclide-based molecular imaging techniques. Because of its strong promoter activity, tumor specificity and capacity for clinical translation, PEG-3 promoter-driven gene expression may represent a practical, new system for facilitating cancer imaging and therapy. [ABSTRACT FROM AUTHOR]

Melanoma differentiation associated gene-7 (mda-7) was cloned using subtraction hybridization from terminally differentiated human melanoma cells. Based on structural and functional properties, mda-7 is now recognized as interleukin-24 (IL-24), a new member of the expanding IL-10 gene family. Unique properties of mda-7/IL-24 include its ability to selectively induce growth suppression, apoptosis and radiosensitization in diverse human cancer cells, without causing similar effects in normal cells. The utility of mda-7/IL-24, administered by means of a replication-incompetent adenovirus, as a gene therapy for cancer has recently received validation in patients, highlighting an important phenomenon initially observed in pancreatic tumor cells, namely a ‘potent bystander apoptosis-inducing effect’ in adjacent tumor cells not initially receiving this gene product. We presently investigated the contribution of mda-7/IL-24 secreted by normal cells in mediating this ‘bystander effect’, and document that normal cells induced to produce mda-7/IL-24 following infection with recombinant adenoviruses expressing this cytokine secrete mda-7/IL-24, which modifies the anchorage-independent growth, invasiveness, survival and sensitivity to radiation of cancer cells that contain functional IL-20/IL-22 receptors, but not in cancer cells that lack a complete set of receptors. Moreover, the combination of secreted mda-7/IL-24 and radiation engenders a ‘bystander antitumor effect’ not only in inherently mda-7/IL-24 or radiation-sensitive cancer cells, but also in tumor cells overexpressing the antiapoptotic proteins bcl-2 or bcl-xL and displaying resistance to either treatment alone. The present studies provide definitive evidence that secreted mda-7/IL-24 from normal cells can induce direct antitumor and radiation-enhancing effects that are dependent on the presence of canonical receptors for this cytokine on tumor cells. Moreover, we now describe a novel means of enhancing mda-7/IL-24's therapeutic potential by targeting normal cells to produce and release this cancer-specific apoptosis-inducing cytokine, a strategy that could be employed as an innovative way of using this unique gene product for treating metastatic disease.Oncogene (2005) 24, 7552–7566. doi:10.1038/sj.onc.1208911; published online 25 July 2005 [ABSTRACT FROM AUTHOR]

Progression Elevated Gene-3 (PEG-3) was cloned using subtraction hybridization as an upregulated transcript associated with transformation and tumor progression of rat embryo fibroblast cells. PEG-3 is a unique gene facilitating tumor progression by modulating multiple pathways in transformed cells, including genomic stability, angiogenesis and invasion. PEG-3 originates from mutation in the growth arrest and DNA damage inducible gene GADD34. A one base deletion in rat GADD34 results in a frame-shift and premature appearance of a stop-codon resulting in a C-terminally truncated molecule that is PEG-3. We now document that mutation in the GADD34 gene is a frequent event during transformation and/or immortalization of rodent cells. Sequencing of the GADD34 gene in a number of independent rat tumor cell lines revealed that in a majority of these the GADD34 gene is mutated to either PEG-3 or a PEG-3-like gene with similar C-terminal truncations. An important function of GADD34 is to inhibit cell growth, predominantly by apoptosis, and we demonstrate that PEG-3 or C-terminal truncations of human GADD34 resembling PEG-3 prevent growth inhibition by both human and rat GADD34. Phosphorylation of p53 by GADD34 is one mechanism by which it inhibits growth and PEG-3 could prevent GADD34-induced p53 phosphorylation. In contrast, PEG-3 was unable to block other GADD34-induced changes, including eIF2adephosphorylation, indicating that its effects on GADD34 may be related more to its effect on cell growth rather than a global inhibitor of all GADD34 functions. We hypothesize that mutational generation of PEG-3 or a similar molecule is a critical event during rodent carcinogenesis. The inherent property of PEG-3 to function as a dominant negative of the growth inhibitory property of GADD34 might rescue cells from DNA damage-induced apoptosis leading to growth independence and tumorigenesis.Oncogene (2005) 24, 2247-2255. doi:10.1038/sj.onc.1208420 Published online 17 January 2005 [ABSTRACT FROM AUTHOR]

Pancreatic cancer is exceptionally aggressive with no long-term effective therapy. Current interventional approaches, including surgery, radiation and/or chemotherapy, have done little to quell the mortality associated with this malignancy. Subtraction hybridization identified a cancer-specific apoptosis-inducing cytokine gene, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), with a broad range of selective antitumor activity in diverse cancers both in vitro and in vivo in nude mice and recently in patients with advanced carcinomas and melanomas. Unlike most neoplasms, pancreatic cancers display innate resistance to mda-7/IL-24-induced apoptosis, which correlates with a diminished capacity to convert mda-7/IL-24 mRNA into protein. We presently demonstrate that this translational block can be reversed by treatment with agents that elevate reactive oxygen species (ROS). Induction of apoptosis in vitro and suppression of tumorigenesis in vivo in nude mice are induced in pancreatic cancers, irrespective of the status of their K-ras gene, only when tumor cells simultaneously express mda-7/IL-24 and are treated with a ROS-inducer, such as arsenic trioxide (ARS), N-(4-hydroxyphenyl) retinamide (HPR) or dithiophene (NSC656240 (NSC)). In pancreatic cancer cells constitutively expressing mda-7/IL-24 mRNA, a single treatment with arsenic trioxide, HPR or NSC656240 induces apoptosis, which correlates with production of MDA-7/IL-24 protein. The specificity of this action is documented by the ability of ROS inhibitors, including N-acetyl-L-cysteine and Tiron, to block this killing effect. Of potential clinical significance, similar treatment of normal cells does not elicit significant changes in growth nor does it induce apoptosis. Analysis of signal transduction changes in pancreatic carcinoma cells infected with Ad.mda-7 in combination with a ROS-inducer indicate that cell death correlates with modulation of discrete cassettes of multiple signaling pathways in a pancreatic cancer cell-specific manner, supporting global signaling dysregulation as a potential mediator of apoptosis induction. These findings suggest a promising combinatorial approach for safely promoting cell death in pancreatic tumors that provides a rational framework for developing a selective and effective therapy for this invariably fatal cancer.Oncogene (2005) 24, 585-596. doi:10.1038/sj.onc.1208183 Published online 6 December 2004 [ABSTRACT FROM AUTHOR]

Glutamate is the principal excitatory neurotransmitter in the nervous system. Inactivation of synaptic glutamate is handled by the glutamate transporter GLT1 (also known as EAAT2; refs 1, 2), the physiologically dominant astroglial protein. In spite of its critical importance in normal and abnormal synaptic activity, no practical pharmaceutical can positively modulate this protein. Animal studies show that the protein is important for normal excitatory synaptic transmission, while its dysfunction is implicated in acute and chronic neurological disorders, including amyotrophic lateral sclerosis (ALS), stroke, brain tumours and epilepsy. Using a blinded screen of 1,040 FDA-approved drugs and nutritionals, we discovered that manyß-lactam antibiotics are potent stimulators of GLT1 expression. Furthermore, this action appears to be mediated through increased transcription of the GLT1 gene.ß-Lactams and various semi-synthetic derivatives are potent antibiotics that act to inhibit bacterial synthetic pathways. When delivered to animals, theß-lactam ceftriaxone increased both brain expression of GLT1 and its biochemical and functional activity. Glutamate transporters are important in preventing glutamate neurotoxicity. Ceftriaxone was neuroprotective in vitro when used in models of ischaemic injury and motor neuron degeneration, both based in part on glutamate toxicity. When used in an animal model of the fatal disease ALS, the drug delayed loss of neurons and muscle strength, and increased mouse survival. Thus these studies provide a class of potential neurotherapeutics that act to modulate the expression of glutamate neurotransmitter transporters via gene activation. [ABSTRACT FROM AUTHOR]

The human mda-7/IL-24 gene product is normally expressed in melanocytes and certain lymphocyte populations. Loss of expression, a distinctive feature of many tumor suppressor genes, has been documented at RNA and protein levels in association with melanoma progression both in vitro as well as in human tumor-derived material. The MDA-7/IL-24 protein undergoes post-translational processing, including removal of an amino-terminal 48-residue signal peptide and extensive glycosylation prior to its secretion by producing cells. Its inherent cytokine properties have been documented in multiple reports, which have identified and characterized its cognate receptors and activation of the JAK/STAT signaling pathway following ligand/receptor docking. A notable and incompletely understood property of MDA-7/IL-24 is its ability to induce apoptosis in transformed cells, while having marginal growth suppressive effects on normal primary or immortalized cell lines. MDA-7/IL-24 has been delivered to cells, tumor xenografts and patients in clinical trials via a nonreplicating adenovirus (Ad.mda-7). Studies utilizing eukaryotically expressed and purified MDA-7/IL-24 protein from several sources have recapitulated some of the molecule's reported properties including receptor binding and JAK/STAT activation. Here, we report the properties and characteristics of a bacterially expressed and purified GST-MDA-7 fusion protein. These studies reveal that GST-MDA-7 retains its cancer-selective apoptosis-inducing properties, thereby providing a new reagent that will assist in defining the mechanism of action of this novel cytokine. In addition, retention of tumor-specific activity of GST-MDA-7 suggests that this protein may also have therapeutic applications.Oncogene (2004) 23, 7679-7690. doi:10.1038/sj.onc.1207958 Published online 30 August 2004 [ABSTRACT FROM AUTHOR]

Melanoma differentiation associated gene-5 (mda-5) was identified by subtraction hybridization as a novel upregulated gene in HO-1 human melanoma cells induced to terminally differentiate by treatment with IFN-ß+MEZ. Considering its unique structure, consisting of a caspase recruitment domain (CARD) and an RNA helicase domain, it was hypothesized that mda-5 contributes to apoptosis occurring during terminal differentiation. We have currently examined the expression pattern of mda-5 in normal tissues, during induction of terminal differentiation and after treatment with type I IFNs. In addition, we have defined its genomic structure and chromosomal location. IFN-ß, a type I IFN, induces mda-5 expression in a biphasic and dose-dependent manner. Based on its temporal kinetics of induction and lack of requirement for prior protein synthesis mda-5 is an early type I IFN-responsive gene. The level of mda-5 mRNA is in low abundance in normal tissues, whereas expression is induced in a spectrum of normal and cancer cells by IFN-ß. Expression of mda-5 by means of a replication incompetent adenovirus, Ad.mda-5, induces apoptosis in HO-1 cells as confirmed by morphologic, biochemical and molecular assays. Additionally, the combination of Ad.mda-5+MEZ further augments apoptosis as observed in Ad.null or uninfected HO-1 cells induced to terminally differentiate by treatment with IFN-ß+MEZ. The mda-5 gene is located on human chromosome 2q24 and consists of 16 exons, without pseudogenes, and is conserved in the mouse genome. Present data documents that mda-5 is a novel type I IFN-inducible gene, which may contribute to apoptosis induction during terminal differentiation and during IFN treatment. The conserved genomic and protein structure of mda-5 in human and mouse will permit analysis of the evolution and developmental aspects of this gene.Oncogene (2004) 23, 1789-1800. doi:10.1038/sj.onc.1207300 Published online 15 December 2003 [ABSTRACT FROM AUTHOR]

Neurodegeneration and dementia caused by human immunodeficiency virus type 1 (HIV-1) infection of the brain are common complications of acquired immunodeficiency syndrome (AIDS). Introduction of highly active antiretroviral therapy (HAART) reduced the incidence of HIV-1-associated dementia, but so far had no effect on the high frequency of milder neurological disorders caused by HIV-1. This indicates that some neuropathogenic processes persist during limited HIV-1 replication in the central nervous system (CNS). The authors are evaluating the hypothesis that interaction of HIV-1 with astrocytes, which bind HIV-1 but support limited productive HIV-1 infection, may contribute to these processes by disrupting astrocyte functions that are important for neuronal activity or survival. Using laser-capture microdissection on brain tissue samples from HIV-1-infected individuals, we found that HIV-1 DNA can be detected in up to 1% of cortical and basal ganglia astrocytes, thus confirming HIV-1 infection in astrocytes from symptomatic patients. Using rapid subtraction hybridization, the authors cloned and identified 25 messenger RNAs in primary human fetal astrocytes either up-regulated or down-regulated by native HIV-1 infection or exposure to gp120 in vitro . Extending this approach to gene microarray analysis using Affymetrix U133A/B gene chips, the authors determined that HIV-1 alters globally and significantly the overall program of gene expression in astrocytes, including changes in transcripts coding for cytokines, G-coupled protein receptors, transcription factors, and others. Focusing on a specific astrocyte function relevant to neuropathogenesis, the authors showed that exposure of astrocytes to HIV-1 or gp120 in vitro impairs the ability of the cells to transport L-glutamate and the authors related this defect to transcriptional inhibition of the EAAT2 glutamate transporter gene. These findings define new pathways through which HIV-1 may contribute to neuropathogenesis under conditions of limited virus replication in the brain. [ABSTRACT FROM AUTHOR]

Subtraction hybridization identified melanoma differentiation associated gene-7, mda-7, in the context of terminally differentiated human melanoma cells. Based on its structure, cytokine-like properties and proposed mode of action, mda-7 has now been classified as IL-24. When expressed by means of a replication-incompetent adenovirus, Ad.mda-7 induces apoptosis in a broad range of cancer cells, without inducing harmful effects in normal fibroblast or epithelial cells. These unique properties of mda-7/IL-24 suggest that this gene will prove beneficial for cancer gene therapy. We now demonstrate that Ad.mda-7 decreases viability by induction of apoptosis in hormone-responsive (LNCaP) and hormone-independent (DU-145 and PC-3) human prostate carcinomas, without altering growth or survival in early-passage normal human prostate epithelial cells (HuPEC). Ad.mda-7 causes G2/M arrest and apoptosis in LNCaP (p53-wildtype), DU-145 (p53 mutant, Bax-negative) and PC-3 (p53-negative) prostate carcinomas, but not in HuPEC. Apoptosis induction correlated with changes in the ratio of pro- to antiapoptotic Bcl-2 protein family members. A potential functional role for changes in bcl-2 family gene expression in Ad.mda-7-induced apoptosis was suggested by the finding that forced overexpression of bcl-xL or bcl-2 differentially diminished the apoptotic effect of Ad.mda-7 in prostate carcinomas. These results confirm that induction of apoptosis by the mda-7/IL-24 gene in prostate cancer cells is Bax- and p53-independent and is mediated by mitochondrial pathways involving bcl-2 family gene members. The mda-7/IL-24 gene represents a new class of cancer-specific apoptosis-inducing genes with obvious potential for the targeted gene-based therapy of human prostate cancer.Oncogene (2003) 22, 8758-8773. doi:10.1038/sj.onc.1206891 [ABSTRACT FROM AUTHOR]

Within the last 15 years, multiple new signal transduction pathways within cells have been discovered. Many of these pathways belong to what is now termed ‘the mitogen-activated protein kinase (MAPK) superfamily.' These pathways have been linked to the growth factor-mediated regulation of diverse cellular events such as proliferation, senescence, differentiation and apoptosis. Based on currently available data, exposure of cells to ionizing radiation and a variety of other toxic stresses induces simultaneous compensatory activation of multiple MAPK pathways. These signals play critical roles in controlling cell survival and repopulation effects following irradiation, in a cell-type-dependent manner. Some of the signaling pathways activated following radiation exposure are those normally activated by mitogens, such as the ‘classical’ MAPK (also known as the ERK) pathway. Other MAPK pathways activated by radiation include those downstream of death receptors and procaspases, and DNA-damage signals, including the JNK and P38 MAPK pathways. The expression and release of autocrine growth factor ligands, such as (transforming growth factor alpha) and TNF-a, following irradiation can also enhance the responses of MAPK pathways in cells and, consequently, of bystander cells. Thus, the ability of radiation to activate MAPK signaling pathways may depend on the expression of multiple growth factor receptors, autocrine factors and Ras mutation. Enhanced basal signaling by proto-oncogenes such as K-/H-/N-RAS may provide a radioprotective and growth-promoting signal. In many cell types, this may be via the PI3K pathway; in others, this may occur through nuclear factor-kappa B or multiple MAPK pathways. This review will describe the enzymes within the known MAPK signaling pathways and discuss their activation and roles in cellular radiation responses.Oncogene (2003) 22, 5885-5896. doi:10.1038/sj.onc.1206701 [ABSTRACT FROM AUTHOR]

Neurodegeneration and human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are the major disease manifestations of HIV-1 colonization of the central nervous system (CNS). In the brain, HIV-1 replicates in microglial cells and infiltrating macrophages and it persists in a low-productive, noncytolytic state in astrocytes. Astrocytes play critical roles in the maintenance of the brain microenvironment, responses to injury, and in neuronal signal transmission, and disruption of these functions by HIV-1 could contribute to HAD. To better understand the potential effects of HIV-1 on astrocyte biology, the authors investigated changes in gene expression using an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue, low-passage cells were infected with HIV-1 or mock infected, and total cellular RNAs were isolated at multiple time points over a period of 1 week. This approach is designed to identify gene products modulated early and late after HIV-1 infection and limits the cloning of genes displaying normal cell-cycle fluctuations in astrocytes. By subtracting temporal cDNAs derived from HIV-1-infected astrocytes from temporal cDNAs made from uninfected cells, 10 genes displaying reduced expression in infected cells, termed astrocyte suppressed genes ( ASGs ), were identified and their suppression was confirmed by Northern blot hybridization. Both known and novel ASGs , not reported in current DNA databases, that are down-regulated by HIV-1 infection are described. Northern blotting confirms suppression of the same panel of ASGs by treatment of astrocytes with recombinant HIV-1 envelope glycoprotein, gp120. These results extend our previous analysis of astrocyte genes induced or enhanced by HIV-1 infection and together they suggest that HIV-1 and viral proteins have profound effects on astrocyte physiology, which may influence their function in the CNS. [ABSTRACT FROM AUTHOR]

Malignant gliomas are extremely aggressive cancers currently lacking effective treatment modalities. Gene therapy represents a promising approach for this disease. A requisite component for improving gene-based therapies of brain cancer includes tumor suppressor genes that exhibit cancer constrained inhibitory activity. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7) as a gene associated with melanoma cell growth, differentiation and progression. Ectopic expression of mda-7 by means of a replication-incompetent adenovirus (Ad), Ad.mda-7, induces growth suppression and apoptosis selectively in diverse human cancers, without producing any apparent harmful effect in normal cells. We presently demonstrate that Ad.mda-7 induces growth inhibition and apoptosis in malignant human gliomas expressing both mutant and wild-type p53, and these effects correlate with an elevation in expression of members of the growth arrest and DNA damage (GADD) gene family. In contrast, infection with a recombinant Ad expressing wild-type p53, Ad.wtp53, specifically affects mutant p53 expressing gliomas. When tested in early passage normal and immortal human fetal astrocytes, growth inhibition resulting from infection with Ad.mda-7 or Ad.wtp53 is significantly less than in malignant gliomas and no toxicity is evident in these normal cells. Moreover, infection of gliomas with Ad.mda-7 or treatment with purified GST-MDA-7 protein sensitizes both wild-type and mutant p53 expressing tumor cells to the growth inhibitory and antisurvival effects of ionizing radiation, and this response correlates with increased expression of specific members of the GADD gene family. Since heterogeneity in p53 expression is common in evolving gliomas, the present findings suggest that Ad.mda-7 may, in many instances, prove more beneficial for the gene-based therapy of malignant gliomas than administration of wild-type p53.Oncogene (2003) 22, 1164-1180. doi:10.1038/sj.onc... [ABSTRACT FROM AUTHOR]

Presents a study which investigated the potential changes in cellular gene expression associated with HIV-1 infection of astrocytes. Methodology; Results of the study; Conclusion.

Analyzes the effects of ectopic expression of mda-7, administered by means of a replication-incompetent adenovirus, on the growth of human melanoma cells. Development of malignant melanoma in humans; Background on human melanoma cells; Infection of metastatic melanoma cells.

We investigated the role of radiation-induced mitogen activated protein kinase (MAPK) pathway activity in the regulation of proliferation, cell survival and vascular endothelial growth factor (VEGF) production in primary astrocytes and in T9 and RT2 glioblastoma cells derived from Fisher 344 rats. In these cells, ionizing radiation (2 Gy) caused activation of the MAPK pathway which was blocked by specific inhibitor drugs. Blunting of radiation-induced MAPK activity weakly enhanced radiation-induced apoptosis 24 h after exposure in RT2 cells. Furthermore, blunting of MAPK activation weakly enhanced the ability of radiation to reduce RT2 cell growth in clonogenic growth assays. These findings argue that inhibition of MAPK signaling reduces proliferation and enhances cell killing by ionizing radiation in transformed astrocytes. Proliferation and survival of cancer cells has been linked in vivo to enhanced expression of angiogenic growth factors. Recently we demonstrated that the gene product of a novel rodent radiation-responsive gene, progression elevated gene 3 (PEG-3), could enhance vascular endothelial growth factor (VEGF) promoter activity in rodent fibroblasts, leading to increased VEGF protein levels and tumorigenic behavior in vivo. Thus PEG-3 and VEGF expression could be expected to directly correlate with the oncogenic potential of transformed cells. RT2 cells expressed more PEG-3 and VEGF protein than T9 cells, and were more tumorigenic in vivo than T9 cells. Radiation activated the PEG-3 promoter via MAPK signaling and ectopic over-expression of PEG-3 enhanced both basal MAPK activity and basal VEGF promoter activity. Basal MAPK activity partially correlated with basal VEGF promoter activity and VEGF protein levels in primary astrocytes, T9 and RT2 cells. Radiation increased the activity of the VEGF promoter and VEGF protein levels in primary astrocytes, T9 and RT2 cells which were dependent upon MAPK function. Furthermore, inhibition of AP-1... [ABSTRACT FROM AUTHOR]

The galectin family of proteins has been associated with several diverse cellular processes. More than 30 years since the discovery of the first member, precise biological functions for the family as a whole, or for individual members has proven elusive. The isolation of Prostate Carcinoma Tumor Antigen-1 (PCTA-1), a cDNA closely related to rat and human Galectin-8, as a surface marker associated with prostate cancer was achieved using a previously described immunological subtraction approach, Surface Epitope Masking (SEM) approach, in combination with expression screening. It appears that PCTA-1 expression is almost ubiquitous in normal human tissues and could alter in specific contexts such as transformation or metastasis. Multiple expression isoforms of PCTA-1 at the mRNA level are observed. PCTA-1 maps to 1q42-43, a locus associated with predisposition to prostate cancer. We have determined the genomic structure of PCTA-1 to account for the several observed isoforms, performed expression analysis to determine distribution in normal and transformed contexts at the RNA and protein level and conducted over-expression studies to determine effects on cellular phenotype. Oncogene (2000) 19, 4405–4416 [ABSTRACT FROM AUTHOR]

Cancer is a progressive disease in which a tumor cell temporally develops qualitatively new transformation related phenotypes or a further elaboration of existing transformation associated properties. Subtraction hybridization identified a novel gene associated with transformation progression in mutant adenovirus type 5, H5ts125, transformed rat embryo cells, progression elevated gene-3 (PEG-3). To define the mechanism by which expression of PEG-3 is enhanced as a function of cancer progression a 5′-flanking promoter region of ∼2.0-kb, PEG-Prom, was isolated, cloned and characterized. The full-length and various mutated regions of the PEG-Prom were linked to a luciferase reporter construct and evaluated for promoter activity during cancer progression. These assays demonstrate a requirement for AP1 and PEA3 sites adjacent to the TATA box region of PEG-3 in mediating basal promoter activity and the enhanced expression of PEG-3 in progressed H5-ts125-transformed rat embryo cells. An involvement of AP1 and PEA3 in PEG-3 regulation was also confirmed by electrophoretic mobility shift assays (EMSA) and transfection studies with cJun and PEA3 expression vectors. Our findings document the importance of both AP1 and PEA3 transcription factors in mediating basal and elevated expression of PEG-3 in H5ts125-transformed rat embryo cells displaying an aggressive and progressed cancer phenotype. Oncogene (2000) 19, 3411–3421 [ABSTRACT FROM AUTHOR]

Induction of irreversible growth arrest and terminal differentiation in human melanoma cells following treatment with recombinant human fibroblast interferon (IFN-β) and mezerein (MEZ) results in elevated expression of a specific melanoma differentiation associated gene, mda-7. Experiments were conducted to define the mechanism involved in the regulation of mda-7 expression in differentiating human melanoma cells. The mda-7 gene is actively transcribed in uninduced HO-1 human melanoma cells and the rate of transcription of mda-7 is not significantly enhanced by treatment with IFN-β, MEZ or IFN-β+MEZ. The high basal activity of the mda-7 promoter in uninduced melanoma cells and the absence of enhancing effect upon treatment with differentiation inducers is corroborated by transfection studies using the promoter region of mda-7 linked to a luciferase reporter gene containing the SV40 polyadenylation signal sequence. RT–PCR analysis detects the presence of low levels of mda-7 transcripts in uninduced and concomitant increases in differentiation inducer treated HO-1 cells. However, steady-state mda-7 mRNA is detected only in IFN-β+MEZ and to a lesser degree in MEZ treated cells. We show that induction of terminal differentiation of HO-1 cells with IFN-β+MEZ dramatically increases the half-life of mda-7 mRNA while treatment with cycloheximide results in detectable mda-7 mRNA in control and inducer treated cells. These observations confirm constitutive activity of the mda-7 promoter in HO-1 cells irrespective of differentiation status suggesting posttranscriptional processes as important determinants of mda-7 expression during terminal differentiation. The 3′ UTR region of mda-7 contains AU-rich elements (ARE) that contribute to rapid mda-7 mRNA turnover during proliferation and reversible differentiation, a process controlled by a labile protein factor(s). Substitution of the SV40 polyadenylation signal sequence in the... [ABSTRACT FROM AUTHOR]

Abnormalities in differentiation are common occurrences in human cancers. Treatment of human melanoma cells with the combination of recombinant human fibroblast interferon (IFN-β) and the antileukemic compound mezerein (MEZ) results in a loss of tumorigenic potential that correlates with an irreversible suppression in proliferative ability and induction of terminal differentiation. It is hypothesized that this is associated with the differential expression of genes that may directly regulate cancer cell growth and differentiation. To define the relevant gene expression changes that correlate with and potentially control these important cellular processes a differentiation induction subtraction hybridization (DISH) scheme is being used. A temporally spaced subtracted differentiation inducer treated (TSS) cDNA library was constructed and differentially expressed DISH clones were isolated and evaluated using a high throughput microchip cDNA (Synteni) array screening approach. Verification of differential gene expression for specific cDNAs was confirmed by Northern blotting. The temporal kinetics of regulation and the expression pattern of DISH genes were also evaluated by microchip cDNA array screening. Using this approach with 1000 DISH cDNA clones (∼10% of the DISH library) has resulted in the identification and cloning of both 26 known and 11 novel cDNAs of potential relevance to growth control and terminal differentiation in human melanoma cells. [ABSTRACT FROM AUTHOR]

The molecular mechanism(s) by which tumor cells survive after exposure to ionizing radiation are not fully understood. Exposure of A431 cells to low doses of radiation (1 Gy) caused prolonged activations of the mitogen activated protein (MAP) kinase and stress activated protein (SAP) kinase pathways, and induced p21Cip-1/WAF1 via a MAP kinase dependent mechanism. In contrast, higher doses of radiation (6 Gy) caused a much weaker activation of the MAP kinase cascade, but a similar degree of SAP kinase cascade activation. In the presence of MAP kinase blockade by the specific MEK1 inhibitor (PD98059) the basal activity of the SAP kinase pathway was enhanced twofold, and the ability of a 1 Gy radiation exposure to activate the SAP kinase pathway was increased ∼sixfold 60 min after irradiation. In the presence of MAP kinase blockade by PD98059 the ability of a single 1 Gy exposure to cause double stranded DNA breaks (TUNEL assay) was enhanced at least threefold over the following 24–48 h. The increase in DNA damage within 48 h was also mirrored by a similar decrease in A431 cell growth as judged by MTT assays over the next 4–8 days following radiation exposure. This report demonstrates that the MAP kinase cascade is a key cytoprotective pathway in A431 human squamous carcinoma cells which is activated in response to clinically used doses of ionizng radiation. Inhibition of this pathway potentiates the ability of low dose radiation exposure to induce cell death in vitro. [ABSTRACT FROM AUTHOR]

Interferons inhibit cell growth in normal and tumor-derived cells. The molecular basis of interferons antiproliferative activity remains to be defined. Using subtraction hybridization, a human melanoma differentiation associated gene, mda-20, has been identified that is down-regulated by treatment with interferon. Sequence analysis indicates that mda-20 is human ribosomal protein L23a (rp L23a). The mRNA levels of rp L23a and growth are diminished in a variety of human tumor cell lines following treatment with human fibroblast interferon, interferon-β (IFN-β). Expression of rp L23a is also reduced in human melanoma cells treated with human leukocyte (IFN-α) and immune (IFN-γ) interferons, but not by growth inhibition resulting from serum starvation. These findings suggest that growth suppression alone is not sufficient to reduce rp L23a expression. Instead, reduced rp L23a mRNA results from biochemical changes mediated by interferons. Ectopic expression of an antisense rp L23a sequence in human HeLa cervical carcinoma cells results in a reduction in colony formation indicating a direct antiproliferative effect by inhibiting rp L23a expression. The mechanism underlying inhibition in rp L23a expression in IFN-β-treated cells may involve antisense rp L23a RNA. These results suggest that rp L23a may be one of the target molecules involved in mediating growth inhibition by interferon. [ABSTRACT FROM AUTHOR]