RhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to ß-catenin and contributes transcriptional regulation of vimentin upon Wnt3A.

In the Wnt canonical pathway, Wnt3A has been known to stabilize ß-catenin. In the non-canonical Wnt signaling pathway, Wnt is known to activate Rho GTPases. The correlation between canonical and non-canonical pathways by Wnt signaling, however, has not been well elucidated. Here, we identified that Wnt3A promoted superoxide generation, leading to Tyr42 phosphorylation of RhoA through activations of c-Src and Rho-dependent coiled coil kinase 2 (ROCK2) and phosphorylation of p47phox, a component of NADPH oxidase. Wnt3A also induced accumulation of ß-catenin along with activations of RhoA and ROCK1. Concurrently, ROCK1 was able to phosphorylate GSK-3ß at Ser9, which phosphorylated Src at Ser51 and Ser492 residues, leading to Src inactivation through dephosphorylation of Tyr416 during the late period of Wnt3A treatment. Meanwhile, p-Tyr42 RhoA bound to ß-catenin via the N-terminal domain of ß-catenin, thereby leading to the nuclear translocation of p-Tyr42 RhoA/ß-catenin complex. Notably, p-Tyr42 RhoA as well as ß-catenin was associated with the promoter of Vim, leading to increased expression of vimentin. In addition, stomach cancer patients harboring higher expressed p-Tyr42 Rho levels revealed the much poorer survival probability. Therefore, we propose that p-Tyr42 RhoA is crucial for transcriptional regulation of specific target genes in the nucleus by binding to their promoters and involved in tumorigenesis.

Anticancer activity of CopA3 dimer peptide in human gastric cancer cells.

CopA3 is a homodimeric α-helical peptide derived from coprisin which is a defensin-like antimicrobial peptide that was identified from the dung beetle, Copris tripartitus. CopA3 has been reported to have anticancer activity against leukemia cancer cells. In the present study, we investigated the anticancer activity of CopA3 in human gastric cancer cells. CopA3 reduced cell viability and it was cytotoxic to gastric cancer cells in the MTS and LDH release assay, respectively. CopA3 was shown to induce necrotic cell death of the gastric cancer cells by flow cytometric analysis and acridine orange/ethidium bromide staining. CopA3-induced cell death was mediated by specific interactions with phosphatidylserine, a membrane component of cancer cells. Taken together, these data indicated that CopA3 mainly caused necrosis of gastric cancer cells, probably through interactions with phosphatidylserine, which suggests the potential utility of CopA3 as a cancer therapeutic.

AEG-1/MTDH/LYRIC, the beginning: initial cloning, structure, expression profile, and regulation of expression.

Since its initial identification as a HIV-1-inducible gene in 2002, astrocyte elevated gene-1 (AEG-1), subsequently cloned as metadherin (MTDH) and lysine-rich CEACAM1 coisolated (LYRIC), has emerged over the past 10 years as an important oncogene providing a valuable prognostic marker in patients with various cancers. Recent studies demonstrate that AEG-1/MTDH/LYRIC is a pleiotropic protein that can localize in the cell membrane, cytoplasm, endoplasmic reticulum (ER), nucleus, and nucleolus, and contributes to diverse signaling pathways such as PI3K-AKT, NF-κB, MAPK, and Wnt. In addition to tumorigenesis, this multifunctional protein is implicated in various physiological and pathological processes including development, neurodegeneration, and inflammation. The present review focuses on the discovery of AEG-1/MTDH/LYRIC and conceptualizes areas of future direction for this intriguing gene. We begin by describing how AEG-1, MTDH, and LYRIC were initially identified by different research groups and then discuss AEG-1 structure, functions, localization, and evolution. We conclude with a discussion of the expression profile of AEG-1/MTDH/LYRIC in the context of cancer, neurological disorders, inflammation, and embryogenesis, and discuss how AEG-1/MTDH/LYRIC is regulated. This introductory discussion of AEG-1/MTDH/LYRIC will serve as the basis for the detailed discussions in other chapters of the unique properties of this intriguing molecule.

Anticancer activity of a synthetic peptide derived from harmoniasin, an antibacterial peptide from the ladybug Harmonia axyridis.

Harmoniasin is a defensin-like antimicrobial peptide identified from the ladybug Harmonia axyridis. Among the synthetic homodimer peptide analogues derived from harmoniasin, HaA4 has been found to have antibacterial activity without hemolytic activity. In this study, we investigated whether HaA4 has anticancer activity against human leukemia cell lines such as U937 and Jurkat cells. HaA4 manifested cytotoxicity and decreased the cell viability of U937 and Jurkat cells in MTS assay and LDH release assay. We found that HaA4 induced apoptotic and necrotic cell death of the leukemia cells using flow cytometric analysis, acridine orange/ethidium bromide staining and nucleosomal fragmentation of genomic DNA. Activation of caspase-7 and -9 and fragmentation of poly (ADP-ribose) polymerase was detected in the HaA4-treated leukemia cells, suggesting induction of a caspase-dependent apoptosis pathway by HaA4. Caspase-dependent apoptosis was further confirmed by reversal of the HaA4-induced viability reduction by treatment of Z-VAD-FMK, a pan-caspase inhibitor. In conclusion, HaA4 caused necrosis and caspase-dependent apoptosis in both U937 and Jurkat leukemia cells, which suggests potential utility of HaA4 as a cancer therapeutic agent.

Antimicrobial activity of the synthetic peptide scolopendrasin ii from the centipede Scolopendra subspinipes mutilans.

The centipede Scolopendra subpinipes mutilans is a medicinally important arthropod species. However, its transcriptome is not currently available and transcriptome analysis would be useful in providing insight into a molecular level approach. Hence, we performed de novo RNA sequencing of S. subpinipes mutilans using next-generation sequencing. We generated a novel peptide (scolopendrasin II) based on a SVM algorithm, and biochemically evaluated the in vitro antimicrobial activity of scolopendrasin II against various microbes. Scolopendrasin II showed antibacterial activities against gram-positive and -negative bacterial strains, including the yeast Candida albicans and antibiotic-resistant gram-negative bacteria, as determined by a radial diffusion assay and colony count assay without hemolytic activity. In addition, we confirmed that scolopendrasin II bound to the surface of bacteria through a specific interaction with lipoteichoic acid and a lipopolysaccharide, which was one of the bacterial cell-wall components. In conclusion, our results suggest that scolopendrasin II may be useful for developing peptide antibiotics.

Effects of the synthetic coprisin analog peptide, CopA3 in pathogenic microorganisms and mammalian cancer cells.

A synthetic coprisin analog peptide, 9-mer dimer CopA3 (CopA3) was designed based on a defensin-like peptide, Coprisin, isolated from the bacteria-immunized dung beetle Copris tripartitus. Here, CopA3 was investigated for its antimicrobial activity and cancer cell growth inhibition. CopA3 showed antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in 2~32 µM ranges, and inhibited the cell viabilities of pancreatic and hepatocellular cancer cells, except MIAPaca2, Hep3B, and HepG2 cells, in a dose-dependent manner. The average IC(50) values of CopA3 against pancreatic and hepatocellular cancer cells were 61.7 µM and 67.8 µM, respectively. The results indicate that CopA3 has potential in the treatments of pancreatic and hepatocellular cancers as well as microorganism infection disease.

Oncogene AEG-1 promotes glioma-induced neurodegeneration by increasing glutamate excitotoxicity.

Aggressive tumor growth, diffuse tissue invasion, and neurodegeneration are hallmarks of malignant glioma. Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, the mechanism(s) controlling this process is poorly understood. Astrocyte elevated gene-1 (AEG-1) is an oncogene that is overexpressed in several types of human cancers, including more than 90% of brain tumors. In addition, AEG-1 promotes gliomagenesis, particularly in the context of tumor growth and invasion, 2 primary characteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-induced neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and samples from glioma patients indicated a strong negative correlation between expression of AEG-1 and a primary glutamate transporter of astrocytes EAAT2. Gain- and loss-of-function studies in normal primary human fetal astrocytes and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional level by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1-mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction of neuronal cell death. These findings were also confirmed in samples from glioma patients showing that AEG-1 expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1 contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2 expression.

Lysyl oxidase like 4, a novel target gene of TGF-beta1 signaling, can negatively regulate TGF-beta1-induced cell motility in PLC/PRF/5 hepatoma cells.

Transforming growth factor-beta1 (TGF-beta1) is a multi-functional cytokine involved in the regulation of cell proliferation, differentiation and extracellular matrix formation. In search for novel genes mediating the TGF-beta1 function at downstream signaling, we performed a cDNA microarray analysis and identified 60 genes whose expression is regulated by TGF-beta1 in the liver cancer cell line PLC/PRF/5. Among them, we report here lysyl oxidase like 4 (LOXL4) as a novel target of TGF-beta1 signaling, and provide experimental evidence for its expression regulation and function. LOXL4 was found to be the only member of LOX family whose expression is induced by TGF-beta1 in hepatoma cells. Deletion mapping of the LOXL4 promoter indicated that the TGF-beta1 regulation of LOXL4 expression is mediated through the binding of AP1 transcription factor to a conserved region of the promoter. This was confirmed by the chromatin immunoprecipitation assay that captured c-Fos-bound chromatin from TGF-beta1-treated cells. Forced expression of LOXL4 in PLC/PRF/5 cells resulted in inhibition of cell motility through Matrigel in the presence of TGF-beta1 treatment. In parallel, LOXL4 suppressed the expression of laminins and alpha3 integrin and the activity of MMP2. These results suggest that LOXL4 may function as a negative feedback regulator of TGF-beta1 in cell invasion by inhibiting the metabolism of extracellular matrix (ECM) components.

Astrocyte elevated gene-1: recent insights into a novel gene involved in tumor progression, metastasis and neurodegeneration.

Tumor progression and metastasis are complex processes involving intricate interplay among multiple gene products. Astrocyte elevated gene (AEG)-1 was cloned as an human immunodeficiency virus (HIV)-1-inducible and tumor necrosis factor-alpha (TNF-alpha)-inducible transcript in primary human fetal astrocytes (PHFA) by a rapid subtraction hybridization approach. AEG-1 down-regulates the expression of the glutamate transporter EAAT2; thus, it is implicated in glutamate-induced excitotoxic damage to neurons as evident in HIV-associated neurodegeneration. Interestingly, AEG-1 expression is elevated in subsets of breast cancer, glioblastoma multiforme and melanoma cells, and AEG-1 cooperates with Ha-ras to augment the transformed phenotype of normal immortal cells. Moreover, AEG-1 is overexpressed in >95% of human malignant glioma samples when compared with normal human brain. Overexpression of AEG-1 increases and siRNA inhibition of AEG-1 decreases migration and invasion of human glioma cells, respectively. AEG-1 contains a lung-homing domain facilitating breast tumor metastasis to lungs. These findings indicate that AEG-1 might play a pivotal role in the pathogenesis, progression and metastasis of diverse cancers. Our recent observations indicate that AEG-1 exerts its effects by activating the nuclear factor kappa B (NF-kappaB) pathway and AEG-1 is a downstream target of Ha-ras and plays an important role in Ha-ras-mediated tumorigenesis. These provocative findings are intensifying interest in AEG-1 as a crucial regulator of tumor progression and metastasis and as a potential mediator of neurodegeneration. In this review, we discuss the cloning, structure and function(s) of AEG-1 and provide recent insights into the diverse actions and intriguing properties of this molecule.

Reciprocal subtraction differential RNA display (RSDD): an efficient technology for cloning differentially expressed genes.

Identification of differentially expressed genes is an essential step in comprehending the molecular basis of complex physiological and pathological processes. Subtraction hybridization and differential RNA display (DDRT-PCR) are two methods that are widely and successfully employed to clone differentially expressed genes. Unfortunately, both methods have inherent problems and limitations requiring improvements in the technique. A combination of these two methods termed reciprocal subtraction differential RNA display is described here that considerably reduces the complexity of DDRT-PCR and facilitates the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes.

Cloning differentially expressed genes using rapid subtraction hybridization (RaSH).

Differential gene expression represents the entry point for comprehending complex biological processes. In this context, identification and cloning of differentially expressed genes represent critical elements in this process. Many techniques have been developed to facilitate achieving these objectives. Although effective in many situations, most currently described approaches are not trouble-free and have limitations, including complexity of performance, redundancy of gene identification (reflecting cloning biases) and false-positive gene identification. A detailed methodology to perform a rapid and efficient cloning approach, called rapid subtraction hybridization is described in this chapter. This strategy has been applied successfully to a number of cell culture systems and biological processes, including terminal differentiation and cancer progression in human melanoma cells, resistance or sensitivity to HIV-1 in human T cells and gene expression changes following infection of normal human fetal astrocytes with HIV-1 or treatment with neutrotoxic agents. Based on its simplicity of performance and high frequency of genuine differential gene identification, the rapid subtraction hybridization (RaSH) approach will allow wide applications in diverse systems and biological contexts.

Complete open reading frame (C-ORF) technique: rapid and efficient method for obtaining complete protein coding sequences.

Several approaches, generally referred to as rapid amplification of cDNA ends, are currently used as a means of obtaining full-length cDNA clones by PCR. However, these protocols are not infallible and in specific instances they have proven unsuccessful, emphasizing a need for further refinement. A novel method, the complete open reading frame (C-ORF) technique, is presently described, which has proven successful in cases, where standard rapid amplification of cDNA ends (RACE) has not worked. In C-ORF, the 5' PCR primer site is provided by a degenerative stem-loop annealing primer, which consists of a stem-loop structure and a 3' random 12-mer. degenerative stem-loop annealing primer is designed to anneal at random sites of the first strand cDNA, while promoting second strand synthesis from the end of given cDNA. Although this technique manifests weak sequence preference for GC-rich regions, in practice it has been successfully applied to clone both known and unknown genes with varying regions of GC-rich content. C-ORF does not use additional enzymes other than reverse transcriptase and Taq polymerase making it a cost-effective and relatively simple method that should be of general utility for gene cloning in multiple laboratories.

Complete open reading frame (C-ORF) technology: simple and efficient technique for cloning full-length protein-coding sequences.

Technical difficulties in full-length cDNA cloning hinder successful characterization of many unknown and potentially novel expressed sequence tags (ESTs). We presently describe improved methods for cDNA cloning. This scheme is based on the polymerase chain reaction and utilizes a degenerate stem-loop annealing primer (dSLAP), consisting of a stem-loop structure followed by 12 random nucleotides, and is called the C-ORF (complete open reading frame) technique. The dSLAP is designed to anneal to first-strand cDNA, while suppressing second-strand synthesis from internal sites because of its bulky stem-loop structure. The C-ORF technique consists of three steps: reverse transcription, dSLAP annealing plus the second-strand synthesis, and PCR amplification. Applications of dSLAP to both known and previously unknown cDNA targets resulted in cloning of their complete open reading frames, in most cases after a single application of the C-ORF method. The currently described protocol is simple and does not require unusual molecular biology reagents, except for reverse transcriptase, Taq polymerase and a DNA primer, which makes it readily amenable for cloning purposes in individual laboratories. Moreover, this approach has wide applicability and in principle can be used to identify the protein-coding region of virtually any gene in which limited or incomplete sequence information is available.

Cloning and characterization of HIV-1-inducible astrocyte elevated gene-1, AEG-1.

We presently describe the full-length cloning and functional characterization of an HIV-1-inducible gene, astrocyte elevated gene (AEG)-1. Additionally, a novel method is outlined for producing tag-free recombinant protein in a baculovirus system and its use in producing AEG-1 protein. AEG-1 mRNA is expressed ubiquitously with higher expression in tissues containing muscular actin and its expression is increased in astrocytes infected with HIV-1 or treated with gp120 or tumor necrosis factor (TNF)-alpha. The mRNA encodes a single pass transmembrane protein of predicted molecular mass of 64-kDa and pI 9.3 that predominantly localizes in the endoplasmic reticulum and perinuclear region. Ectopic expression of AEG-1 inhibits excitatory amino acid transporter 2 (EAAT2) promoter activity with the potential to promote glutamate excitotoxicity and consequently HIV-1-associated dementia (HAD). AEG-1 expression is elevated in subsets of breast carcinomas, malignant gliomas and melanomas and it synergizes with oncogenic Ha-ras to enhance soft agar colony forming ability of non-tumorigenic immortalized melanocytes, documenting its tumor promoting activity. AEG-1 may affect tumor progression in multiple cell lineages by augmenting expression of the transformed phenotype and/or by inducing glutamate excitotoxicity in malignant glioma. In these contexts, an HIV-1-inducible gene, AEG-1, may contribute to multiple brain abnormalities, including HAD and tumor formation, by both common and distinct mechanisms.

Identification and cloning of genes displaying elevated expression as a consequence of metastatic progression in human melanoma cells by rapid subtraction hybridization.

Although extensively investigated, the complete repertoire of genes associated with and causative of metastasis remain largely unknown. We developed an efficient approach for identifying differentially expressed genes that involves rapid subtraction hybridization (RaSH) of cDNA clones prepared from two cell populations, a driver and a tester. This RaSH approach has previously documented high sensitivity and effectiveness in identifying genes that are differentially expressed as a function of induction of terminal differentiation in human melanoma cells, resistance or sensitivity to human immunodeficiency virus-1 (HIV-1) infection of human T cells and perturbation in gene expression in normal human fetal astrocytes infected with HIV-1 or treated with HIV-1 gp120 viral envelope glycoprotein or tumor necrosis factor-alpha (TNF-alpha). In the present study, RaSH has been applied to a metastatic melanoma model, which mimics the early events of metastasis in humans, comprising weakly metastatic vs. immunosuppressed newborn rat-selected highly metastatic variants. This has now resulted in the identification of eight genes displaying elevated expression in the high metastatic variants vs. normal immortal melanocytes or weakly metastatic parental clones. These include six known genes, 67-kDa laminin receptor (67LR), endothelin receptor B (ENDRB), Na+/K+-ATPase, Ku antigen, interleukin-receptor-associated kinase-1 (IRAK-1) and ribosomal protein RPLA, which may contribute to the complex process of melanoma metastasis. Additionally, two unknown genes (not reported in current databases) that may also impact on the metastatic phenotype have also been identified. These studies provide additional support of the use of the RaSH approach, in this application in the context of closely related variant cell lines with different metastatic potential, for effective differential gene identification and elucidate eight previously unrecognized genes whose role in melanoma progression to metastatic competence can now be scrutinized.

Human polynucleotide phosphorylase (hPNPaseold-35): a potential link between aging and inflammation.

Chronic inflammation is a characteristic feature of aging, and the relationship between cellular senescence and inflammation, although extensively studied, is not well understood. An overlapping pathway screen identified human polynucleotide phosphorylase (hPNPase(old-35)), an evolutionary conserved 3',5'-exoribonuclease, as a gene up-regulated during both terminal differentiation and cellular senescence. Enhanced expression of hPNPase(old-35) via a replication-incompetent adenovirus (Ad.hPNPase(old-35)) in human melanoma cells and normal human melanocytes results in a characteristic senescence-like phenotype. Reactive oxygen species (ROS) play a key role in the induction of both in vitro and in vivo senescence. We now document that overexpression of hPNPase(old-35) results in increased production of ROS, leading to activation of the nuclear factor (NF)-kappaB pathway. Ad.hPNPase(old-35) infection promotes degradation of IkappaBalpha and nuclear translocation of NF-kappaB and markedly increases binding of the transcriptional activator p50/p65. The generation of ROS and activation of NF-kappaB by hPNPase(old-35) are prevented by treatment with a cell-permeable antioxidant, N-acetyl-l-cysteine. Infection with Ad.hPNPase(old-35) enhances the production of interleukin (IL)-6 and IL-8, two classical NF-kappaB-responsive cytokines, and this induction is inhibited by N-acetyl-l-cysteine. A cytokine array reveals that Ad.hPNPase(old-35) infection specifically induces the expression of proinflammatory cytokines, such as IL-6, IL-8, RANTES, and matrix metalloproteinase (MMP)-3. We hypothesize that hPNPase(old-35) might play a significant role in producing pathological changes associated with aging by generating proinflammatory cytokines via ROS and NF-kappaB. Understanding the relationship between hPNPase(old-35) and inflammation and aging provides a unique opportunity to mechanistically comprehend and potentially intervene in these physiologically important processes.

The origin of polynucleotide phosphorylase domains.

In this report, we document the presence of polynucleotide phosphorylase (PNPase) in the animal eukaryotes. These proteins contain several domains, including 2 RNase PH domains (PNPase 1 and PNPase 2) which are closely related functionally and in sequence similarity to ribonuclease PH (RPH) protein. Phylogenetic analysis of the gene genealogy of these three domains suggests that PNPase was formed via a duplication event that also produced the RNase PH protein. Given the current distribution of these domains in the tree of life, these duplication events most likely occurred in the common ancestor of the three organismal superkingdoms, Archaea, Eukarya, and Bacteria. In particular, PNPase 2 and RPH are more closely related to each other than either one is to PNPase 1, suggesting a deeper differentiation of PNPase 1 in the common organismal ancestor. In addition, while PNPase 1 and PNPase 2 appear to have the same evolutionary signal as determined by the incongruence length difference (ILD) test, RPH appears to have an incongruent signal with both of the PNPase domains. This result suggests that RPH experienced different evolutionary divergence patterns than the PNPase domains, consistent with the linked nature of the two PNPase domains.

Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: potential role in neuropathogenesis.

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.

Expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda-5: a novel type I interferon-responsive apoptosis-inducing gene.

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-beta+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-beta, 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-beta. 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-beta+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.

Expression regulation and genomic organization of human polynucleotide phosphorylase, hPNPase(old-35), a Type I interferon inducible early response gene.

An overlapping pathway screening (OPS) approach designed to identify and clone genes displaying parallel expression profiles as a function of induction of terminal differentiation and cellular senescence in human cells identified a novel gene old-35. Sequence and functional analysis indicates that old-35 encodes human polynucleotide phosphorylase, hPNPase(old-35). Polynucleotide phosphorylases comprise a family of phosphate dependent 3'-5' RNA exonucleases implicated in RNA regulation. Treatment of HO-1 human melanoma and additional diverse normal and tumor-derived human cell types with Type I interferon (IFN), IFN-beta or IFN-alpha, induces hPNPase(old-35) expression. To provide insights into the regulation of hPNPase(old-35), we cloned and analyzed the promoter region of this gene. These studies demonstrate that IFN-beta controls hPNPase(old-35) expression by transcriptional modulation rather than by altering mRNA stability. Transcriptional activation of hPNPase(old-35) by IFN-beta is primarily mediated by the interferon stimulatory response element (ISRE) present in its promoter. Analysis of hPNPase(old-35) expression in cell lines defective in various IFN signaling molecules confirms that hPNPase(old-35) expression is dependent upon the Janus activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Furthermore, gel shift analyses document that hPNPase(old-35) is a direct target of the interferon stimulated gene factor 3 (ISGF3) complex. The hPNPase(old-35) gene spans approximately 54 kb of genomic DNA and is distributed on 28 exons and 27 introns. hPNPase(old-35) maps to 2p15-2p16.1, a region implicated in hereditary nonpolyposis colorectal cancer, Carney complex, Doyne's honeycomb retinal dystrophy and several other diseases. To provide insights into PNPase function in vivo, we have also cloned the mouse PNPase(old-35) cDNA, mPNPase(old-35). Induction of hPNPase(old-35) by IFN treatment as well as during differentiation and senescence suggest that this gene may play a significant role in regulating cellular growth and that overlapping gene expression changes, also induced by IFN, may contribute to these important physiological processes.