"Differentiation therapy" provides a unique and potentially effective, less toxic treatment paradigm for cancer. Moreover, combining "differentiation therapy" with molecular approaches presents an unparalleled opportunity to identify and clone genes mediating cancer growth control, differentiation, senescence, and programmed cell death (apoptosis). Subtraction hybridization applied to human melanoma cells induced to terminally differentiate by treatment with fibroblast interferon (IFN-beta) plus mezerein (MEZ) permitted cloning of melanoma differentiation associated (mda) genes. Founded on its novel properties, one particular mda gene, mda-7, now classified as a member of the interleukin (IL)-10 gene family (IL-24) because of conserved structure, chromosomal location, and cytokine-like properties has become the focus of attention of multiple laboratories. When administered by transfection or adenovirus-transduction into a spectrum of tumor cell types, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) induces apoptosis, whereas no toxicity is apparent in normal cells. mda-7/IL-24 displays potent "bystander antitumor" activity and also has the capacity to enhance radiation lethality, to induce immune-regulatory activities, and to inhibit tumor angiogenesis. Based on these remarkable attributes and effective antitumor therapy in animal models, this cytokine has taken the important step of entering the clinic. In a Phase I clinical trial, intratumoral injections of adenovirus-administered mda-7/IL-24 (Ad.mda-7) was safe, elicited tumor-regulatory and immune-activating processes, and provided clinically significant activity. This review highlights our current understanding of the diverse activities and properties of this novel cytokine, with potential to become a prominent gene therapy for cancer.

Glutamate is an essential neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters primarily expressed in astroglial cells. Dysfunction of EAAT2 is implicated in acute and chronic neurological disorders, including stroke/ischemia, temporal lobe epilepsy, amyotrophic lateral sclerosis, Alzheimer disease, human immunodeficiency virus 1-associated dementia, and growth of malignant gliomas. Ceftriaxone, one of the ␤-lactam antibiotics, is a stimulator of EAAT2 expression with neuroprotective effects in both in vitro and in vivo models based in part on its ability to inhibit neuronal cell death by glutamate excitotoxicity. Based on this consideration and its lack of toxicity, ceftriaxone has potential to manipulate glutamate transmission and ameliorate neurotoxicity. We investigated the mechanism by which ceftriaxone enhances EAAT2 expression in primary human fetal astrocytes (PHFA). Ceftriaxone elevated EAAT2 transcription in PHFA through the nuclear factor-B (NF-B) signaling pathway. The antibiotic promoted nuclear translocation of p65 and activation of NF-B. The specific NF-B binding site at the ؊272 position of the EAAT2 promoter was responsible for ceftriaxone-mediated EAAT2 induction. In addition, ceftriaxone increased glutamate uptake, a primary function of EAAT2, and EAAT2 small interference RNA completely inhibited ceftriaxone-induced glutamate uptake activity in PHFA. Taken together, our data indicate that ceftriaxone is a potent modulator of glutamate transport in PHFA through NF-Bmediated EAAT2 promoter activation. These findings suggest a mechanism for ceftriaxone modulation of glutamate transport and for its potential effects on ameliorating specific neurodegenerative diseases through modulation of extracellular glutamate.Glutamate plays a central role in brain physiology and pathology (1). It is the major mediator of excitatory signal transduction in the mammalian central nervous system and is implicated in most aspects of normal brain function, including cognition, memory, and learning (2). Glutamate exerts its signaling role by acting on glutamate receptors located on the surface of target cells. Accordingly, it is the glutamate concentration in the surrounding extracellular fluid that determines the extent of receptor stimulation. It is clinically relevant that the extracellular glutamate concentration is maintained at a low level, because excessive activation of glutamate receptors is harmful and glutamate is a potent neurotoxin at high concentration (2). The brain contains large quantities of glutamate, but only a small fraction of this glutamate is normally present in the extracellular fluid. Glutamate is constantly being released from cells and is continually being removed from the extracellular fluid (3). Glutamate transporters, also known as excitatory amino acid transporters, regulate this removal of glutamate from the extracellular fluid (3). Five excitatory amino acid transporter (EAAT) 4 cDNAs have been identified an...

NAC proteins are plant-specific transcriptional regulators. ATAF1 was one of the first identified NAC proteins in Arabidopsis. In present study, we characterized the ATAF1 expression and biological function in response to water deficit stress. ATAF1 mRNA expression was strongly induced by dehydration and abscisic acid (ABA) treatment, but inhibited by water treatment, suggesting a general role in drought stress responses. Transient expression analysis in onion epidermal cells indicated the nuclear localization for the ATAF1::GFP fusion protein. Yeast transactivation analysis showed that ATAF1 had ability to activate reporter gene expression. Furthermore, domain deletion analysis revealed that the ATAF1 transactivation activity was conferred by its C-terminal domain. When ATAF1 gene was knocked out by T-DNA insertions, Arabidopsis ataf1-1 and ataf1-2 mutants displayed a recovery rate about seven times higher than wild-type plants in drought response test. This ataf1 phenotype was coincident with the enhanced expression of stress responsive marker genes, such as COR47, ERD10, KIN1, RD22 and RD29A under drought stress. Above evidences suggest that ATAF1, as a transcriptional regulator, negatively regulates the expression of stress responsive genes under drought stress in Arabidopsis.

angiogenesis-related molecules ͉ tumor progression

Limitations of current viral-based gene therapies for malignant tumors include lack of cancer-specific targeting and insufficient tumor delivery. To ameliorate these problems and develop a truly effective adenovirus gene-based therapy for cancer, we constructed a conditionally replication competent adenovirus (CRCA) manifesting the unique properties of tumor-specific virus replication in combination with production of a cancer-selective cytotoxic cytokine, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), which embodies potent bystander antitumor activity. Cancer cell selective tropism was ensured by engineering the expression of the adenoviral E1A protein, necessary for viral replication, under the control of a minimal promoter region of progression elevated gene-3 (PEG-3), which functions selectively in diverse cancer cells with minimal activity in normal cells. In the E3 region of this CRCA, we introduced the mda-7/IL-24 gene, thereby mediating robust production of this cytokine as a function of adenovirus replication. Infection of this CRCA (designated Ad.PEG-E1A-mda-7) in normal mammary epithelial cells and breast cancer cells confirmed cancer cell selective adenoviral replication, mda-7/ IL-24 expression, growth inhibition, and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 into human breast cancer xenografts in athymic nude mice completely eradicated not only the primary tumor but also distant tumors (established on the opposite flank of the animal) thereby implementing a cure. This dual cancer-specific targeting strategy provides an effective approach for treating breast and other human neoplasms with the potential for eradicating both primary tumors and metastatic disease. Additionally, these studies support the potential use of mda-7/IL-24 in the therapy of malignant cancers.bystander antitumor activity ͉ conditionally replication competent adenovirus ͉ PEG-Prom ͉ mda-7͞IL-24 ͉ in vivo tumorigenesis

Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 10(6) pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split-green flourescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.

The mda-7/IL-24 cDNA was isolated almost a decade ago in a screen for genes differentially upregulated following growth arrest and terminal differentiation of a human melanoma cell line employed as an in vitro cell differentiation model. The underlying rationale for the screen was that oncogenesis arises from a cellular dedifferentiation process culminating in uncontrolled proliferation and acquisition of invasive and metastatic potential. Identification of genes upregulated during the process of reactivation of faulty or inoperational differentiation maintenance programs was postulated to have cancer gene therapeutic potential. In this context, it is heartening to note that mda-7/IL-24 has made a methodical and progressive journey, from an unidentified novel sequence with little homology to known genes at its time of isolation to currently having the status of a molecule belonging to the IL-10-related family of cytokines, with considerable cancer gene therapeutic potential. Extensive in vitro and in vivo human tumor xenograft studies have established its transformed cell apoptosis-inducing capacity in various model systems. It has recently taken an important step for a candidate cancer gene therapeutic molecule, in the ultimate goal of benchtop to clinic, by being currently utilized in human Phase I/II clinical trials. This review provides a current perspective of our understanding of mda-7/IL-24, including established and more recent information about the molecular properties, specificity of anti-tumor-cell apoptosis-inducing activity, and underlying mechanisms of this action relative to its cancer gene therapeutic potential.

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