A Key role for cyclic AMP-responsive element binding protein in hypoxia-mediated activation of the angiogenesis factor CCN1 (CYR61) in Tumor cells.

Hypoxia is a prominent feature of solid tumors known to contribute to malignant progression and therapeutic resistance. Cancer cells adapt to hypoxia using various pathways, allowing tumors to thrive in a low oxygen state. Induction of new blood vessel formation via the secretion of proangiogenic factors is one of the main adaptive responses engaged by tumor cells under hypoxic conditions. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays a pivotal role in mediating such responses. In addition, several other transcription factors have also been implicated in hypoxic gene regulation, either independently or in cooperation with HIF-1. In this work, we show that the expression of the angiogenesis-related, immediate early gene CCN1 (formerly known as CYR61), considered to be involved in tumor growth and invasiveness, is enhanced upon hypoxia stress primarily in a protein kinase A and cyclic AMP-responsive element binding protein (CREB) and CRE-dependent manner in various cell lines. The hypoxia-mediated activation of the CCN1 promoter is independent of HIF-1 and HIF-2, as shown by small interfering RNA knockdown. We identify the cis element in the mouse CCN1 promoter responsible for CREB binding to be one of two partial CRE sites present in the promoter. Moreover, we report for the first time that CREB-mediated CCN1 transcription is enhanced in hypoxic regions of tumors in vivo. Identifying and characterizing the molecular mechanisms that govern the response of tumors to hypoxia may be instrumental to identify the tumors that will respond favorably to inhibition of angiogenesis and thus lead to the development of treatments that could complement hypoxia-inducing treatment modalities.

Adenovirus expressing a bioluminescence reporter gene and cMAGI cell assay for the detection of HIV-1.

We report a fast, highly sensitive method for detecting and testing drug resistance of M-tropic and T-tropic laboratory and primary HIV-1 isolates. cMAGI cells are infected with an adenovirus vector harboring the luciferase reporter gene controlled by HIV-1 Tat-responsive element, TAR. HIV-1 Tat production by HIV-1 chronically infected cells, or by cMAGI cells as early as two days after being acutely infected with HIV-1, is readily monitored in the presence or absence of antiviral drugs. This method is more sensitive than HIV-1 Tat dependant production of beta-galactosidase in the cMAGI cells. The fast answer, ease and sensitivity as well as the possibility of using this method in high throughput screening, makes it an very attractive tool for phenotypic detection of HIV-1 in clinical samples as well as a sensitive assay for monitoring drug resistant HIV-1 variants. This method can also be used for discovery of novel anti HIV-1 drugs.

A pivotal role of cyclic AMP-responsive element binding protein in tumor progression.

Tumor microenvironment controls the selection of malignant cells capable of surviving in stressful and hypoxic conditions. The transcription factor, cyclic AMP-responsive element binding (CREB) protein, activated by multiple extracellular signals, modulates cellular response by regulating the expression of a multitude of genes. Previously, we have demonstrated that two cystein residues, at the DNA binding domain of CREB, mediate activation of CREB-dependent gene expression at normoxia and hypoxia. The construction of a dominant-positive CREB mutant, insensitive to hypoxia cue (substitution of two cystein residues at position 300 and 310 with serine in the DNA binding domain) and of a dominant negative CREB mutant (addition of a mutation in serine(133)), enabled a direct assessment, in vitro and in vivo, of the role of CREB in tumor progression. In this work, we demonstrate both in vitro and in vivo that CREB controls hepatocellular carcinoma growth, supports angiogenesis, and renders resistance to apoptosis. Along with the identification, by DNA microarray, of the CREB-regulated genes in normoxia and hypoxia, this work demonstrates for the first time that in parallel to other hypoxia responsive mechanisms, CREB plays an important role in hepatocellular carcinoma tumor progression.

Intracellular expression of a functional short peptide confers resistance to apoptosis.

Expression of free short peptides could potentially be used to modulate biochemical cascades and consequently to change cellular phenotypes. Here we demonstrate that expression of a short peptide of 15 amino acids, including the pseudo-substrate site of the baculovirus-apoptosis inhibitor P35, Asp-Gln-Met-Asp (DQMD), leads to abrogation of the apoptotic cascade. Treatment of cells, expressing the DQMD peptide with two apoptosis inducers, etoposide and sodium nitroprusside, (SNP) results in blocking of the apoptotic cascade, indicated by DNA fragmentation and caspase activation. Consequently, stable expression of the DQMD peptide led to protection of cells, following induction of apoptosis and to the outgrowth and enrichment of resistant cell colonies. The results presented in this work demonstrate for the first time the feasibility of expressing in cells functional short peptides that block apoptotic cascade, and to rescue the phenotypically altered cells in a stable fashion. This approach is general and could be applied to the study of other peptides and the respective biochemical cascades.

Femtosecond infrared laser-an efficient and safe in vivo gene delivery system for prolonged expression.

The major advantages of "naked DNA gene therapy" are its simplicity and a low or negligible immune response. Gene delivery by DNA electroporation (EP) involves injection of DNA and the application of a brief electric pulse to enhance cellular permeability. Although EP is an efficient gene transduction technique in rodents, it requires much higher voltages (>500 V) in larger animals, and hence, in practice it would be hazardous for human patients, as it would cause serious tissue damage. To overcome the obstacles associated with EP-mediated gene delivery in vivo, we developed a new method of gene transduction that uses laser energy. The femtosecond infrared titanium sapphire laser beam was developed specifically for enhancing in vivo gene delivery without risks of tissue damage. System optimization revealed that injection of 10 micro g naked DNA into the tibial muscle of mice followed by application of the laser beam for 5 s, focused to 2 mm depth upon an area of 95 x 95 micro m(2), resulted in the highest intensity and duration of gene expression with no histological or biochemical evidence of muscle damage. We assessed the potential clinical application of LBGT technology by using it to transfer the murine erythropoietin (mEpo) gene into mice. LBGT-mediated mEpo gene delivery resulted in elevated (>22%) hematocrit levels that were sustained for 8 weeks. Gene expression following LBGT was detected for >100 days. Hence, LBGT is a simple, safe, effective, and reproducible method for therapeutic gene delivery with significant clinical potential.