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Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise ‘undruggable’ targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

Recombinant adeno-associated viral (rAAV) vectors containing an improved tetracycline (tet) system of transcriptional regulation are an efficient strategy for the control of long-term therapeutic gene expression. In vivo studies with the original tet-off and tet-on vectors, while promising, have failed to demonstrate complete repression in the uninduced state. To address this issue, we incorporated the tTSkid fusion of the tet repressor and a KRAB-derived transcriptional silencer into the tet-on system in the context of rAAV vectors. The tTSkid repressor and rtTA activator were expressed constituitively from a regulator vector, and the repressor and an erythropoietin (Epo) transgene were expressed inducibly via a second vector. Following intramuscular co-injection of these vectors, we observed repeated induction of serum Epo protein following drug administration and undetectable levels after its withdrawal. Four cycles of regulation were achieved over a 32-week period. Thus, the tet-on system plus the tTSkid repressor delivered via nonpathogenic rAAV vectors is a powerful tool for controlling the in vivo expression of therapeutic transgenes. In a clinical setting, the repressor could provide a mechanism for abolishing transgene expression if it were no longer needed or if the safety of a patient became compromised.

Background.Anemia is a significant problem in many disease states. Erythropoietin (Epo) has been used in the treatment of anemia associated with numerous chronic diseases. This study investigates the dose–response profiles of a single intramuscular (im) injection of a recombinant adeno-associated virus vector (rAAV) containing the Epo gene with the goal of achieving a sustained elevation of hematocrit (Hct).

AbstractThe TNF family is involved in the regulation of the immune system, and its members have been implicated in a variety of biological events such as apoptosis, cell proliferation, differentiation and survival. Here we present a new member of the TNF family, tumor necrosis factor super family member 20 (TNFSF20) that we have identified from the expressed sequence tag (EST) database and characterized. The human protein is a 285 amino acid long type II transmembrane protein and is 19% homologous to TNF in its extra-cellular domain. TNFSF20 is expressed at the surface of antigen presenting cells such as cells of the macrophagemonocyte lineage and dendritic cells. After treatment with bacterial lipopolysaccharide (LPS), TNFSF20 expression is downregulated at the surface of the expresssing cells, suggesting that the membrane-bound protein gets cleaved, and that a soluble factor is released in the extra-cellular compartment. The soluble form of the recombinant TNFSF20 induces proliferation of resting peripheral blood monocytes (PBMC) and cell death of activated lymphocytes. TNFSF20 might therefore play a critical role in the regulation of cell-mediated immune responses.

We describe a transcription-based assay system to screen for antiviral drugs in vivo. The system consists of two transcription units, a cytomegalovirus promoter driving a reporter gene physically linked to an HIV-1 promoter oriented in the opposite direction. Based on the arrangement of the transcription units, enhanced HIV-1 promoter activity in the presence of the viral transactivating Tat protein downregulates reporter gene expression initiated from the CMV promoter. Inhibitors of HIV-1 transcription relieve the suppression and are identified by an increase in reporter gene expression. This positive selection system allows discrimination between drugs that nonspecifically block cellular functions by cytotoxicity and molecules that specifically repress HIV-1 promoter activity.

The angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors belong to the seven transmembrane receptor superfamily. Previous studies have suggested that the AT1R couples to a Gqsignaling pathway, whereas the AT2R does not associate with Gq. To identify the role that individual intracellular domains play in AT1R function, AT1R/AT2R chimeric receptors were prepared by substitution of intracellular loops. CHO cells expressing these chimeras were used to test angiotensin II-induced c-fosexpression and Ca2+mobilization which are involved in the AT1R signaling pathway through Gqcoupling. Substitution of the second intracellular loop (IC2) and the cytoplasmic tail between the two receptors did not affect AT1R function. However, exchange of the third intracellular loop (IC3) resulted in the loss of function in the AT1R and conferred to the AT2R the ability to constitutively activate the fospromoter. These findings suggest that the third intracellular loop of the AT1R is critical for Gqcoupling. Substitution of discrete amino acid sequences of the third intracellular loop indicate that its N-terminal and C-terminal portions, especially the seven amino acids 219-225 in the N-terminal portion, are important for AT1R function, and that the intermediate portion of this loop is not required for Gqcoupling.

Tumor cells may stimulate their own proliferation through an autocrine mechanism by simultaneously producing growth factors and growth factor receptors. We now report that numerous human tumor-derived cell lines simultaneously express the genes for platelet-derived growth factor (PDGF) A and B chains and the PDGF receptor (PDGF-R). Measurement of mRNA transcribed from these genes showed that among 16 malignant glioma cell lines tested, 15 expressed the PDGF A gene, 12 expressed the PDGF B gene, and 13 expressed the PDGF-R gene. Of three osteosarcoma lines, three expressed PDGF A, two expressed PDGF B, and three expressed PDGF-R. For eight malignant melanoma lines, seven expressed PDGF A, five expressed PDGF B, and three expressed PDGF-R genes. Thus, 13 of 16 malignant glioma, 3 of 3 osteosarcomas, and 3 of 8 malignant melanoma cell lines expressed the PDGF receptor gene and either or both PDGF genes. Five cell lines were tested for production of biologically active PDGF and PDGF receptor protein. Media conditioned by each of the five cell lines induced tyrosine phosphorylation of a protein identical in size to the PDGF receptor. These five cell lines also produced PDGF receptor protein as measured by Western blot analysis or metabolic labeling and immunoprecipitation using PDGF-R antibodies. The PDGF receptors of these cell lines were activated by human platelet PDGF or by recombinant AA or BB homodimers. Intracellular interaction of these receptors with the growth factor simultaneously produced may provide continuous stimulation to the proliferation of these cells.

To investigate possible pathways leading to egg activation at fertilization, the ability of exogenously introduced tyrosine kinase and G-protein-coupled recaptors to mimic events of fertilization was examined. Oocytes of the starfish Asterina miniatawere injected with RNA for a chimeric receptor consisting of the extracellular domain of the β form of the mouse platelet-derived growth factor (PDGF) receptor and the transmembrane/intracellular domain of the human fibroblast growth factor (FGF) receptor, or with RNA for the rat serotonin 1c receptor. These oocytes were cultured for 1 to 3 days and then matured with 1-methyladenine. In response to PDGF or serotonin, the injected eggs underwent responses like those at fertilization: cortical granule exocytosis, a rise in intracellular free calcium, and DNA synthesis. Some of these artificially activated eggs cleaved, and some of the PDGF-activated eggs were observed to form larvae, A PDGF/FGF receptor with a point mutation which eliminated its ability to interact with phospholipase C-γ did not cause fertilization-like responses. Thus components of a signaling pathway involving phospholipase C-γ, characteristic of tyrosine kinase receptors, as well as components of a pathway involving a G-protein and phospholipase C-β, characteristic of G-protein-coupled receptors, appear to be present in starfish eggs. Either or both could function in egg activation at fertilization.

ABSTRACTAdeno-associated viral (AAV) vectors have much promise in gene therapy. Among the many properties that make AAV an ideal vector for gene therapy are its ability to infect both dividing and nondividing cells and the longevity of expression in tissues such as brain, skeletal muscle, and liver. However, like other viral vectors, readministration of vector is limited because of the host's immune response to viral components of the vector. Using class I, class II, and CD40 ligand (CD40L)-deficient mice, we demonstrate that neutralizing antibodies to the viral capsid proteins prevent transgene expression following readministration of rAAV vectors. Transient immunosuppression of mice by treatment with antibody to CD4 at the time of primary infection allowed transgene expression after readministration of rAAV vectors to animals. Transient immunosuppression with antibody to CD40L had only a modest effect on the efficacy of readministration. The ability to readminister virus was inversely correlated with both AAV capsid enzyme-linked immunosorbent assay titers and AAV neutralizing antibody titers. These studies demonstrate that readministration of rAAV can be accomplished by down regulating the anti-AAV immune response and suggest the use of repeated administration of rAAV as a viable form of therapy for the treatment of chronic diseases.

The receptors for mesenchymal growth factors contain tyrosine kinase coding sequences and exhibit ligand-activated tyrosine kinase activity. A variety of mutations of the epidermal growth factor1,2, insulin3,4and platelet-derived growth factor (PDGF) (manuscript in preparation) receptors that have resulted in a loss of tyrosine kinase activity have produced a concomitant loss of growth factor-stimulated DNA synthesis. Comparison of amino acid sequences of tyrosine kinases shows that these regions in the PDGF receptors in mouse5and human6contain an insert of unknown function. We have deleted this region, and expressed the altered form of the receptor in fibroblasts which lack PDGF receptors. This had no effect on a number of responses to PDGF, but cells bearing the mutant receptor did not proliferate or synthesize DNA in response to PDGF. This demonstrates that the insert is essential in PDGF-induced mitogenesis, and that PDGF stimulation of the mutant receptor tyrosine kinase and phosphatidy-linositol turnover are not sufficient to elicit a mitogenic response to PDGF.

No relevant conflicts of interest to declare.

Members of the Bromodomain and Extra-Terminal (BET) family of bromodomain-containing proteins (BRD2, BRD3, BRD4, and BRDT) bind to acetylated lysine residues on histone tails and act as epigenetic readers to regulate chromatin structure and gene expression. In particular, BET family member BRD4 has been shown to positively regulate the expression of MYCand other critical cancer-associated genes through the localization of BRD4 to super-enhancer regulatory elements. Results from preclinical studies conducted using the small molecule tool BET inhibitor (+)-JQ1, and emerging data from clinical trials of BET inhibitors in leukemia and lymphoma patients, have provided support for the therapeutic potential of BET inhibitors in hematologic malignancies.