Gene therapy for glioblastoma [correction of gliobestome] multiform: in vivo tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir.

Clinical Trial

A phase 1-2 clinical trial of gene therapy for recurrent glioblastoma multiforme by tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir.

This study has investigated the effects of herpes simplex thymidine kinase gene (HSV-tk) transfer followed by ganciclovir treatment as adjuvant gene therapy to surgical resection in patients with recurrent glioblastoma multiforme (GBM). The study was open and single-arm, and aimed at assessing the feasibility and safety of the technique and indications of antitumor activity. In 48 patients a suspension of retroviral vector-producing cells (VPCs) was administered by intracerebral injection immediately after tumor resection. Intravenous ganciclovir was infused daily 14 to 27 days after surgery. Patients were monitored for adverse events and for life by regular biosafety assaying. Tumor changes were monitored by magnetic resonance imaging (MRI). Reflux during injection was a frequent occurrence but serious adverse events during the treatment period (days 1-27) were few and of a nature not unexpected in this population. One patient experienced transient neurological disorders associated with postganciclovir MRI enhancement. There was no evidence of replication-competent retrovirus in peripheral blood leukocytes or in tissue samples of reresection or autopsy. Vector DNA was shown in the leukocytes of some patients but not in autopsy gonadal samples. The median survival time was 8.6 months, and the 12-month survival rate was 13 of 48 (27%). On MRI studies, tumor recurrence was absent in seven patients for at least 6 months and for at least 12 months in two patients, one of whom remains recurrence free at more than 24 months. Treatment-characteristic images of injection tracks and intracavity hemoglobin were apparent. In conclusion, the gene therapy is feasible and appears to be satisfactorily safe as an adjuvant to the surgical resection of recurrent GBM, but any benefit appears to be marginal. Investigation of the precise effectiveness of this gene therapy requires prospective, controlled studies.

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway.

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.

Characterization of the human and mouse ETV1/ER81 transcription factor genes: Role of the two alternatively spliced isoforms in the human

The Ets transcription factors of the PEA3 group - E1AF/PEA3, ETV1/ER81 and ERM - are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1α) or the absence (ETV1β) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1β) and the full-length isoform (human ETV1α) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1α and human ETV1β expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

ERM transactivation is up-regulated by the repression of DNA binding after the PKA phosphorylation of a consensus site at the edge of the ETS domain

The final step of the transduction pathway is the activation of gene transcription, which is driven by kinase cascades leading to changes in the activity of many transcription factors. Among these latter, PEA3/E1AF, ER81/ETV1, and ERM, members of the well conserved PEA3 group from the Ets family are involved in these processes. We show here that protein kinase A (PKA) increases the transcriptional activity of human ERM and human ETV1, through a Ser residue situated at the edge of the ETS DNA-binding domain. PKA phosphorylation does not directly affect the ERM transactivation domains but does affect DNA binding activity. Unphosphorylated wild-type ERM bound DNA avidly, whereas after PKA phosphorylation it did so very weakly. Interestingly, S367A mutation significantly reduced the ERM-mediated transcription in the presence of the kinase, and the DNA binding of this mutant, although similar to that of unphosphorylated wild-type protein, was insensitive to PKA treatment. Mutations, which may mimic a phosphorylated serine, converted ERM from an efficient DNA-binding protein to a poor DNA binding one, with inefficiency of PKA phosphorylation. The present data clearly demonstrate a close correlation between the capacity of PKA to increase the transactivation of ERM and the drastic down-regulation of the binding of the ETS domain to the targeted DNA. What we thus demonstrate here is a relatively rare transcription activation mechanism through a decrease in DNA binding, probably by the shift of a non-active form of an Ets protein to a PKA-phosphorylated active one, which should be in a conformation permitting a transactivation domain to be active.

Expression of the Ets transcription factor Erm is regulated through a conventional PKC signaling pathway in the Molt4 lymphoblastic cell line.

Erm, a member of the PEA3 group within the Ets family of transcription factors, is expressed in murine and human lymphocytes. Here, we show that in the human Molt4 lymphoblastic cell line, the erm gene expression is regulated by the conventional PKC (cPKC) pathway. To better characterize the molecular mechanism by which cPKC regulates Erm transcription in Molt4 cells, we tested proximal promoter deletions of the human gene, and identified a specific cPKC-regulated region between positions -420 and -115 upstream of the first exon.

The methyl-CpG-binding protein MECP2 is required for prostate cancer cell growth.

The incidence of prostate cancer is increasing in western countries because of population aging. Prostate cancer begins as an androgen-dependent disease, but it can become androgen independent at a later stage or in tumors recurring after an antihormonal treatment. Although many genetic events have been described to be involved in androgen-dependent and/or -independent prostate cancer growth, little is known about the contribution of epigenetic events. Here we have examined the possibility that the methyl-CpG-binding protein MECP2 might play a role in controlling the growth of prostate cancer cells. Inhibition of MECP2 expression by stable short hairpin RNA stopped the growth of both normal and cancer human prostate cells. In addition, ectopic expression of the MECP2 conferred a growth advantage to human prostate cancer cells. More importantly, this expression allowed androgen-dependent cells to grow independently of androgen stimulation and to retain tumorigenic properties in androgen-depleted conditions. Analysis of signaling pathways showed that this effect is independent of androgen receptor signaling. Instead, MECP2 appears to act by maintaining a constant c-myc level during antihormonal treatment. We further show that MECP2-expressing cells possess a functional p53 pathway and are still responsive to chemotherapeutic drugs.

Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

Physiological levels of PTEN control the size of the cellular Ins(1,3,4,5,6)P5 pool

To understand how a signaling molecule's activities are regulated, we need insight into the processes controlling the dynamic balance between its synthesis and degradation. For the Ins(1,3,4,5,6)P5 signal, this information is woefully inadequate. For example, the only known cytosolic enzyme with the capacity to degrade Ins(1,3,4,5,6)P5 is the tumour-suppressor PTEN [J.J. Caffrey, T. Darden, M.R. Wenk, S.B. Shears, FEBS Lett. 499 (2001) 6 ], but the biological relevance has been questioned by others [E.A. Orchiston, D. Bennett, N.R. Leslie, R.G. Clarke, L. Winward, C.P. Downes, S.T. Safrany, J. Biol. Chem. 279 (2004) 1116 ]. The current study emphasizes the role of physiological levels of PTEN in Ins(1,3,4,5,6)P5 homeostasis. We employed two cell models. First, we used a human U87MG glioblastoma PTEN-null cell line that hosts an ecdysone-inducible PTEN expression system. Second, the human H1299 bronchial cell line, in which PTEN is hypomorphic due to promoter methylation, has been stably transfected with physiologically relevant levels of PTEN. In both models, a novel consequence of PTEN expression was to increase Ins(1,3,4,5,6)P5 pool size by 30-40% (p<0.01); this response was wortmannin-insensitive and, therefore, independent of the PtdIns 3-kinase pathway. In U87MG cells, induction of the G129R catalytically inactive PTEN mutant did not affect Ins(1,3,4,5,6)P(5) levels. PTEN induction did not alter the expression of enzymes participating in Ins(1,3,4,5,6)P5 synthesis. Another effect of PTEN expression in U87MG cells was to decrease InsP6 levels by 13% (p<0.02). The InsP6-phosphatase, MIPP, may be responsible for the latter effect; we show that recombinant human MIPP dephosphorylates InsP6 to D/L-Ins(1,2,4,5,6)P5, levels of which increased 60% (p<0.05) following PTEN expression in U87MG cells. Overall, our data add higher inositol phosphates to the list of important cellular regulators [Y. Huang, R.P. Wernyj, D.D. Norton, P. Precht, M.C. Seminario, R.L. Wange, Oncogene, 24 (2005) 3819 ] the levels of which are modulated by expression of the highly pleiotropic PTEN protein.

Pathogenicity of Salmonella: SopE-mediated membrane ruffling is independent of inositol phosphate signals

Studies [Zhou, D. Chen, L.-M. Hernandez, L. Shears, S.B. and Galán, J.E. (2001) A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host-cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39, 248-259] with engineered Salmonella mutants showed that deletion of SopE attenuated the pathogen's ability to deplete host-cell InsP5 and remodel the cytoskeleton. We pursued these observations: In SopE-transfected host-cells, membrane ruffling was induced, but SopE did not dephosphorylate InsP5, nor did it recruit PTEN (a cytosolic InsP5 phosphatase) for this task. However, PTEN strengthened SopE-mediated membrane ruffling. We conclude SopE promotes host-cell InsP5 hydrolysis only with the assistance of other Salmonella proteins. Our demonstration that Salmonella-mediated cytoskeletal modifications are independent of inositolphosphates will focus future studies on elucidating alternate pathogenic consequences of InsP5 metabolism, including ion channel conductance and apoptosis.