Protein disulphide isomerase (PDI) is protective against amyotrophic lateral sclerosis (ALS)-related mutant Fused in Sarcoma (FUS) in in vitro models.

Mutations in Fused in Sarcoma (FUS) are present in familial and sporadic cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS is localised in the nucleus where it has important functions in DNA repair. However, in ALS/FTD, mutant FUS mislocalises from the nucleus to the cytoplasm where it forms inclusions, a key pathological hallmark of neurodegeneration. Mutant FUS also inhibits protein import into the nucleus, resulting in defects in nucleocytoplasmic transport. Fragmentation of the neuronal Golgi apparatus, induction of endoplasmic reticulum (ER) stress, and inhibition of ER-Golgi trafficking are also associated with mutant FUS misfolding in ALS. Protein disulphide isomerase (PDI) is an ER chaperone previously shown to be protective against misfolding associated with mutant superoxide dismutase 1 (SOD1) and TAR DNA-binding protein-43 (TDP-43) in cellular and zebrafish models. However, a protective role against mutant FUS in ALS has not been previously described. In this study, we demonstrate that PDI is protective against mutant FUS. In neuronal cell line and primary cultures, PDI restores defects in nuclear import, prevents the formation of mutant FUS inclusions, inhibits Golgi fragmentation, ER stress, ER-Golgi transport defects, and apoptosis. These findings imply that PDI is a new therapeutic target in FUS-associated ALS.

The expression of HMGB1 on microparticles from Jurkat and HL-60 cells undergoing apoptosis in vitro.

HMGB1 is a highly conserved nuclear protein that displays important biological activities inside as well as outside the cell and serves as a prototypic alarmin to activate innate immunity. The translocation of HMGB1 from inside to outside the cell occurs with cell activation as well as cell death, including apoptosis. Apoptosis is also a setting for the release of cellular microparticles (MPs), which are small membrane-bound vesicles that represent an important source of extracellular nuclear molecules. To investigate whether HMGB1 released from cells during apoptosis is also present on MPs, we determined the presence of HMGB1 on particles released from Jurkat and HL-60 cells induced to undergo apoptosis in vitro by treatment with either etoposide or staurosporine; MPs released from cells undergoing necrosis by freeze-thaw were also characterized. As shown by both Western blot analysis and flow cytometry, MPs from apoptotic cells contain HMGB1, with binding by antibodies indicating an accessible location in the particle structure. These results indicate that HMGB1, like other nuclear molecules, can translocate into MPs during apoptosis and demonstrate another biochemical form of this molecule that may be immunologically active.

The effects of heparins on the liver: application of mechanistic serum biomarkers in a randomized study in healthy volunteers.

Heparins have been reported to cause elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) but have not been associated with clinically significant liver injury. The mechanisms underlying these benign laboratory abnormalities are unknown. Forty-eight healthy men were randomized to receive subcutaneous injections of unfractionated heparin (UFH; 150 U/kg), enoxaparin sodium (1 mg/kg), dalteparin sodium (120 IU/kg), or adomiparin sodium (125 IU/kg; a novel heparin) every 12 h for 4.5 days. Asymptomatic elevations in serum ALT or AST were observed in >90% of the subjects. Elevations were also observed in the levels of serum sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH), miR-122, high-mobility group box-1 protein (including the acetylated form), full-length keratin 18, and DNA. Keratin 18 fragments, which are apoptosis biomarkers, were not detected. Biomarker profiles did not differ significantly across heparin treatments. We conclude that heparins as a class cause self-limited and mild hepatocyte necrosis with secondary activation of an innate immune response.

Death switch for gene therapy: application to erythropoietin transgene expression

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv’-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Death switch for gene therapy: application to erythropoietin transgene expression.

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv'-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 microg/50 g ptet-mEpoD and 0.5 microg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 microg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65% for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30% of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50% of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Barminomycin, a model for the development of new anthracyclines.

Barminomycin is a member of the anthracycline class of anticancer agents and was originally discovered as a pink/red complex with DNA and RNA and named SN-07. The chromophore was subsequently separated from the nucleic acids by nuclease digestion and contained the four-membered anthraquinone ring system characteristic of anthracyclines, but with an unusual eight membered ring that contained a carbinolamine which readily interconverted to an imine. The imine form is analogous to the formaldehyde-activated form of other anthracyclines such as doxorubicin. The imine form confers exceptional activity to barminomycin which is 1,000-fold more cytotoxic than doxorubicin. Barminomycin rapidly forms adducts with DNA, reacting with the exocyclic amino group of guanine residues and with high selectivity for 5'-GC-3' sequences. The coupling to DNA appears to be identical to the N-C-N aminal linkage formed between doxorubicin and DNA where the carbon derives from formaldehyde for doxorubicin-DNA adducts, whereas this "activated carbon" is an inherent component of the imine group in the eight membered ring of barminomycin. Although the linkage of both drugs to DNA appears to be identical, barminomycin-DNA complexes are essentially irreversible compared to the labile doxorubicin-DNA adducts which have an in vitro (purified DNA) half-life of 25 h at 37 degrees C. A 3D model of the barminomycin-DNA complex has been defined from 307 NOE distance constraints. The enhanced stability of barminomycin-DNA adducts appears to be due primarily to protection of the aminal linkage from hydrolysis and this has provided insight into the design of new anthracycline derivatives with enhanced stability and activity. Strategies for harnessing the extreme reactivity and activity of barminomycin are also presented.

Lipid raft-targeted Akt promotes axonal branching and growth cone expansion via mTOR and Rac1, respectively.

The molecular mechanisms by which extracellular guidance cues regulate axonal morphology are not fully understood. Recent findings suggest that increased activity of the protein kinase Akt promotes dendritic branching and elongation in hippocampal neurons. We tested whether expression of constitutively active Akt (CA-Akt) in primary sensory neurons would promote axonal branching and whether targeting CA-Akt to lipid rafts, common sites of Akt function, would differentially regulate axonal morphology. Biolistic transduction of sensory neurons induced a rapid expression of CA-Akt, resulting in increased axonal branching, cell hypertrophy, and growth cone expansion. Additionally, we found that targeting of CA-Akt to lipid rafts significantly potentiated growth cone expansion compared with expression of CA-Akt throughout the neuron. Because lipid rafts are concentrated within the growth cone, this finding suggests that signaling of expansion is likely regulated locally. We found that CA-Akt-mediated growth cone expansion, but not axonal branching, was attenuated by coexpression of dominant-negative Rac1. In contrast, blockade of mammalian target of rapamycin (mTOR) prevented axonal branching and hypertrophy in response to CA-Akt, but not growth cone expansion. These data indicate that Akt activity can regulate growth cone expansion via localized Rac1 signaling and regulate axonal branching and soma size via activation of mTOR.

Conditional activation of FGFR1 in the prostate epithelium induces angiogenesis with concomitant differential regulation of Ang-1 and Ang-2.

The expression of fibroblast growth factor receptor (FGFR)-1 correlates with angiogenesis and is associated with prostate cancer (CaP) progression. To more precisely define the molecular mechanisms whereby FGFR1 causes angiogenesis in the prostate we exploited a transgenic mouse model, JOCK-1, in which activation of a conditional FGFR1 allele in the prostate epithelium caused rapid angiogenesis and progressive hyperplasia. By labeling the vasculature in vivo and applying a novel method to measure the vasculature in three dimensions, we were able to observe a significant increase in vascular volume 1 week after FGFR1 activation. Although vessel volume and branching both continued to increase throughout a 6-week period of FGFR1 activation, importantly, we discovered that continued activation of FGFR1 was not required to maintain the new vasculature. Exploring the molecular mediators of the angiogenic phenotype, we observed consistent upregulation of HIF-1alpha, vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2), whereas expression of Ang-1 was lost. Further analysis revealed that loss of Ang-1 expression occurred in the basal epithelium, whereas the increase in Ang-2 expression occurred in the luminal epithelium. Reporter assays confirmed that the Ang-2 promoter was regulated by FGFR1 signaling and a small molecule inhibitor of FGFR activity, PD173074, could abrogate this response. These findings establish a method to follow spontaneous angiogenesis in a conditional autochthonous system, implicate the angiopoietins as downstream effectors of FGFR1 activation in vivo, and suggest that therapies targeting FGFR1 could be used to inhibit neovascularization during initiation and progression of CaP.

Antiangiogenic gene therapy: disruption of neovascular networks mediated by inducible caspase-9 delivered with a transcriptionally targeted adenoviral vector.

The activation of an inducible caspase (iCaspase-9) mediates apoptosis of neovascular endothelial cells, and overcomes the prosurvival effect of vascular endothelial growth factor or basic fibroblast growth factor. The potential utilization of direct activation of caspases as an antiangiogenic strategy for treatment of angiogenesis-dependent diseases (eg cancer) requires expression of the inducible caspase primarily in the tumor endothelium. The objective of this work was to develop and characterize a transcriptionally targeted adenoviral vector that mediates expression of iCaspase-9 specifically in neovascular endothelial cells. We observed that adenoviral vectors containing the human VEGFR2 promoter induced reporter gene expression primarily in proliferating human dermal microvascular endothelial cells (HDMEC). HDMEC transduced with recombinant adenoviral vectors containing iCaspase-9 under regulation of the VEGFR2 promoter (Ad-hVEGFR2-iCaspase-9) and exposed to a cell-permeable dimerizer drug (AP20187), presented higher caspase-3 activity and apoptosis than controls (P < or = 0.05). Using the SCID Mouse Model of Human Angiogenesis, we observed that local delivery of Ad-hVEGFR2-iCaspase-9 followed by intraperitoneal injection of AP20187 resulted in endothelial cell apoptosis and local ablation of microvessels. We believe that this constitutes the first report of a transcriptionally targeted antiangiogenic adenoviral vector that mediates neovascular disruption upon activation of a caspase-based artificial death switch.

Suicide genes as safety switches in T lymphocytes.

Broader application of adoptive transfer of tumor-specific T-lymphocytes is accompanied by the need for effective suicide genes to ensure the safety of this cell-based therapy. In vivo elimination of T-lymphocytes expressing the herpes simplex virus-derived thymidine kinase gene has demonstrated the feasibility of this suicide gene as safety switch. However, improvements are required to overcome initial problems, such as immunogenicity. Here, newly developed suicide genes, including inducible Fas, inducible caspase and CD20 are discussed. In addition, problems of clinical application of marker genes and gene transfer techniques, which are prerequisites for suicide gene therapy, are addressed.

Ablation of microvessels in vivo upon dimerization of iCaspase-9.

Anti-angiogenic therapies based on targeted disruption of the tumor microvascular network have been proposed for cancer treatment. Inhibitors of the endothelial cell pro-survival pathway mediated by VEGF were shown to activate caspases and cause microvascular regression, but the efficacy of this strategy can be hindered by the engagement of redundant survival pathways. Alternatively, if direct activation of an apical pro-apoptotic caspase is sufficient to disrupt microvessels in vivo, such a strategy could potentially override upstream endothelial cell survival inputs and disrupt tumor neovascular networks. Here, we fused caspase-9 to a mutated FKBP12 domain to express an inducible caspase-9 molecule (iCaspase-9) that can be activated by a cell-permeable dimerizer drug, and transduced this construct into primary endothelial cells. We found that drug-induced dimerization of iCaspase-9 is sufficient to activate endogenous caspase-3 and trigger apoptosis even when endothelial cells are treated with the pro-survival factors VEGF or bFGF. A single intraperitoneal injection of the dimerizer drug induced apoptosis of endothelial cells expressing iCaspase-9 and elimination of human microvessels engineered in immunodeficient mice. These results demonstrate that the activation of iCaspase-9 disrupts microvessels in vivo, and suggest a novel anti-angiogenic strategy based on the expression and controlled activation of an inducible death gene in neovascular endothelial cells.

A novel conditional Akt 'survival switch' reversibly protects cells from apoptosis.

The anti-apoptotic Akt kinase is commonly activated by survival factors following plasma membrane relocalization attributable to the interaction of its pleckstrin homology (PH) domain with phosphatidylinositol 3-kinase (PI3K)-generated PI3,4-P(2) and PI3,4,5-P(3). Once activated, Akt can prevent or delay apoptosis by phosphorylation-dependent inhibition or activation of multiple signaling molecules involved in apoptosis, such as BAD, caspase-9, GSK3, and NF-kappaB and forkhead family transcription factors. Here, we describe and characterize a novel, conditional Akt controlled by chemically induced dimerization (CID). In this approach, the Akt PH domain has been replaced with the rapamycin (and FK506)-binding domain, FKBP12, to make F3-DeltaPH.Akt. To effect membrane recruitment, a myristoylated rapamycin-binding domain from FRAP/mTOR, called M-FRB, binds to lipid permeable rapamycin (and non-bioactive synthetic 'rapalogs'), leading to reversible heterodimerization of M-FRB with FKBP-DeltaPH.Akt. Like endogenous c-Akt, we show that the kinase activity of membrane-localized F3-DeltaPH.Akt correlates strongly with phosphorylation at T308 and S473; however, unlike c-Akt, phosphorylation and activation of inducible Akt (iAkt) is largely PI3K independent. CID-mediated activation of iAkt results in phosphorylation of GSK3, and contributes to NF-kappaB activation in vivo in a dose-sensitive manner. Finally, in Jurkat T cells stably expressing iAkt, CID-induced Akt activation rescued cells from apoptosis triggered by multiple apoptotic stimuli, including staurosporine, anti-Fas antibodies, PI3K inhibitors and the DNA damaging agent, etoposide. This novel inducible Akt should be useful for identifying new Akt substrates and for reversibly protecting tissue from apoptosis due to ischemic injury or immunological attack.

Caspase-10 is an initiator caspase in death receptor signaling.

A role for caspase-10, previously implicated in the autoimmune lymphoproliferative syndrome, in death receptor signaling has not been directly shown. Here we show that caspase-10 can function independently of caspase-8 in initiating Fas- and tumor necrosis factor-related apoptosis-inducing ligand-receptor-mediated apoptosis. Moreover, Fas crosslinking in primary human T cells leads to the recruitment and activation of caspase-10. Fluorescent resonance energy transfer analysis indicates that the death-effector domains of caspase-8 and -10 both interact with the death-effector domain of FADD. Nonetheless, we find that caspase-8 and -10 may have different apoptosis substrates and therefore potentially distinct roles in death receptor signaling or other cellular processes.

Adenovirus-mediated tissue-targeted expression of a caspase-9-based artificial death switch for the treatment of prostate cancer.

Clinical experience with suicide gene therapy for prostate cancer using first-generation approaches has provided a basis for developing improved strategies. Given the low proliferation rate exhibited by prostate cancer, one improvement would be to develop suicide genes that effectively kill both dividing and nondividing cells. A second improvement would be to restrict cytotoxicity to prostate cancer cells, limiting injury of nondiseased tissue. Here we describe a novel approach to achieving both goals based on: (a) the use of a small, but potent, prostate-specific composite promoter, ARR(2)PB, based on the rat probasin gene; and (b) the use of a powerful artificial death switch, called inducible caspase-9 (iCaspase-9). ARR(2)PB includes two copies of the androgen response region (ARR), each containing two androgen receptor (AR)-binding sites, placed upstream of the probasin promoter elements necessary for basal transcription. Because iCaspase-9 contains two binding sites for the dimeric ligand, AP20187, administration of chemical inducers of dimerization leads to aggregation and caspase activation, followed by rapid apoptosis in both dividing and nondividing cells. Using both reagents, we constructed two novel adenoviruses (ADVs), ADV.ARR(2)PB-iCasp9 expressing iCaspase-9 and control ADV.ARR(2)PB-EGFP expressing enhanced green fluorescent protein (EGFP). We demonstrate that tissue specificity is not sacrificed in an ADV backbone because the marker protein, EGFP, is expressed in R1881-stimulated ADV.ARR(2)PB-EGFP-transduced LNCaP cells but not in AR(-) PC-3, 293, HuH-7, U-87, and MCF-7 cells. Similarly, Pro-iCaspase-9 is expressed in ADV.ARR(2)PB-iCasp9-infected LNCaP cells after R1881 administration and is activated after AP20187 administration. In vitro experiments revealed rapid and efficient iCaspase-9-induced apoptosis of LNCaP cells in both an R1881- and AP20187-dependent manner. Only 28, 8, and 0.5% survival of LNCaP cells was seen at multiplicities of infection of 2, 10, and 25, respectively. Furthermore, at a multiplicity of infection of 10, extraordinary sensitivity to AP20187 was seen (IC(50), approximately 3 pM). In vivo experiments showed that ADV.ARR(2)PB-iCasp9 induced apoptosis in LNCaP but not in HuH-7 xenograft tumors in an AP20187-dependent manner. Furthermore, a simple i.p. injection of AP20187 dramatically suppressed LNCaP tumor growth in nude mice and led to a significantly increased host survival. This study demonstrates the feasibility of using tissue-specific expression of cell cycle-independent iCaspases as a nonmutagenic alternative modality for prostate cancer suicide gene therapy.

Cell proliferation through forced engagement of c-Kit and Flt-3.

To investigate the potential for functional interactions between heterologous receptors, the cytoplasmic domains of 2 different receptors (c-Kit and Flt-3) were coexpressed in the interleukin-3-dependent cell line Ba/F3. The receptor signaling domains were presented in the context of fusion proteins, with c-Kit linked to the FK506 binding protein (FKBP12) and Flt-3 linked to the FRB domain of the FKBP12-rapamycin-associated protein. The fusions were brought into apposition with the use of chemical inducers of dimerization (CIDs). Two classes of CID were employed. FK1012 and its synthetic analogue AP1510 bring together 2 copies of the FKBP12 domain, thereby inducing homodimerization of the c-Kit(FKBP12) fusion. A second type of CID, rapamycin, brings together one FKBP12 domain and one FRB domain, resulting in heterodimerization of the c-Kit(FKBP12) and Flt-3(FRB) fusions. Ba/F3 cell growth was promoted not only by FK1012- or AP1510-induced homodimerization of the c-Kit(FKBP12) fusion (as reported previously), but also by rapamycin-induced c-Kit(FKBP12)-Flt-3(FRB) heterodimerization. These findings demonstrate the potential for a direct functional interaction between c-Kit and Flt-3. (Blood. 2001;97:3662-3664)

Intracellular calcium signals regulating cytosolic phospholipase A2 translocation to internal membranes.

Increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) promote cytosolic phospholipase A(2) (cPLA(2)) translocation to intracellular membranes. The specific membranes to which cPLA(2) translocates and the [Ca(2+)](i) signals required were investigated. Plasmids of EGFP fused to full-length cPLA(2) (EGFP-FL) or to the cPLA(2) C2 domain (EGFP-C2) were used in Ca(2+)/EGFP imaging experiments of cells treated with [Ca(2+)](i)-mobilizing agonists. EGFP-FL and -C2 translocated to Golgi in response to sustained [Ca(2+)](i) greater than approximately 100-125 nm and to Golgi, ER, and perinuclear membranes (PNM) at [Ca(2+)](i) greater than approximately 210-280 nm. In response to short duration [Ca(2+)](i) transients, EGFP-C2 translocated to Golgi, ER, and PNM, but EGFP-FL translocation was restricted to Golgi. However, EGFP-FL translocated to Golgi, ER, and PNM in response to long duration transients. In response to declining [Ca(2+)](i), EGFP-C2 readily dissociated from Golgi, but EGFP-FL dissociation was delayed. Agonist-induced arachidonic acid release was proportional to the [Ca(2+)](i) and to the extent of cPLA(2) translocation. In summary, we find that the differential translocation of cPLA(2) to Golgi or to ER and PNM is a function of [Ca(2+)](i) amplitude and duration. These results suggest that the cPLA(2) C2 domain regulates differential, Ca(2+)-dependent membrane targeting and that the catalytic domain regulates both the rate of translocation and enzyme residence.

Adenovirus-mediated transfer of inducible caspases: a novel "death switch" gene therapeutic approach to prostate cancer.

In patients with localized prostate cancer, radical prostatectomy and radiation therapy, although effective in controlling localized disease, are often associated with significant side effects attributable to injury of adjacent tissues. Moreover, patients with metastatic disease eventually fail systemic hormonal or chemotherapy because of the development of progressive, refractory disease. In this study, we evaluated the safety and efficacy of a novel suicide gene therapy that could potentially spare normal tissue while bypassing molecular mechanisms of apoptosis resistance by using chemically inducible effector caspases to trigger apoptosis in prostate cancer cells. Initially, we compared the ability of a panel of inducible Fas signaling intermediates to kill human and murine prostate cancer cell lines. On the basis of the superior killing by downstream caspase-1 and caspase-3, replication-deficient adenoviral vectors expressing conditional caspase-1 (Ad-G/iCasp1) or caspase-3 (Ad-G/iCasp3), regulated by nontoxic, lipid-permeable, chemical inducers of dimerization (CID), were constructed. Upon vector transduction followed by CID administration, aggregation and activation of these recombinant caspases occur, leading to rapid apoptosis. In vitro, both human (LNCaP and PC-3) and murine (TRAMP-C2 and TRAMP-C2G) prostate cancer cell lines were efficiently transduced and killed in a CID-dependent fashion. In vivo, direct injection of Ad-G/iCasp1 into s.c. TRAMP-C2 tumors caused focal but extensive apoptosis without evidence for a bystander effect at the maximal viral dose (i.e., 2.5 x 10(10) viral particles/25 microl) in host animals that also received CID compared with control animals. Treatment with Ad-G/iCasp1 plus CID resulted in a transient, yet significant, reduction both in tumor growth and volume compared with tumors treated with vector but not CID (P < 0.035) or vector-diluent plus CID (P < 0.022), both of which grew more rapidly. These results demonstrate that CID-regulated, caspase-based suicide gene therapy is safe and can inhibit the growth of experimental prostate cancer in vitro and in vivo through potent induction of apoptosis, providing a rationale for further development.

Robust prostate-specific expression for targeted gene therapy based on the human kallikrein 2 promoter.

Tissue-specific transcriptional regulatory elements can increase the safety of gene therapy vectors. Unlike prostate-specific antigen (PSA/hK3), whose expression displays an inverse correlation with prostate cancer grade and stage, human glandular kallikrein 2 (hK2) is upregulated in higher grade and stage disease. Therefore, our goal was to develop a strong and prostate-specific hK2-based promoter for targeted gene therapy. We identified the minimum "full-strength" hK2 enhancer and built transcriptional regulatory elements composed of multiple tandem copies of this 1.2-kb enhancer, fused to the hK2 minimal promoter. Relative to the weak induction of the minimal hK2 promoter by androgen analog (R1881) in androgen receptor (AR)-positive LNCaP cells, transcriptional activity was increased by 25-, 44-, 81-, and 114-fold when one to four enhancers were spliced to the hK2 promoter, respectively. In contrast, the enhancer/promoter elements were inactive in the AR(-) prostate cancer line PC-3 and in a panel of nonprostate lines, including 293, U87, MCF-7, HuH-7, and HeLa cells. Furthermore, we generated a recombinant adenovirus, ADV.hK2-E3/P-EGFP, expressing enhanced green fluorescent protein (EGFP) under the control of the hK2 triplicate enhancer/promoter, and compared its properties with ADV.CMV-EGFP expressing EGFP under the control of the cytomegalovirus (CMV) enhancer/promoter. Unlike the CMV promoter, the hK2-E3/P promoter was at least 100-fold inducible by R1881 in the adenoviral backbone. Compared with in situ injection of subcutaneous LNCaP tumors with ADV.CMV-EGFP, which led to detectable EGFP expression in tumor, liver, and brain tissue, ADV.hK2-E3/P-EGFP injection led to robust but tumor-restricted EGFP expression. These results suggest that the hk2 multienhancer/promoter should be a powerful novel reagent for safer targeted gene therapy of prostate cancer.

Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that Do and Do not mobilize calcium. Novel role of mitogen-activated protein kinase pathways in cytosolic phospholipase A2 regulation.

The 85-kDa cytosolic phospholipase A(2) (cPLA(2)) mediates agonist-induced arachidonic acid release and eicosanoid production. Calcium and phosphorylation on Ser-505 by mitogen-activated protein kinases (MAPKs) regulate cPLA(2). Arachidonic acid release and eicosanoid production induced by stimuli that do (A23187, zymosan) or do not (phorbol myristate acetate (PMA), okadaic acid) mobilize calcium were quantitatively suppressed in cPLA(2)-deficient mouse peritoneal macrophages. The contribution of MAPKs to cPLA(2)-mediated arachidonic acid release was investigated. Both extracellular signal-regulated kinases (ERKs) and p38 contributed to cPLA(2) phosphorylation on Ser-505. However, although ERK inhibition did not affect A23187-induced arachidonic acid release, it suppressed zymosan-, PMA-, and okadaic acid-induced arachidonic acid release under conditions where phosphorylation of cPLA(2) on Ser-505 was unaffected. This indicates an additional regulatory mechanism for the ERK pathway. A role for transcriptional regulation is suggested by data showing that cycloheximide and actinomycin D inhibited arachidonic acid release induced by zymosan, PMA and, okadaic acid but not by A23187. Our results show that MAPK pathways contribute to arachidonic acid release in macrophages through alternative mechanisms in addition to their ability to phosphorylate cPLA(2) on Ser-505 and suggest a role for new protein synthesis.