Tetracycline-responsive Transactivator Research Articles

Hypoxia-responsive transgene expression system using RTP801 promoter and synthetic transactivator fused with oxygen-dependent degradation domain

Precise control of gene expression using an artificial gene circuit is a major challenge in the application of synthetic biology. Here, we designed a hypoxia-responsive transgene expression system by combining a hypoxia-inducible RTP801 promoter and a tetracycline-responsive transactivator fused with an oxygen-dependent degradation domain (TA-ODD). The reporter gene expression was highly induced by hypoxia when a transactivator-expression plasmid, pRTP801/TA-ODD, harboring a TA-ODD gene driven by the RTP801 promoter, was cotransfected with a reporter plasmid, pTRE/EGFP, harboring an EGFP gene controlled under the transactivator-responsive promoter. A stable cell line into which the expression units RTP801/TA-ODD and TRE/EGFP had been introduced responded to hypoxia and expressed the reporter gene in an oxygen-concentration-dependent manner. Moreover, the cells demonstrated potential as sensors to detect hypoxic conditions in a three-dimensional tissue culture invitro. These results indicate that the hypoxia-responsive transgene expression system is useful for constructing cell-based hypoxia detection systems.

Hsp72 overexpression protects from acetaminophen and DDC-associated liver injury but enhances protein aggregation

Heat shock protein (Hsp) 72 is a molecular chaperone which is upregulated in response to a variety of stress situations and possesses broad cytoprotective functions. However its hepatic function remains largely unknown. To study the importance of Hsp72 in the liver, we generated transgenic mice overexpressing Hsp72 under the control of a tissue-specific tetracycline-inducible system and crossed them with animals carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor (Hsp72-LAP mice). Acute liver injury was induced by a single intraperitoneal injection of acetaminophen (800 mg/kg). Long-term feeding (12 weeks) with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was used to induce Mallory-Denk-body (MDB) formation. Hsp72-LAP mice displayed doxycycline-regulated, robust Hsp72 overexpression in hepatocytes, but not in the other tissues or cell types tested. 18 hours after acetaminophen injection, significantly lower liver transaminase levels were noted in Hsp72-LAP mice compared to single transgenes (ALT: 933 vs. 1977, p < 0.05). No differences in acetaminophen metabolism were seen, but Hsp72 overexpression protected from formation of APAP protein adducts (p = 0.027). After DDC-feeding, Hsp72-LAP mice experienced a lower weight loss (p = 0.03) and lower ALT levels (650 vs. 875, p = 0.03). On the other hand, DDC-feeding resulted in a more abundant MDB formation in Hsp72-LAP mice vs. single transgenes. Conclusions: Our results suggest that Hsp72 overexpression protects from liver injury in two independent models but precipitates aggregate formation.

Inducible, tightly regulated and non-leaky neuronal gene expression in mice

The Tetracycline (Tet)-controlled inducible system is the most widely used reversible system for transgene expression in mice with over 500 lines created to date. Although this system has been optimized over the years, it still has limitations such as residual transgene expression when turned off, referred to as leakiness. Here, we present a series of new Tet-OFF transgenic mice based on the second generation tetracycline-responsive transactivator system. The tTA-Advanced (tTA2(S)) is expressed under control of the neuron-specific Thy1.2 promoter (Thy-OFF), to regulate expression in the mouse brain. In addition, we generated a lacZ reporter line, utilizing the P tight Tet-responsive promoter (P(tight)-lacZ), to test our system. Two Thy-OFF transgenic lines displaying two distinct patterns of expression were selected. Oral doxycycline treatment of Thy-OFF/P tight-lacZ mice demonstrated tight transgene regulation with no leak expression. These new Thy-OFF mice are valuable for studies in a broad range of neurodegenerative diseases such as Alzheimer's disease and related forms of dementia, where control of transgene expression is critical to understanding mechanisms underlying the disease. Furthermore, P tight-lacZ reporter mice may be widely applicable.

288 Hsp72 OVEREXPRESSION PROTECTS FROM ACETAMINOPHEN-, BUT EXACERBATES Fas-INDUCED LIVER INJURY

Background: Heat shock protein (Hsp) 72 is a molecular chaperone which is upregulated in response to a variety of stress situations and possesses broad cytoprotective functions. However its hepatic function remains largely unknown. Aims and Methods: To study the importance of Hsp72 in the liver, we generated transgenic mice overexpressing Hsp72 under the control of a tissue-specific tetracycline-inducible system and crossed them with animals carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor (Hsp72-LAP mice). Acute liver injury was induced by a single intraperitoneal injection of acetaminophen (800mg/kg) or the anti-CD95 antibody Jo2 (0.30mg/g). The severity of experimental liver injury was determined via serological and histological analyses. Results: Hsp72-LAP mice displayed doxycycline-regulated, robust Hsp72 overexpression in hepatocytes, but not in the other tissues tested. 18 hours after acetaminophen injection, a significantly lower liver injury was noted in Hsp72-LAP mice compared to single transgenes (ALT: 933 vs. 1977, p < 0.05). No differences in acetaminophen metabolism were seen. On the other hand, injection of anti-CD95 antibody resulted in a more profound injury in Hsp72-LAP vs. single transgenic animals (ALT level: 6435 vs. 2574, p = 0.004). Conclusions: Hsp72-LAP mice represent a unique tool to study the role of Hsp72 in the liver in a timeand cell-type-specific manner. Our results suggest that Hsp72, dependent on context, both ameliorates and promotes liver injury.

Abstract C84: Application of sensitive EMT reporter to identification of breast cancer stem cell-related genes

Abstract Epithelial-mesenchymal transition (EMT) is a key feature in breast cancer stem cell. In order to identify genes responsible for EMT/stemness, we have established a novel reporter system, in which mammary epithelial cells undergoing EMT can be visualized and positively selected by expression of GFP and blasticidine-resistant (BsR) genes, respectively. By incorporating tetracycline-responsive transactivator (rTA) and tetracycline-responsive element (TRE), vimentin promoter activity could be successfully amplified, which make it possible to induce sufficient Bsr/GFP fusion protein. Snail introduction can induce GFP+ CD44hi population by 30%. Using this reporter cell line, cDNA libraries derived from aggressive breast cancer cell lines MDA-MB-231 and BT549 were screened. Snail cDNA was isolated from several clones with GFP+ CD44hi. An shRNA library screening identified several candidate genes, which might be responsible for maintenance of epithelial phenotype. GFP-positive epithelial cells were identified in the original cells, which enable us to identify early EMT markers. By replacing the vimentin promoter with E-cadherin promoter, we could successfully establish a reporter system for mesenchymal-epithelial transition (MET). Through these attempts, we are aiming to identify molecular targets in breast cancer stem cell and triple-negative breast cancer. Citation Format: Kiyotsugu Yoshikawa, Yoshiaki Matsumoto, Junji Ito, Yoshie Shiojiri, Ellis Reinherz, Masakazu Toi. Application of sensitive EMT reporter to identification of breast cancer stem cell-related genes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C84.

Heat-inducible transgene expression with transcriptional amplification mediated by a transactivator

Purpose: Control of therapeutic gene expression in tumours is a major goal of gene therapy research, as it can restrict cytotoxic gene expression in cancer cells. In addition, the combination of hyperthermia with gene therapy through the application of heat-inducible vectors can result in considerable improvements in therapeutic efficiency. In this study, to combine heat-inducibility with high-level transgene expression, we developed a heat-inducible transgene expression system with transcriptional amplification mediated by a tetracycline-responsive transactivator.Materials and methods: A hybrid promoter was generated by placing the heat shock protein (HSP) 70B′ promoter under the tetracycline-repressor responsive element sequence, and a reporter/therapeutic gene expression plasmid was constructed by placing a reporter/therapeutic gene under the control of this hybrid promoter.Results: When the transactivator expression plasmid harbouring an expression cassette of the tetracycline-responsive transactivator gene was co-transfected with a reporter gene expression plasmid, the reporter gene expression was controlled by heat treatment. With this system, high levels of heat-induced transgene expression were observed compared to that from the HSP promoter alone without the transactivator. Evaluation of in vitro therapeutic effects using cancer cell lines revealed that therapeutic gene expression effectively caused cell death in a greater percentage of the cells.Conclusion: These findings indicate that this strategy improves the efficacy of cancer gene therapy.

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-κB (NF-κB) system is activated in response to several of these stresses, we hypothesized that NF-κB activation in hepatocytes may contribute to fibrosis development. To activate the NF-κB signaling pathway in a time- and cell-type-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-κB-induced liver fibrosis in a liver-injury-independent manner. Our results revealed that hepatic activation of IKK/NF-κB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases.

399 HEPATIC ACTIVATION OF IKK/NF-KB SIGNALING INDUCES LIVER FIBROSIS VIA MACROPHAGE-MEDIATED CHRONIC INFLAMMATION

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-jB (NF-jB) system is activated in response to several of these stresses, we hypothesized that NF-jB activation in hepatocytes may contribute to fibrosis development. To activate the NF-jB signaling pathway in a timeand celltype-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-jB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-jB-induced liver fibrosis in a liver-injury-independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF-jB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-jB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117-1128)

Persistence of High-Grade Cervical Dysplasia and Cervical Cancer Requires the Continuous Expression of the Human Papillomavirus Type 16 E7 Oncogene

Several mucosotropic human papillomaviruses (HPV), including HPV type 16 (HPV-16), are etiologic agents of a subset of anogenital cancers and head and neck squamous cell carcinomas. In mice, HPV-16 E7 is the most potent of the papillomaviral oncogenes in the development of cervical disease. Furthermore, interfering specifically with the expression of E7 in HPV-positive cell lines derived from human cervical cancers inhibits their ability to proliferate, indicating that the expression of E7 is important in maintaining the transformed phenotype in vitro. To assess the temporal role of E7 in maintaining HPV-associated tumors and precancerous lesions in vivo, we generated Bi-L E7 transgenic mice that harbor a tetracycline-inducible transgene that expresses both HPV-16 E7 and firefly luciferase. When we crossed Bi-L E7 mice to a K5-tTA transgene-inducing line of mice, which expresses a tetracycline-responsive transactivator selectively in the stratified squamous epithelia, the resulting Bi-L E7/K5-tTA bitransgenic mice expressed E7 and luciferase in the skin and cervical epithelium, and doxycycline repressed this expression. Bitransgenic mice displayed several overt and acute epithelial phenotypes previously shown to be associated with the expression of E7, and these phenotypes were reversed on treatment with doxycycline. Repressing the expression of E7 caused the regression of high-grade cervical dysplasia and established cervical tumors, indicating that they depend on the continuous expression of E7 for their persistence. These results suggest that E7 is a relevant target not only for anticancer therapy but also for the treatment of HPV-positive dysplastic cervical lesions.

The β-Propensity of Tau Determines Aggregation and Synaptic Loss in Inducible Mouse Models of Tauopathy

Neurofibrillary lesions are characteristic for a group of human diseases, named tauopathies, which are characterized by prominent intracellular accumulations of abnormal filaments formed by the microtubule-associated protein Tau. The tauopathies are accompanied by abnormal changes in Tau protein, including pathological conformation, somatodendritic mislocalization, hyperphosphorylation, and aggregation, whose interdependence is not well understood. To address these issues we have created transgenic mouse lines in which different variants of full-length Tau are expressed in a regulatable fashion, allowing one to switch the expression on and off at defined time points. The Tau variants differ by small mutations in the hexapeptide motifs that control the ability of Tau to adopt a beta-structure conformation and hence to aggregate. The "pro-aggregation" mutant DeltaK280, derived from one of the mutations observed in frontotemporal dementias, aggregates avidly in vitro, whereas the "anti-aggregation" mutant DeltaK280/PP cannot aggregate because of two beta-breaking prolines. In the transgenic mice, the pro-aggregation Tau induces a pathological conformation and pre-tangle aggregation, even at low expression levels, the anti-aggregation mutant does not. This illustrates that abnormal aggregation is primarily controlled by the molecular structure of Tau in vitro and in the organism. Both variants of Tau become mislocalized and hyperphosphorylated independently of aggregation, suggesting that localization and phosphorylation are mainly a consequence of increased concentration. These pathological changes are reversible when the expression of Tau is switched off. The pro-aggregation Tau causes a strong reduction in spine synapses.

Constitutive IKK2 activation in acinar cells is sufficient to induce pancreatitis in vivo

Activation of the inhibitor of NF-kappaB kinase/NF-kappaB (IKK/NF-kappaB) system and expression of proinflammatory mediators are major events in acute pancreatitis. However, the in vivo consequences of IKK activation on the onset and progression of acute pancreatitis remain unclear. Therefore, we modulated IKK activity conditionally in pancreatic acinar cells. Transgenic mice expressing the reverse tetracycline-responsive transactivator (rtTA) gene under the control of the rat elastase promoter were generated to mediate acinar cell-specific expression of IKK2 alleles. Expression of dominant-negative IKK2 ameliorated cerulein-induced pancreatitis but did not affect activation of trypsin, an initial event in experimental pancreatitis. Notably, expression of constitutively active IKK2 was sufficient to induce acute pancreatitis. This acinar cell-specific phenotype included edema, cellular infiltrates, necrosis, and elevation of serum lipase levels as well as pancreatic fibrosis. IKK2 activation caused increased expression of known NF-kappaB target genes, including mediators of the inflammatory response such as TNF-alpha and ICAM-1. Indeed, inhibition of TNF-alpha activity identified this cytokine as an important effector of IKK2-induced pancreatitis. Our data identify the IKK/NF-kappaB pathway in acinar cells as being key to the development of experimental pancreatitis and the major factor in the inflammatory response typical of this disease.

A genetic time‐delay circuitry in mammalian cells

Gene expression circuitries with time-delayed expression profiles regulate key events, such as oscillating systems, noise elimination, and coordinated multi-step processes, in all organisms from bacteria to mammalian cells. We present the rational synthesis of a genetic circuit displaying time-delayed expression in silico and in mammalian cells. The network is based on a time-delay circuit, where the tetracycline-responsive transactivator (tTA) induces expression of the pristinamycin-responsive repressor PIP-KRAB, which silences expression of the terminal human placental secreted alkaline phosphatase (SEAP). While the addition of pristinamycin I inactivates PIP-KRAB and results in the immediate resumption of SEAP expression, addition of tetracycline abolishes PIP-KRAB synthesis. Consequently, SEAP production remains repressed until the PIP-KRAB buffer in the cell is eliminated. We characterized in silico and in vivo the time-delayed expression properties and analyzed the impact of the size and stability of the PIP-KRAB buffer on fine-tuning of the response kinetics. This tunable time-delay circuitry represents a biologic building block for emulating a fundamental circuit topology in integrated artificial synthetic gene networks for the design of tailor-made cell types and organisms.

Performance of a tetracycline-responsive transactivator system for regulating transgenes in the oomycete Phytophthora infestans

The oomycete genus Phytophthora includes many important plant pathogens for which extensive genome data exist, but lacking is an inducible expression system to study contributions of their genes to growth and pathogenicity. Here the adaptation of the reverse tetracycline transactivator (rtTA) system to P. infestans is described. Vectors were developed containing rtTA expressed from an oomycete promoter, and beta-glucuronidase (GUS) controlled by TetR binding sites fused to a minimal oomycete promoter. Transformants were obtained in which GUS was expressed in a dose-dependent manner by the rtTA inducer doxycycline, indicating that the gene switch functions in P. infestans. However, toxicity of rtTA hindered the isolation of transformants if expressed on the same plasmid as the nptII selection marker. Better results were obtained by cotransforming those genes on separate plasmids, with 92% of transformants acquiring both DNAs although only 4% expressed rtTA at detectable levels. Low levels of reporter activity were measured in such transformants, suggesting that rtTA activated transcription weakly. Also, significant variation in the sensitivity of isolates to doxycycline and tetracycline was observed. These results are useful both in terms of developing tools for functional genomics and understanding the fate of DNA during Phytophthora transformation.

Animal models for the study of squamous cell carcinoma of the upper aerodigestive tract: A historical perspective with review of their utility and limitations part B. Transgenic mouse models

Animal models for the study of squamous cell carcinoma of the upper aerodigestive tract: A historical perspective with review of their utility and limitations part B. Transgenic mouse models

Transgenic targeting with regulatory elements of the humanCD34 gene

The human CD34 gene is expressed on early progenitor and stem cells in the bone marrow. Here we report the isolation of the human CD34 locus from a human P1 artificial chromosome (PAC) library and the characterization and evaluation of this genomic fragment for expression of reporter genes in stable cell lines and transgenic mice. We show that a 160-kb fragment spanning 110 kb of the 5' flanking region and 26 kb of the 3' flanking region of the CD34 gene directs expression of the human CD34 gene in the bone marrow of transgenic mice. The expression of human CD34 transgenic RNA in tissues was found to be similar to that of the endogenous murine CD34 gene. Colony-forming cell assays showed that bone marrow cells staining positive for human CD34 consist of early progenitor cells in which expression of CD34 decreased with cell maturation. In order to test the construct for its ability to express heterologous genes in vivo, we used homologous recombination in bacteria to insert the tetracycline-responsive transactivator protein tTA. Analysis of transgenic human CD34-tTA mice by cross breeding with a strain carrying Cre recombinase under control of a tetracycline-responsive element demonstrated induction of Cre expression in mice in a pattern consistent with the expression of the human CD34 transgene.

A Model System for Studying Postnatal Myogenesis with Tetracycline-Responsive, Genetically Engineered Clonal Myoblasts in Vitro and in Vivo

The aim of this work was to introduce a tetracycline-responsive (Tet-off) gene expression system into myoblasts in order to regulate a reporter gene not only in vitro but also particularly in muscles implanted with these engineered myoblasts. Mouse myoblasts from a long-term culture (i28 cells) were transfected initially to generate and characterize two stable master clones expressing tetracycline-responsive transactivator protein tTA. Like parental i28 myoblasts, these clones differentiated well in vitro. The second step introduced the firefly (Photinus pyralis) luciferase gene into one of the stable tTA clones producing double transfectants expressing luciferase in the absence of tetracycline. Addition of tetracycline (1 μg ml−1) resulted in at least 100-fold decreases in luciferase activity within 8 h in both growing and differentiating myoblast cultures. Enzyme activity was rapidly restored after tetracycline was removed (8 h). After successful implantation of these myoblasts into damaged mouse muscles, luciferase expression in the matured progeny cells could be regulated by oral application of doxycycline for at least 1 month. The tetracycline-responsive master clones are potentially powerful tools for studying the function of various genes in postnatal myogenesis.

Evaluation of the tetracycline- and ecdysone-inducible systems for expression of neurotransmitter receptors in mammalian cells.

Establishing a stable cell line that expresses a particular protein of interest is often a laborious and time-consuming experience. With constitutive expression systems, a gradual loss of the highly expressing clones over a given time span and/or a severe counter-selection due to toxicity of the expressed protein for the host cell line are major drawbacks. In both cases, inducible expression systems offer a valuable alternative. Over the years, many regulated expression systems have been developed and evaluated. In the present study, we compare the efficiency, the advantages and the drawbacks of a tetracycline- and an ecdysone-inducible system for expression of the reporter protein chloramphenicol acetyltransferase and of different G-protein-coupled serotonin (5-HT) receptors. A high level of expression of different 5-HT receptors was obtained with the tetracycline-inducible system. In the cell line L929, which stably expresses the tetracycline-responsive transactivator, a maximum ligand binding of 20,000 and 9500 fmol/mg protein was measured for the h5-HT(1B) and h5-ht(1F) receptors, respectively. In the HEK293rtTA cell line, levels of 15,700, 3000, and 9100 fmol bound ligand/mg protein were obtained for the h5-HT(1B), h5-ht(1F) and h5-HT(4b) receptors, respectively. These high expression levels remained stable for several months of continuous culture. Although the ecdysone-inducible expression system was useful for tightly regulated expression, the levels were far lower than those obtained with the tetracycline system (e.g. 640 fmol bound ligand/mg protein for the h5-ht(1F) receptor in HEK293EcR).

Influence of a transiently transfected gene on apoptosis, measurements guided by cotransfected GFP

A rapid and quantitative flow cytometric method simultaneously identifying cells that incorporated a gene of interest in a transient electroporation and discriminating between dead, live and apoptotic states in a single measurement is presented. An expression vector encoding the gene of interest was cotransfected with a plasmid carrying the green fluorescent protein (GFP). Subsequently, the cultured cells were stained with 7-amino-actinomycin D (7-AAD) without fixation and were subjected to a multivariate analysis. The value of the method and its high reproducibility were demonstrated on Jurkat cells. Those cells were transiently transfected with a construct expressing a short C-terminal fragment of presenilin 1 (PS1-f) known to show anti-apoptotic properties. The PS1-f gene was under the control of the tetracycline-responsive transactivator.

Mdx mice inducibly expressing dystrophin provide insights into the potential of gene therapy for duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by the lack of expression of the dystrophin protein in muscle tissues. We genetically engineered a mouse model (mdx) of DMD that allowed for the high level and inducible transcription of a dystrophin mini-gene. This was achieved via the tetracycline-responsive transactivator (tTA) system. Multiple analyses confirmed that dystrophin expression in the mice was: (i) tTA dependent; (ii) correctly localized to the sarcolemmal membranes; (iii) capable of preventing the onset of dystrophy; and (iv) effectively blocked by the oral administration of tetracyclines. The model allowed us to somatically extinguish or induce dystrophin gene transcription. Somatic induction of dystrophin transcription prevented the onset of muscular dystrophy in some muscle groups. The levels of phenotypic rescue were influenced, however, by the age of the animals at the time of dystrophin induction. We also found that despite somatic termination of dystrophin gene transcription, the dystrophin protein was found to be associated with the sarcolemmal membrane for at least 26 weeks. Persistent detection of dystrophin was also accompanied by a prolonged protection of the muscle cells from the onset of dystrophy. The findings demonstrated that somatic transfer of the dystrophin gene not only may allow for the prevention of muscular dystrophy in multiple muscle groups, but also may be accompanied by persistent efficacy, secondary to the long-term functional stability of the dystrophin protein in vivo. This model should be useful in future studies concerning the potential of genetic therapy for DMD, as well as other muscle disorders.

Regulation of macropinocytosis by p21-activated kinase-1.

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.