Biomedical subjects

B Massie

Publications and source records attributed to B Massie.

At least 37 records · Page 2Linked to original sources

PubMed2000

The chaperone function of hsp70 is required for protection against stress-induced apoptosis.

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.

Adaptation, Physiological↗

PubMed1999

Induction of humoral and cellular immune responses against the nucleocapsid of bovine viral diarrhea virus by an adenovirus vector with an inducible promoter.

A new recombinant adenovirus was constructed that expressed the nucleocapsid (C protein or p14) of the bovine viral diarrhea virus (BVDV) under the control of a tetracycline-regulatable promoter. Mice covaccinated with this recombinant adenovirus, accompanied by another recombinant adenovirus expressing the trans-activator protein, induced a strong humoral immune response to the BVDV/C protein as detected by ELISA. Splenocytes from mice immunized with the recombinant adenovirus showed a specific proliferation response to both genotypes (type 1 and 2) of BVDV. High levels of IFN-gamma were detected in the supernatant of murine mononuclear cells of mice immunized by the recombinant adenovirus when stimulated in vitro by both genotypes of BVDV. These results indicate that this recombinant adenovirus is highly immunogenic and stimulates both cellular and humoral immune responses against the nucleocapsid of BVDV.

Adenoviruses, Human↗

PubMed1999

Recombinant adenoviruses expressing the E2 protein of bovine viral diarrhea virus induce humoral and cellular immune responses.

The E2 protein of bovine viral diarrhea virus (BVDV) is a major viral glycoprotein and an attractive target for BVDV vaccines. Three replication defective recombinant adenoviruses expressing the BVDV/E2 protein (rAds/E2) were constructed. Two contain a constitutive promoter, and one an inducible promoter. All three recombinant adenoviruses induced very strong BVDV specific antibody responses in a mouse model as detected by enzyme-linked immunosorbant assay (ELISA) and neutralization tests. Induction of cellular immune responses was investigated in two recombinant adenoviruses with a constitutive promoter. The mononuclear cells from the immunized mice demonstrated a proliferative response after in vitro stimulation with an homologous BVDV strain, but only one of them induced the production of IFN-gamma.

Adenoviridae↗

PubMed1999

Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myeloma cells transfected with the E1B-19K adenoviral gene.

It is now well documented that apoptosis represents the prevalent mode of death in lymphoid cultures and occurs spontaneously in late-exponential phase of batch cultures following nutrient exhaustion. In an attempt to enhance the cell survival of these cell lines, we have initially engineered nonproducing NS/0 myeloma cells with a vector expressing the adenoviral E1B-19K protein. NS/0 cells transfected with E1B-19K were found to be more resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. In this study, we have characterised a number of NS/0 subclones constitutively expressing different levels of E1B-19K, as well as several subclones in which the expression of E1B-19K was regulated by a tetracycline-controllable gene switch. We have found that a threshold E1B-19K level was required in order to achieve protection against apoptosis. The extent of resistance against cell death induced by nutrient deprivation in glutamine-free medium and in the late phase of batch cultures correlated with the level of E1B-19K expression up to an optimal level where further increases in E1B-19K levels did not result in significant additional protection. To assess the effects of E1B-19K on antibody productivity, an apoptosis-resistant NS/0 clone was then transfected with a chimeric antibody construct. Despite their improved viability, the antibody productivity of E1B-19K clones in batch culture was not significantly improved. Moreover, while the use of E1B-19K considerably delayed cell death, cells eventually died by apoptosis. Surprisingly, E1B-19K had no beneficial effect on the efficiency of fusion of NS/0 myelomas and splenocytes for the generation of hybridoma cells. Furthermore, the resulting hybridomas, although expressing E1B-19K at levels comparable to the myeloma parent, were no longer resistant to apoptosis. This indicates that the ability of E1B-19K to prevent apoptosis is not only dose-dependent but also seems to be cell-type dependent.

Adenovirus E1B Proteins↗

PubMed1999

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions.

We have shown previously that recombinant NS/0 myelomas expressing sufficient amounts of E1B-19K were resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. However, no significant increase in monoclonal antibodies (MAb) was observed during the prolonged stationary phase of these batch cultures. Here, we show that E1B-19K can enhance cell survival and improve MAb productivity in high cell density perfusion culture. Typically, lymphoid cells grown under steady state in perfusion exhibit decreasing viabilities with concomitant accumulation of apoptotic cells. By modulating the ability of these cells to resist to induction of apoptosis in low nutrient environment, a 3-fold decrease in specific death rate from 0.22 day-1 for NS/0 control to 0.07 day-1 for E1B-19K cells was achieved, resulting in a significant improvement in cell viability throughout perfusion. E1B-19K cells at the perfusion plateau phase also exhibited a 3-fold reduction in specific growth rate concomitant with a lower percentage of S and higher percentage of G1 phase cells. This was associated with a 40% decrease in specific oxygen consumption rate, likely related to a reduction in the specific consumption rates of limiting nutrient(s). Expression of E1B-19K consequently had a significant impact on the steady-state viable cell density, allowing maintenance of 11.5 x 10(6) E1B-19K cells/mL versus 5.9 x 10(6) control NS/0 cells/mL for the same amount of fresh medium brought into the system (half a volume per day). Whereas MAb concentrations found in perfusion culture of control NS/0 myelomas were almost 3-fold higher than those found in batch culture; in the case of E1B-19K-expressing myelomas, the MAb concentration in perfusion was more than 7-fold higher than in batch. This was attributable to the 2-fold increase in viable cell plateau and to a 40% increase in the perfusion to batch ratio of specific MAb productivity (2.2-fold for E1B-19K myelomas versus 1.6-fold for NS/0 control).

Adenovirus E1B Proteins↗

PubMed1999

Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles.

Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.

Adenoviridae↗

PubMed1999

Baseline clinical characteristics of patients recruited into the assessment of treatment with lisinopril and survival study.

BACKGROUND: The beneficial effect of ACE inhibitors on mortality has been established in a series of trials. However, in clinical practice, ACE inhibitors are commonly administered in doses much lower than those shown to be effective in the landmark trials. AIMS: This report describes the baseline characteristics of the patients recruited into the ATLAS study by age and gender sub-groups. METHODS: The ATLAS study compared the effects of 'low' dose (2.5-5.0 mg/day) to 'high' dose (32.5-35.0 mg/day) lisinopril in a double-blind study of 3164 patients with moderate to severe heart failure and left ventricular ejection fraction < 30% during a mean follow-up period of 46 months. The primary end-point was all cause mortality and the principal secondary end-point a composite of all-cause hospitalisation or all-cause mortality. RESULTS: Among patients with heart failure selected for the presence of left ventricular systolic function there were few differences among age groups or between genders. Older patients were not so heavy, were more likely to have ischaemic heart disease, hypertension and atrial fibrillation contributing to their heart failure and had a higher blood urea. Women were not so heavy as men. Age and gender had no major influence on mean ejection fraction or baseline treatment in the ATLAS study. CONCLUSIONS: Weight and renal function may alter the plasma concentration of any given dose of an ACE inhibitor. Potential interactions between dose of lisinopril, weight and renal function will be explored after the study is completed.

Adult↗

PubMed1998

Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells.

This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.

Adenoviridae↗

PubMed1998

High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.

Replication-deficient adenovirus vectors (AdV) have been successfully used to transfer a truncated human dystrophin cDNA to skeletal muscle of dystrophin-deficient mdx mice. A dystrophin-deficient golden retriever dog model (GRMD) has been identified, which, unlike the mouse model, leads to a clinicopathological phenotype similar to that of Duchenne muscular dystrophy (DMD). We show for the first time that high-level dystrophin expression in skeletal muscle of GRMD dogs can be achieved by AdV-mediated gene transfer. However, a humoral and cellular immune response of the host against antigens of viral and transgene origin (similar to that occurring in mdx mice after AdV-mediated dystrophin gene transfer) leads to a decline of dystrophin expression over a 2-month period. Immunosuppression by cyclosporin significantly prolonged transgene expression. The GRMD model may help to solve the open questions pertaining to dystrophin gene transfer such as systemic delivery and improvement of muscle function before human trials for gene replacement therapy in DMD may be considered.

Adenoviridae↗

PubMed1998

The R1 subunit of herpes simplex virus ribonucleotide reductase is a good substrate for host cell protein kinases but is not itself a protein kinase.

The N terminus of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is believed to be a protein kinase domain mainly because the R1 protein was phosphorylated in a protein kinase assay on blot. Using Escherichia coli and adenovirus expression vectors to produce R1, we found that, whereas the reductase activity of both recombinant proteins was similar, efficient phosphorylation of R1 and casein in the presence of Mg2+ was obtained only with the R1 purified from eukaryotic cells. Phosphorylation of this R1, in solution or on blot, results mainly from the activity of casein kinase II (CKII), a co-purifying protein kinase. Labeling on blot occurs from CKII leakage off the membrane and its subsequent high affinity binding to in vivo CKII-phosphorylated R1. CKII target sites were mapped to an acidic serine-rich segment of the R1 N terminus. Improvement in purification of the R1 expressed in eukaryotic cells nearly completely abolished its phosphorylation potential. An extremely low level of phosphorylation observed in the presence of Mn2+ with the R1 produced in E. coli was probably due to an unidentified prokaryotic protein kinase. These results provide evidence that the herpes simplex virus type 2 R1 does not possess an intrinsic protein kinase activity.

Adenosine Triphosphate↗

PubMed1998

Efficient utrophin expression following adenovirus gene transfer in dystrophic muscle.

Utrophin is a homologue of dystrophin, the protein whose absence is responsible for Duchenne muscular dystrophy (DMD). As a first step toward clarifying if adenovirus (AV)-mediated utrophin transfer is a possible option to treat DMD, we have constructed an AV expressing utrophin (AdCMV-Utr) and studied utrophin expression after intramuscular injection of mdx mice, the mouse DMD model. Overexpression of utrophin by AdCMV-Utr was marked and nontoxic. The recombinant utrophin was distributed homogeneously at the surface of the muscle fibers. Its expression was sufficient to restore the normal histochemical pattern of alpha-sarcoglycan and beta-dystroglycan at this site. These two proteins are members of the dystrophin associated protein complex whose distribution is greatly reduced at the surface of the DMD muscle. These data indicate that AV-mediated utrophin transfer is an efficient way of utrophin upregulation in muscle and has the potential of becoming a treatment for DMD.

Adenoviridae↗

PubMed1998

A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus.

Open reading frame 3 (ORF3) of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), Quebec strain IAF-Klop, was reverse-transcribed and cloned into the procaryotic expression vector pGEX-4T-1, then subcloned into the eucaryotic expression vector pAdCMV5 which was used as a shuttle vector to generate a replication-defective recombinant adenovirus. The procaryotic GST-ORF3 recombinant fusion protein was used to raise a monospecific antiserum in rabbits. By Western-immunoblotting with PRRSV-infected cell extracts, the ORF3 encoded protein had an estimated molecular mass (M(r)) of 42 kDa, similar to that of the protein expressed by the adenovirus vector. Endoglycosidase F digestion showed that the ORF3 encoded protein occurs in an highly glycosylated form (GP3) in the infected MARC-145 cells. Pulse-chase and radioimmunoprecipitation experiments revealed that the GP3 protein was present in amounts equivalent to those of the N, M, and GP5 proteins in the infected cells, whereas no GP3 could be detected in purified virions. During the first 30 min of chase, the GP3 undergoes a gradual downward shift of its apparent M(r), thought to result from trimming of the mannose-rich glycan structures. Tested convalescent pig sera that were found to be seropositive to PRRSV by indirect immunofluorescence reacted positively with the recombinant GST-ORF3 fusion protein by immunoblotting. Data indicated that the ORF3 protein of the Quebec reference strain of PRRSV is a highly glycosylated and antigenic protein, which is nonstructural.

Adenoviridae↗

PubMed1998

Adenovirus-mediated dystrophin minigene transfer improves muscle strength in adult dystrophic (MDX) mice.

Duchenne muscular dystrophy (DMD) and murine X-linked muscular dystrophy (mdx) are both due to absence of the subsarcolemmal protein dystrophin. Recombinant adenovirus vectors (AdV) are considered a promising means for delivering a functional dystrophin gene to muscle. However, the usefulness of AdV for this purpose is limited by vector toxicity as well as immune-mediated elimination of infected fibers. In addition, studies to date of AdV-mediated dystrophin gene transfer have either failed to examine effects on muscle strength or been performed in immunologically immature neonatal animals with little baseline abnormality of force-generating capacity. In the present study, AdV-mediated dystrophin gene transfer was performed in adult mdx mice with pre-existent dystrophic pathology and muscle weakness. The main findings are as follows: (1) acute myofiber toxicity and gene transfer efficiency are both AdV dose-dependent, such that the therapeutic margin of safety is fairly narrow; (2) immunosuppressive therapy (FK506) prevents immune-mediated elimination of dystrophin-positive fibers but not the dose-dependent toxic effects; (3) at the optimal vector dosage and with effective immunosuppression, AdV-mediated dystrophin minigene transfer is capable of alleviating the loss of force-generating capacity as well as histopathological evidence of disease progression normally seen in adult mdx muscles over a 2-month period. These findings have important implications for the eventual application of AdV-mediated dystrophin gene transfer in DMD patients.

Adenoviridae↗

PubMed1998

Inducible overexpression of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette.

We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(delta2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce deltaR1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5 deltaR1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy.

Adenoviridae↗

PubMed1998

Combinatorial blockade of calcineurin and CD28 signaling facilitates primary and secondary therapeutic gene transfer by adenovirus vectors in dystrophic (mdx) mouse muscles.

Recombinant adenovirus vectors (AdV) have been considered a potential vehicle for performing gene therapy in patients suffering from Duchenne muscular dystrophy but are limited by a cellular and humoral immune response that prevents long-term transgene expression as well as effective transduction after AdV readministration. Conventional immunosuppressive agents such as cyclosporine and FK506, which act by interfering with CD3-T-cell receptor-mediated signaling via calcineurin, are only partially effective in reversing these phenomena and may also produce substantial organ toxicity. We hypothesized that activation of redundant T-cell activation pathways could limit the effectiveness of these drugs at clinically tolerable doses. Therefore, we have tested the ability of immunomodulatory immunoglobulins (Ig) with different modes of action to facilitate AdV-mediated gene transfer to adult dystrophic (mdx) mice. When used in isolation, immunomodulatory Ig (anti-intercellular adhesion molecule-1, anti-leukocyte function-associated antigen-1, anti-CD2, and CTLA4Ig) were only mildly effective in mitigating cellular and/or humoral immunity against adenovirus capsid proteins and the therapeutic transgene product, dystrophin. However, the combination of FK506 plus CTLA4Ig abrogated the immune response against adenovirus proteins and dystrophin to a degree not achievable with the use of either agent alone. At 30 days after AdV injection, >90% of myofibers could be found to express dystrophin with little or no evidence of a cellular immune response against transduced fibers. In addition, the humoral immune response was markedly suppressed, and this was associated with increased transduction efficiency following vector readministration. These data suggest that by facilitating both primary and secondary transduction after AdV administration, combined targeting of CD3-T-cell receptor-mediated signaling via calcineurin and the B7:CD28 costimulatory pathway could greatly increase the potential utility of AdV-mediated gene transfer as a therapeutic modality for genetic diseases such as Duchenne muscular dystrophy that will require long-term transgene expression and repeated vector delivery.

Adenoviridae↗

PubMed1998

A subset of porcine reproductive and respiratory syndrome virus GP3 glycoprotein is released into the culture medium of cells as a non-virion-associated and membrane-free (soluble) form.

The GP3 protein of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in 293 cells by a recombinant human type 5 adenovirus carrying the open reading frame 3 gene. The protein exhibited a molecular mass of 42 kDa and comigrated with GP3 expressed in PRRSV-infected MARC-145 cells. Removal of N-linked glycans from GP3 resulted in a 27-kDa protein (P3), confirming its highly glycosylated nature. Pulse-chase experiments carried out either in the context of PRRSV infection or upon individual expression of GP3 in 293 cells showed that the protein remains completely endo-beta-N-acetylglucosaminidase H-sensitive even after 4 h of synthesis. Thus, the transport of GP3 was restricted to the premedial Golgi compartment, presumably the endoplasmic reticulum (ER). However, a minor fraction of GP3 was found to be secreted in the culture medium as a soluble membrane-free form. This released protein (sGP3) was readily identified upon individual expression of GP3 in 293 cells as well as in the context of PRRSV infection, albeit at lower levels. The sGP3 migrated as a smear and displayed a molecular mass ranging from 43 to 53 kDa. The unglycosylated form of sGP3 comigrated with its intracellular deglycosylated counterpart, suggesting that the release from the cell of a subset of GP3 did not result from cleavage of a putative membrane-anchor sequence. Strikingly, unlike GP3, the sGP3 acquired Golgi-specific modifications of its carbohydrate side chains and folded into a disulfide-linked homodimer. Brefeldin A treatment completely abolished the release of sGP3, suggesting that the ER-to-Golgi compartment is an obligatory step in cellular secretion of sGP3. In contrast, 10 mM monensin did not prevent sGP3 release but inhibited the terminal glycosylation that confers on the protein its diffuse pattern. Since GP3 was found to be nonstructural in the case of the North American strain, secretion of a minor fraction of GP3 might be an explanation for its high degree of immunogenicity in infected pigs. Furthermore, this secreted protein might be relevant as a model for further studies on the cellular subcompartments involved in the sorting of proteins to the extracellular milieu.

Adenoviridae↗

PubMed1998

Reduced thermotolerance in aged cells results from a loss of an hsp72-mediated control of JNK signaling pathway.

Aged organisms exhibit a greatly decreased ability to induce the major heat shock protein, Hsp72, in response to stresses, a phenomenon that can also be observed in cell cultures (Heydari AR, Takahashi R, Gutsmann A, You S and Richardson A (1994) Hsp70 and aging. Experientia 50: 1092-1098). Hsp72 was shown to protect cells from a variety of stresses. The protective function of Hsp72 has been commonly ascribed to its chaperoning ability. However, recently we showed that Hsp72 protects cells from heat shock by suppression of a stress-kinase JNK, an essential component of the heat-induced apoptotic pathway (Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S, Shifrin VI and Sherman MY (1997) Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 272: 18033-18037). Here we demonstrate that because of the diminished inducibility of Hsp72 in aged cells, Hsp72-mediated control of JNK signaling pathway is compromised. This results in increased rate of apoptotic cell death following heat shock. We show that forced expression of Hsp72 in aged cells from an adenovirus-based vector completely suppresses activation of JNK by heat shock and consequently protects from heat-induced apoptosis. We also demonstrate for the first time that it is possible to restore endogenous expression of Hsp72 in aged cells. This can be achieved by treatment with the proteasome inhibitor MG132. Induction of Hsp72 in aged cells under these conditions leads to suppression of JNK activation by a heat shock and restoration of thermotolerance manifested in a lower rate of apoptosis.

Adenoviridae↗

PubMed1997

Study of adenovirus-mediated dystrophin minigene transfer to skeletal muscle by combined microscopic display of adenoviral DNA and dystrophin.

In situ DNA hybridization of an E4 adenoviral sequence amplified by in situ polymerase chain reaction (PCR) was used to mark adenovirus-containing myonuclei in muscles of immunocompetent and immunosuppressed mdx mice following intramuscular injection of adenoviral recombinants. The adenoviral recombinants contained a 6.3-kb dystrophin cDNA (minigene) driven by a cytomegalovirus (CMV) promoter/enhancer and thus, immunostaining for dystrophin of the same sections permitted correlation of adenoviral recombinant-containing myonuclei with dystrophin positivity of the same muscle fiber segments. As early as 2 hr post-injection of adenoviral recombinant, an appreciable number of adenoviral recombinant-positive (AVR+) myonuclei, and some partial dystrophin positive (pdys+) fibers were observed. Some fully dystrophin-positive (dys+) muscle fibers were present as early as 6 hr. The maximum number of fibers containing AVR+ myonuclei (observed by 72 hr) was maintained until 60 days in immunosuppressed, but not in immunocompetent, animals. In immunocompetent animals, the maximum number of dys+ fibers was observed at 10 days. The vast majority of these fibers contained AVR+ myonuclei; however, by 60 days, dys+ fibers disappeared with some AVR+ myonuclei persisting. Our studies suggest that widespread delayed inactivation of the dystrophin expression cassette is probably unlikely. Thus, optimization of immunosuppression could assure successful long-term dystrophin gene transfer for gene therapy.

Adenoviridae↗