Protection of Neuronal Cells from Apoptosis by Hsp27 Delivered with a Herpes Simplex Virus-based Vector
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.5061
ISSN1083-351X
AutoresMarcus Wagstaff, Yollanda Collaço-Moraes, Jill Smith, Jaqueline S. de Belleroche, Robert S. Coffin, David S. Latchman,
Tópico(s)Bee Products Chemical Analysis
ResumoOverexpression of the gene encoding the 70-kDa heat shock protein (hsp70) has previously been shown to protect neuronal cells against subsequent thermal or ischemic stress. It has no protective effect, however, against stimuli that induce apoptosis, although a mild heat shock (sufficient to induce hsp synthesis) does have a protective effect against apoptosis. We have prepared disabled herpes simplex virus-based vectors that are able to produce high level expression of individual hsps in infected neuronal cells without damaging effects. We have used these vectors to show that hsp27 and hsp56 (which have never previously been overexpressed in neuronal cells) as well as hsp70 can protect dorsal root ganglion neurons from thermal or ischemic stress. In contrast, only hsp27 can protect dorsal root ganglion neurons from apoptosis induced by nerve growth factor withdrawal, and hsp27 also protects the ND7 neuronal cell line from retinoic acid-induced apoptosis. However, hsp70 showed no protective effect against apoptosis in contrast to its anti-apoptotic effect in non-neuronal cell types. These results thus identify hsp27 as a novel neuroprotective factor and show that it can mediate this effect when delivered via a high efficiency viral vector. Overexpression of the gene encoding the 70-kDa heat shock protein (hsp70) has previously been shown to protect neuronal cells against subsequent thermal or ischemic stress. It has no protective effect, however, against stimuli that induce apoptosis, although a mild heat shock (sufficient to induce hsp synthesis) does have a protective effect against apoptosis. We have prepared disabled herpes simplex virus-based vectors that are able to produce high level expression of individual hsps in infected neuronal cells without damaging effects. We have used these vectors to show that hsp27 and hsp56 (which have never previously been overexpressed in neuronal cells) as well as hsp70 can protect dorsal root ganglion neurons from thermal or ischemic stress. In contrast, only hsp27 can protect dorsal root ganglion neurons from apoptosis induced by nerve growth factor withdrawal, and hsp27 also protects the ND7 neuronal cell line from retinoic acid-induced apoptosis. However, hsp70 showed no protective effect against apoptosis in contrast to its anti-apoptotic effect in non-neuronal cell types. These results thus identify hsp27 as a novel neuroprotective factor and show that it can mediate this effect when delivered via a high efficiency viral vector. The heat shock proteins (hsps) 1The abbreviations hsp(s)heat shock protein(s)HSVherpes simplex virusDRGdorsal root ganglionBHKbaby hamster kidneyNGFnerve growth factorm.o.i.multiplicity of infection were originally identified on the basis of their increased synthesis following exposure to elevated temperature. Subsequently, however, they were shown to be induced by a wide variety of other stresses in many different cell types (for review see Refs. 1Lindquist S. Annu. Rev. Biochem. 1986; 55: 1151-1191Crossref PubMed Google Scholar, 2Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4419) Google Scholar, 3Latchman D.S. J. R. Coll. Physicians Surg. 1991; 25: 295-300PubMed Google Scholar) including neuronal cells exposed to ischemia (4Nowak T.S.J. J. Neurochem. 1985; 45: 1635-1641Crossref PubMed Scopus (230) Google Scholar), amphetamine treatment (5Miller E. Raese J.B. Morrison-Bogorad M. J. Neurochem. 1991; 56: 2060-2071Crossref PubMed Scopus (120) Google Scholar), or to sodium arsenite (6Lowenstein D.H. Chan P.H. Miles M.F. Neuron. 1991; 7: 1053-1060Abstract Full Text PDF PubMed Scopus (256) Google Scholar). Such induction of the hsps has been shown to have a protective effect against exposure to a subsequent stress in a variety of cell types. Thus, for example, exposure of primary neuronal cultures to a mild heat stress or ischemic stress sufficient to induce the hsps has been shown to be protective against subsequent exposure to the excitotoxin glutamate or to severe heat or ischemic stress (6Lowenstein D.H. Chan P.H. Miles M.F. Neuron. 1991; 7: 1053-1060Abstract Full Text PDF PubMed Scopus (256) Google Scholar, 7Rordorf G. Koroshetz W.J. Bonventre J.V. Neuron. 1991; 7: 1043-1052Abstract Full Text PDF PubMed Scopus (192) Google Scholar, 8Amin V. Cumming D.V.E. Coffin R.S. Latchman D.S. Neurosci. Lett. 1995; 200: 85-88Crossref PubMed Scopus (39) Google Scholar), whereas similar exposure in vivo can protect against damaging effects caused by subsequent exposure to light (9Barbe M.F. Tytell M. Gower D.J. Welch W.J. Science. 1988; 241: 1817-1820Crossref PubMed Scopus (388) Google Scholar) or ischemia (10Chopp M. Chen H. Ho K.-L. Dereski M.O. Brown E. Hetzel F.W. Welch K.M. Neurology. 1989; 39: 1396-1398Crossref PubMed Google Scholar, 11Kitagawa K. Matsumoto M. Tagaya M. Hata R. Keda H. Ninobe M. Handa N. Fukunaga R. Kimura K. Mikshiba K. Kamada T. Mol. Brain Res. 1990; 528: 21-24Crossref Scopus (1056) Google Scholar). heat shock protein(s) herpes simplex virus dorsal root ganglion baby hamster kidney nerve growth factor multiplicity of infection In a number of cases the protective effect of a mild hsp-inducing stress can be reproduced by the artificial overexpression of a single hsp. Thus, for example, dorsal root ganglion (DRG) neurons can be protected against thermal or ischemic stress by overexpression of either the 70-kDa hsp (hsp70) or the 90-kDa hsp (hsp90) (12Uney J.B. Kew C.N.N. Staley K. Tyers P. Sofroniew M.V. FEBS Lett. 1993; 334: 313-317Crossref PubMed Scopus (59) Google Scholar, 13Amin V. Cumming D.V.E. Latchman D.S. Neurosci. Lett. 1996; 206: 45-48Crossref PubMed Scopus (92) Google Scholar, 14Wyatt S. Mailhos C. Latchman D.S. Mol. Brain Res. 1996; 39: 52-56Crossref PubMed Scopus (39) Google Scholar), and a similar protective effect of hsp70 and hsp90 has also been observed in the ND7 immortalized cell line derived from sensory neurons (15Mailhos C. Howard M.K. Latchman D.S. J. Neurochem. 1994; 63: 1787-1795Crossref PubMed Scopus (115) Google Scholar). Interestingly, Fink et al. (16Fink S.L. Chang L.K. Ho D.Y. Sapolsky R.M. J. Neurochem. 1997; 68: 961-969Crossref PubMed Scopus (116) Google Scholar) were able to protect cultured hippocampal neurons against subsequent heat shock using a herpes simplex virus (HSV)-derived amplicon vector expressing hsp70 indicating that this effect applies to neurons derived from both the central and peripheral nervous systems. Moreover, the use of an HSV-based vector opens up the possibility of testing the protective effect of the hsps in vivo and of their ultimate therapeutic use in man. Such vectors can effectively deliver genes to neuronal cells following in vivo injection and may be useful for gene therapy procedures (for review see Refs. 17Coffin R.S. Latchman D.S. Latchman D.S. Genetic Manipulation of the Nervous System. Academic Press, Lndon1995: 99-114Crossref Google Scholar and 18Fink D.J. DeLuca N.A. Goins W.F. Glorioso J.C. Annu. Rev. Neurosci. 1996; 19: 265-287Crossref PubMed Scopus (148) Google Scholar). Such a possibility is of particular interest in view of the finding that a prior mild heat shock can protect neonatal DRG neurons against apoptosis induced by withdrawal of nerve growth factor (15Mailhos C. Howard M.K. Latchman D.S. J. Neurochem. 1994; 63: 1787-1795Crossref PubMed Scopus (115) Google Scholar) and similarly protects ND7 cells against apoptosis induced by serum withdrawal and addition of retinoic acid (19Mailhos C. Howard M.K. Latchman D.S. Neuroscience. 1993; 55: 621-627Crossref PubMed Scopus (154) Google Scholar). Thus the ability to manipulate the rate of apoptosis would be of use in neurological diseases such as muscular dysgenesis (20Oppenheim R.W. Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2759) Google Scholar) or spinal muscular atrophy (21Gamstrop I. Sarnet H.B. Progressive Spinal Muscular Atrophies. Raven Press, Ltd., New York1984Google Scholar) which involve changes in the normal level of neuronal cell apoptosis during development as well as in later onset diseases such as Alzheimer's or Parkinson's diseases where the excessive neuronal cell death may be apoptotic in nature (20Oppenheim R.W. Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2759) Google Scholar). Unfortunately, however, it has not proved possible thus far to identify a single hsp whose overexpression in neuronal cells can protect them from apoptosis. Thus, overexpression of hsp70 can protect several different non-neuronal cell types from apoptosis including fibrosarcoma cells (22Samali A. Cotter T.G. Exp. Cell Res. 1996; 223: 163-170Crossref PubMed Scopus (481) Google Scholar), normal fibroblasts (23Li W.X. Chen C.H. Ling C.C. Li G.C. Radiat. Res. 1996; 145: 324-330Crossref PubMed Scopus (84) Google Scholar), and T cell leukemia cell lines (24Mosser D.D. Caron A.W. Bourget L. Denis-Larose C. Massie B. Mol. Cell. Biol. 1997; 17: 5317-5327Crossref PubMed Scopus (869) Google Scholar, 25Wei Y.-Q. Zhao X. Kariya Y. Teshigawara K. Uchida A. Cancer Immunol. Immunother. 1995; 40: 73-78Crossref PubMed Scopus (177) Google Scholar). In contrast, however, overexpression of hsp70 or hsp90 in ND7 cells (15Mailhos C. Howard M.K. Latchman D.S. J. Neurochem. 1994; 63: 1787-1795Crossref PubMed Scopus (115) Google Scholar) or DRG neurons (14Wyatt S. Mailhos C. Latchman D.S. Mol. Brain Res. 1996; 39: 52-56Crossref PubMed Scopus (39) Google Scholar) does not reproduce the protective effect of mild heat shock against subsequent apoptotic stimuli. Similarly, in the experiments of Fink et al. (16Fink S.L. Chang L.K. Ho D.Y. Sapolsky R.M. J. Neurochem. 1997; 68: 961-969Crossref PubMed Scopus (116) Google Scholar) overexpression of hsp70 with an HSV vector did not protect hippocampal neurons against glutamate toxicity which may act by inducing apoptosis (26Kure S. Tuminaga T. Yoshimoto T. Tada K. Narisawa K. Biochem. Biophys. Res. Commun. 1991; 179: 39-45Crossref PubMed Scopus (230) Google Scholar). It is clear therefore that the protective effect of heat shock against apoptosis in neuronal cells cannot be reproduced by hsp70 alone despite its protective effect in other cell types. The protective effect could involve another hsp such as hsp27 which has also been shown to protect non-neuronal cell types against apoptosis (22Samali A. Cotter T.G. Exp. Cell Res. 1996; 223: 163-170Crossref PubMed Scopus (481) Google Scholar, 27Mehlen P. Schulze-Osthoff K. Arrigo A.-P. J. Biol. Chem. 1996; 271: 16510-16514Crossref PubMed Scopus (582) Google Scholar) but has not been artificially overexpressed in neuronal cells. Alternatively, it could require a combination of several hsps or some other effect of heat shock not involving hsp synthesis. Such protective effects not involving hsp synthesis are likely to account for cases in non-neuronal cells where tolerance to stress can be enhanced in the absence of hsp synthesis (28Watson K. Dunlop G. Caviccholi R. FEBS Lett. 1984; 169: 267-273Crossref PubMed Scopus (37) Google Scholar, 29Wiedlitz R.B. Magun B.E. Gerner E.W. Mol. Cell. Biol. 1986; 6: 1088-1094Crossref PubMed Scopus (72) Google Scholar) or where cells with the same hsp levels exhibit different levels of thermotolerance (30Ferrini U. Falcioni R. Delpino A. Cavaliere R. Zupi G. Natali P.G. Int. J. Cancer. 1984; 34: 651-655Crossref PubMed Scopus (30) Google Scholar). In this case it would evidently not be possible to produce an effective anti-apoptotic effect using gene therapy procedures involving the overexpression of one or more hsp genes. To resolve these possibilities and attempt to identify the hsp involved in the protective effect against apoptosis, we have constructed HSV-based vectors expressing individual hsps and used these to investigate their protective effects in vitro. These viruses offer a high efficiency means of delivering specific genes to primary neuronal cells and could be used, for example, to overexpress several different hsps in the same neuronal cell. Moreover, they could also ultimately be used to direct similar overexpression of hsps in vivo, in gene therapy procedures. Hsp cDNAs or a control β-galactosidase gene (from pCH110; Pharmacia) under the control of the cytomegalovirus immediate early promoter (from pJ7; Ref. 54Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 1068Crossref PubMed Scopus (257) Google Scholar) were inserted into a plasmid containing the region of the HSV-1 genome encoding the latency associated transcript, between the twoBstXI sites immediately downstream of the latency-associated transcript promoter region (HSV-1 strain 17+; GenBankTMaccession number HE1CG). Hsp cDNAs were Chinese hamster hsp27 (from Jacques Landry (31Lavoie J. Chretien P. Landry J. Nucleic Acids Res. 1990; 18: 1637Crossref PubMed Scopus (24) Google Scholar)), rabbit hsp56 (from Marie-Claire Lebeau), inducible human hsp70 (32Wu B. Hunt C. Morimoto R. Mol. Cell. Biol. 1985; 5: 330-341Crossref PubMed Scopus (367) Google Scholar), and human hsp90 (33Twomey B.M. Dhillon V.B. McCallum S. Isenberg D.A. Latchman D.S. J. Autoimm. 1993; 6: 495-506Crossref PubMed Scopus (34) Google Scholar, 41Twomey B.M. McCallum S. Isenberg D.A. Latchman D.S. Clin. Exp. Immunol. 1993; 93: 178-183Crossref PubMed Scopus (42) Google Scholar). Hsps were introduced into the latency-associated transcript region of an HSV vector deleted for ICP27 (34Coffin R.S. Maclean A. Latchman D.S. Brown S.M. Gene Ther. 1995; 3: 886-891Google Scholar, 49Howard M.K. Kershaw T. Gibb B. Storey W. Maclean A.R. Zeng B.-W. Jenner P. Brown S.M. Woolf C.J. Anderson P.N. Coffin R.S. Latchman D.S. Gene Ther. 1998; 5: 1137-1147Crossref PubMed Scopus (54) Google Scholar) directly replacing thelacZ gene in the control virus described above by standard homologous recombination. Here a blue/white selection for recombinant plaques was performed after 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside staining, white Hsp-expressing plaques being picked and plaque-purified five times followed by Western blotting to confirm expression of the appropriate Hsp from each virus. ICP27 deleted viruses were grown in B130/2 BHK cells that have been stably transfected to express ICP27 (49Howard M.K. Kershaw T. Gibb B. Storey W. Maclean A.R. Zeng B.-W. Jenner P. Brown S.M. Woolf C.J. Anderson P.N. Coffin R.S. Latchman D.S. Gene Ther. 1998; 5: 1137-1147Crossref PubMed Scopus (54) Google Scholar) allowing lytic growth of the virus. There is no overlap between sequences inserted into the cell line to complement ICP27 and the ICP27-deleted virus in this virus cell line combination, and thus homologous recombination and repair of the ICP27 deletion is undetectable (reversion frequency <1 in 109 plaque-forming units (49Howard M.K. Kershaw T. Gibb B. Storey W. Maclean A.R. Zeng B.-W. Jenner P. Brown S.M. Woolf C.J. Anderson P.N. Coffin R.S. Latchman D.S. Gene Ther. 1998; 5: 1137-1147Crossref PubMed Scopus (54) Google Scholar)). B130/2 cells, parental BHK cells (35Macpherson I. Stoker M. Virology. 1962; 16: 147-151Crossref PubMed Scopus (598) Google Scholar), ND7 cells (36Wood J.N. Bevan S.J. Coote P. Darn P.M. Hogan P. Latchman D.S. Morrison C. Rougon G. Theveniau M. Wheatley S.C. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1990; 241: 187-194Crossref PubMed Scopus (226) Google Scholar), and neonatal rat primary dorsal root ganglion cells were infected in each case at a multiplicity of infection (m.o.i.) of 10 in serum-free media for 1 h. Virus containing media was then replaced with full growth media and incubation continued for a further 16 h prior to the induction of cell stress or harvesting of the cells for Western blotting. Cultures were prepared from 2-day-old Sprague-Dawley rats. Spinal ganglia were removed aseptically, digested with 0.3% collagenase (Boehringer Mannheim) for 90 min, and then mechanically dissociated through a 1-ml Gilson tip. Routinely cultures contain approximately 90% neurons and 10% glial cells as assayed by morphology and staining with appropriate antibodies. Cells were plated and grown overnight in defined media (37Davies A.M. Cohen J. Wilkin G.P. Neural Cell Culture: A Practical Approach. Oxford University Press, New York1995: 153-175Google Scholar) on sterile glass coverslips and subsequently transferred to 24-well plates (Nunc, Roskilde, Denmark). Cells were infected/mock-infected over 1 h in defined media in the absence of bovine serum albumin. After overnight incubation the cells were either exposed to heat shock (see below), simulated ischemia (see below), or stimulated to undergo apoptosis by prolonged incubation in defined media in the absence of nerve growth factor (NGF). Viable neurons were assessed at 12 and 24 h recovery after insult (heat shock and ischemia) and at 24 and 48 h after prolonged NGF withdrawal by visualization at 400 × magnification under light microscopy. The parameters used to define viable neurons were phase bright bodies, agranular appearance, and non-ruffled membranes. Non-viable neurons were defined by phase dark bodies, lack of neurites, granular/vacuolated appearance, and ruffled membranes (14Wyatt S. Mailhos C. Latchman D.S. Mol. Brain Res. 1996; 39: 52-56Crossref PubMed Scopus (39) Google Scholar). ND7 cells were originally generated as a fusion of primary sensory DRG neurons and neuroblastoma cells and display many of the characteristics of sensory neurons (36Wood J.N. Bevan S.J. Coote P. Darn P.M. Hogan P. Latchman D.S. Morrison C. Rougon G. Theveniau M. Wheatley S.C. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1990; 241: 187-194Crossref PubMed Scopus (226) Google Scholar). ND7 cells were grown in Leibovitz L15 medium with 10% fetal calf serum and infected as above before exposure to either lethal heat shock (see below) or simulated ischemia (see below) prior to assay of cell viability by trypan blue exclusion (see below). To induce the cells to cease dividing and undergo morphological differentiation or apoptosis, they were transferred to serum-free medium consisting of a 1:1 mix of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and nutrient mix Ham's F12 (Life Technologies, Inc.) supplemented with human transferrin (5 μg/ml), bovine insulin (250 ng/ml), and sodium selenite (30 nm). All-trans- retinoic acid was added to a final concentration of 1 μm to increase the proportion of cells undergoing apoptosis. This procedure has previously been shown to induce cell death by apoptosis in these cells (43Howard M.K. Burke L.C. Mailhos C. Pizzey A. Gilbert C.S. Lawson W.D. Collins M.K.L. Thomas N.S.B. Latchman D.S. J. Neurochem. 1993; 60: 1783-1791Crossref PubMed Scopus (94) Google Scholar). ND7 cells were seeded into 8-well chamber slides and infected at an m.o.i. of 10 with each of the HSV vectors expressing lacZ (control) or each of the Hsps individually. After serum withdrawal/addition of retinoic acid, the cultures were incubated for 48 h at 37 °C, 5% CO2. Media were removed, and the cells were fixed with 1% paraformaldehyde for 30 min. Slides were then washed twice in phosphate-buffered saline at 37 °C and TUNEL reactions (Boehringer Mannheim) performed according to the manufacturer's instructions. The samples were visualized using fluorescein optics, and the numbers of positive staining, apoptotic cells were counted in three confluent fields of view for each sample. Growth media was pre-warmed to 48 °C and 1 ml/well added to ND7 cells or primary rat DRG cells in vitro. The plates were wrapped in parafilm and incubated in a water bath at 48 °C (lethal heat shock) for 20 (ND7 cells) or 30 min (rat primary DRG cultures) (16Fink S.L. Chang L.K. Ho D.Y. Sapolsky R.M. J. Neurochem. 1997; 68: 961-969Crossref PubMed Scopus (116) Google Scholar) followed by transfer to a 37 °C, 5% CO2 incubator for recovery for a period of 1 (ND7 cells) or 24 h (primary neurons) as in our previous experiments (15Mailhos C. Howard M.K. Latchman D.S. J. Neurochem. 1994; 63: 1787-1795Crossref PubMed Scopus (115) Google Scholar,19Mailhos C. Howard M.K. Latchman D.S. Neuroscience. 1993; 55: 621-627Crossref PubMed Scopus (154) Google Scholar). The ND7 cells were gently harvested and gently resuspended in 100 μl of ice-cold phosphate-buffered saline before trypan blue exclusion assay for cell viability (see below). Primary DRG neurons were assessedin situ at 0, 12, and 24 h time points, similar to work reported previously (14Wyatt S. Mailhos C. Latchman D.S. Mol. Brain Res. 1996; 39: 52-56Crossref PubMed Scopus (39) Google Scholar). Ischemia can be simulated in vitro by incubating the cells in a physiological buffer containing raised levels of lactic acid, high potassium, and decreased pH and inhibitors of electron transport and glycolysis (38Esumi K. Nishida M. Shaw D. Smith T.W. Marsh J.D. Am. J. Physiol. 1991; 260: H1743-H1752PubMed Google Scholar). ND7 and rat DRG cells were cultured in a 24-well plate and mock-infected or infected with the viruses 24 h prior to ischemia. The cells were incubated for 4 h at 37 °C, 5% CO2 in either control buffer (pH 7.4) or ischemic buffer (38Esumi K. Nishida M. Shaw D. Smith T.W. Marsh J.D. Am. J. Physiol. 1991; 260: H1743-H1752PubMed Google Scholar). Viable ND7 cells were harvested as above and counted immediately after insult. Viable primary DRG neurons were visualized and counted after 12 and 24 h recovery in defined media at 37 °C, 5% CO2. The extent of cell death was quantitated by measuring the percentage of viable cells able to exclude trypan blue by adding an equal volume of 0.4% trypan blue in phosphate-buffered saline to cells that had been gently harvested by centrifugation (ND7s) or by direct addition to the cell culture (DRGs). Mixtures were incubated at room temperature for 5 min, and the proportion of cells able to exclude trypan blue was assessed. Protein samples were electrophoresed on polyacrylamide gels, transferred to nitrocellulose filters as described previously (19Mailhos C. Howard M.K. Latchman D.S. Neuroscience. 1993; 55: 621-627Crossref PubMed Scopus (154) Google Scholar, 39Dhillon V.B. McCallum S. Norton P.M. Twomey B.M. Eskeller-Yuksel F. Lydyard P. Isenberg D.A. Latchman D.S. Ann. Rheum. Dis. 1993; 52: 436-442Crossref PubMed Scopus (74) Google Scholar), and probed with either an anti-hsp90 antibody (AC88; see Ref. 40Riehl R.M. Sullivan W.P. Vroman B.T. Bauer V.J. Pearson G.R. Toft D.O. Biochemistry. 1985; 24: 6586-6591Crossref PubMed Scopus (114) Google Scholar), or antibodies to the other hsps purchased from StressGen Ltd. Cells were harvested 16 h after infection as described above, and in each case the degree of hsp induction was determined in comparison to cells infected with the lacZexpressing control virus. Equal loading of protein samples was confirmed by Coomassie staining of duplicate gels, in each case protein extracted from ∼1 × 105 cells run/lane. Previously an HSV-based amplicon vector overexpressing hsp70 has been reported (16Fink S.L. Chang L.K. Ho D.Y. Sapolsky R.M. J. Neurochem. 1997; 68: 961-969Crossref PubMed Scopus (116) Google Scholar). Here a plasmid containing an HSV origin of replication and packaging sequence is propagated by transfection into susceptible cells and co-infection with a helper virus (for review of HSV vectors see Refs. 17Coffin R.S. Latchman D.S. Latchman D.S. Genetic Manipulation of the Nervous System. Academic Press, Lndon1995: 99-114Crossref Google Scholar and 18Fink D.J. DeLuca N.A. Goins W.F. Glorioso J.C. Annu. Rev. Neurosci. 1996; 19: 265-287Crossref PubMed Scopus (148) Google Scholar). Progeny virions consist of a mixture of helper virus and packaged amplicon vector. This system suffers from the disadvantage that it produces a mixed viral population consisting of packaged amplicon and helper virus whose ratio varies between different stocks, although more recently a technique allowing low efficiency helper virus-free amplicon growth has been reported (55Fraefel C. Song S. Lim F. Lang P. Yu L. Wang Y. Wild P. Geller A.I. J. Virol. 1996; 70: 7190-7197Crossref PubMed Google Scholar). We have used the alternative system in which hsps are recombined directly into a disabled virus vector thus allowing reproducible stocks to be prepared for quantitive experiments. Here cDNAs for hsp90, hsp70, hsp56, and hsp27 were introduced into a disabled HSV strain lacking the viral gene encoding ICP27. ICP27 is essential for HSV replication (42Sacks W.R. Greene C.C. Aschmann D.P. Schaffer P.A. J. Virol. 1985; 55: 796-805Crossref PubMed Google Scholar) and thus ICP27 deletion results in an efficient gene transfer vector (34Coffin R.S. Maclean A. Latchman D.S. Brown S.M. Gene Ther. 1995; 3: 886-891Google Scholar, 49Howard M.K. Kershaw T. Gibb B. Storey W. Maclean A.R. Zeng B.-W. Jenner P. Brown S.M. Woolf C.J. Anderson P.N. Coffin R.S. Latchman D.S. Gene Ther. 1998; 5: 1137-1147Crossref PubMed Scopus (54) Google Scholar). Following plaque purification, viruses were tested for their ability to express the appropriate hsp following infection of B130/2 cells (49Howard M.K. Kershaw T. Gibb B. Storey W. Maclean A.R. Zeng B.-W. Jenner P. Brown S.M. Woolf C.J. Anderson P.N. Coffin R.S. Latchman D.S. Gene Ther. 1998; 5: 1137-1147Crossref PubMed Scopus (54) Google Scholar) which are BHK fibroblast cells stably transfected to express ICP27 and allow growth of the virus. Clear overexpression of hsp70, hsp56, and hsp27 was seen with the appropriate virus (Fig.1) at levels comparable to those observed in stressed cells (data not shown). In contrast, the hsp90 virus did not show significant overexpression of hsp90 above endogenous levels. Similar results were also seen in parental BHK cells (data not shown) and in ND7 cells which are of neuronal origin (Fig.2), confirming that hsps were also overexpressed without virus replication, and in particular in cells of neuronal origin.Figure 2Hsp expression in recombinant HSV infected ND7 cells. Western blotting was carried out with antibody to hsp27 (a), hsp56 (b), or hsp70 (c) using extracts from uninfected cells (control, C), cells infected with virus expressing β-galactosidase (lac), or virus expressing the appropriate hsp as indicated. RM, molecular mass in kilodaltons.View Large Image Figure ViewerDownload (PPT) As the hsp90 virus did not direct expression of hsp90 significantly above endogenous levels, it was not used further for these experiments. However, the other viruses were used to infect initially ND7 cells and subsequently primary DRG cultures. ND7 cells provide a convenient cell type of neuronal origin to initially assess the protective effect of the viruses against various insults. In these experiments, cells infected with the control virus expressing β-galactosidase showed comparable survival either before or after stress to that observed in uninfected ND7 cells (Figs. 3 and4), showing that the virus itself did not have any significant cytopathic effect in these cells. Moreover, the hsp70-expressing virus was able to protect ND7 cells from subsequent exposure to both a severe thermal (Fig. 3) or ischemic (Fig. 4) stress, confirming our previous results obtained with ND7 cells stably transfected with a plasmid-expressing hsp70 (15Mailhos C. Howard M.K. Latchman D.S. J. Neurochem. 1994; 63: 1787-1795Crossref PubMed Scopus (115) Google Scholar). A similar protective effect was also observed with the virus expressing hsp27, but not with the virus expressing hsp56 (Figs. 3 and 4), providing for the first time data on the protective effect of overexpressing these proteins in neuronal cells.Figure 4ND7 cell survival following simulated ischemia when infected with HSV vectors expressing hsps. Black bars, proportion of surviving mock-infected (ND7) and virus-infected ND7 cells incubated at 37 °C, 5% CO2 in control buffer for 4 h. Striped bars, proportion of surviving mock-infected (ND7) and virus-infected ND7 cells after 4 h incubation in ischemic buffer at 37 °C, 5% CO2. Cell survival was assessed by trypan blue exclusion assay. Barsrepresent S.E. of the means calculated from the means of three counts for each sample (n). n for all experiments = 6. *, significant difference in survival compared with mock-infected ND7 cells (p < 0.001). +, significant difference in survival compared with lacZvirus-infected ND7 cells (p < 0.001). Significance calculated using a Boniferroni Multiple Comparison t test after one-way analysis of variance.View Large Image Figure ViewerDownload (PPT) We next tested the effect of each of these viruses on the ability of ND7 cells to survive the removal of serum together with the addition of retinoic acid. We have previously shown this treatment to induce apoptosis in ND7 cells on the basis of a number of criteria such as the morphology of cells under light and electron microscopy and DNA fragmentation (43Howard M.K. Burke L.C. Mailhos C. Pizzey A. Gilbert C.S. Lawson W.D. Collins M.K.L. Thomas N.S.B. Latchman D.S. J. Neurochem. 1993; 60: 1783-1791Crossref PubMed Scopus (94) Google Scholar). In these experiments (Fig.5) cells infected with viruses expressing β-galactosidase or hsp56 showed comparable rates of survival to untreated cells, whereas cells infected with the hsp70 virus showed a marginally protective effect, but only at the 48-h time point (p < 0.05). In contrast, cells infected with the hsp27-expressing virus showed a highly statistically significant protection at both 24 and 48 h after the onset of treatment (p < 0.001 at both time points). Hence the hsp27-expressing virus had a protective effect that was specific to this virus and not observed with the other viruses. This indicates that protection was not due to a nonspecific effect of viral infection, such as an alteration in the rate of cell division. To confirm that the
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