Pharmacokinetics of combined gene therapy expressing constitutive human GM-CSF and hyperthermia-regulated human IL-12.

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by  Wei, Fang

Weietal.JournalofExperimental&ClinicalCancerResearch2013,32:5
http://www.jeccr.com/content/32/1/5
RESEARCH Open Access
Pharmacokinetics of combined gene therapy
expressing constitutive human GM-CSF and
hyperthermia-regulated human IL-12
Fang Wei1†, Huiping Wang1†, Jufeng Zhang1, Xiafang Chen1, Chuanyuan Li2* and Qian Huang1*
Abstract
Background: Anadenovirus that expresses both interleukin (IL)-12 and granulocyte-macrophage
colony-stimulating-factor (GM-CSF) has been proven to be very effective in treating several tumors, but causes
serious normal tissue toxicities.
Methods: In this study, a novel adenoviral vector was constructed by placingthe human GM-CSF gene under the
control of theCMV-IE promoter and human IL-12 gene under the control of heatshockprotein 70Bgene
promoter. Both hGM-CSF and hIL-12 expressionsin virus-infectedtumor cells were analyzedin vitro and in vivo
when underlying single or multiple rounds ofhyperthermia.
Results: We observed constitutive high expression of humanGM-CSF and heat-induced expression ofhumanIL-12
after a single round ofhyperthermia post viral infection. The heat-induced hIL-12 expression exhibited a pulse-like
pattern with a peakat 24 hrsfollowed bya decline 48 hrspost heat stress. Repeated heattreatmentwas more
effective ininducing hIL-12 expression than a one-time heattreatment. Interestedly, wealso observed that
constitutive expression ofhGM-CSFcould be stimulated by heat stress in testedtumor cells.
Conclusion: Our study provided a novel strategy for combined gene therapy that allows constitutive expression of
a non-toxic gene such as GM-CSFand heat-induced expression of a toxic gene such as IL-12. Inaddition, our study
also showed that hyperthermia can be used to trigger gene expression in temporal and special manner.
Keywords: GM-CSF, IL-12, Adenovirus,Hsp70, Hyperthermia
Introduction protein therapy, gene therapy proved to be as efficient
Theuniqueabilityofcancertoexploittheimmunesystem while inducing less toxicity [3]. Among these cytokine-
in order to promote tumor growth and suppress immune based gene therapies, an adenovirus that expresses both
response makes cancer therapy difficult. However, modu- interleukin (IL)-12 and granulocyte-macrophage colony-
lation of the immune system should provide promising stimulating-factor (GM-CSF) has been proven to be very
results. Cytokines are a large family of intercellular signa- effectiveintreatingseveraltumors[4,5].However,current
ling peptides that function in the regulation of immune adenoviruses deliver constitutive IL-12 gene expression,
response. Cytokine therapy has been reported to be an whichcausesseriousnormaltissuetoxicity[6].
effective strategy at inducing strong antitumor immune GM-CSF is a growth factor capable of enhancing antitu-
response[1]. However, initial studiesusingsystemictreat- mor activity by activating dendritic cells (DCs) to improve
ment with recombinant cytokines produced discouraging antigen presentation. GM-CSF can also activate macro-
results due to dose-limiting toxicities [2]. Compared to phages and induce the release of tumor necrosis factor
(TNF) [7] to achieve an antitumor effect. In addition,
*Correspondence:[email protected];[email protected] GM-CSF can indirectly stimulate T-cell activation via
†Equalcontributors interleukin-1 release [8]. However, increased cellular GM-
2DepartmentofDermatology,DukeUniversityMedicalCenter,Durham,NC
CSF expression also leads to counter-regulatory immune
27710,USA
1ExperimentalResearchCenter,FirstPeople’sHospital,SchoolofMedicine, responses to decrease the expansion of cytotoxic T cells
ShanghaiJiaotongUniversity,85WujinRoad,Shanghai200080,China
©2013Weietal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Weietal.JournalofExperimental&ClinicalCancerResearch2013,32:5 Page2of7
http://www.jeccr.com/content/32/1/5
(Tc), thereby limiting its antitumor activity [7]. In contrast, Adenoviruspreparation
IL-12 has been shown to exert potent immunostimulatory The adenovirus used to establish constitutively high
effectsoncertainhelperTcellsaswellascytotoxicTlym- expression of human GM-CSF and heat-inducible ex-
phocytes(CTL)andnaturalkiller(NK)cells[9].Therefore, pression of human IL-12 was constructed according to
the combined use of GM-CSF and IL-12 can counteract established protocols [12] using commercially available
thecounter-regulatoryroleofGM-CSFonTcandincrease plasmids (Microbix,Toronto, Canada). To construct the
theimmunebenefitsofGM-CSF. heat-inducible IL-12 expression cassette, cDNAs for
Human IL-12 is a disulfide-linked heterodimer com- both the p40 and p35 subunits of human IL-12 were
posed of 35 and 40 kDa subunits. Preclinical studies and inserted into the E1 region under control of the human
clinicaltrialsofIL-12genetherapyshowedthatthistreat- hsp70B gene promoter [13,14]. The p40 and p35 subu-
ment can induce remarkable anti-tumor response in nits were connected using an internal ribosome entry
various tumors, including melanoma, sarcoma, and site sequence [15] so that both subunits could be tran-
adenocarcinoma[3].However,bothpreclinicalandclinical scribed under the control of the same promoter. The
tests revealed that IL-12 gene therapy can induce highly human GM-CSF expression cassette was constructed by
toxic side effects [3]. This is because high constitutive placing the human GM-CSF gene under the control of a
IL-12 expression increases IFN-γ production [10]. Thus, constitutively active CMV-IE promoter in the E1 region
IL-12 expression in gene therapy requires regulation. [16] (see Figure 1). The completed adenovirus called
However, the current adenovirus coexpressing GM-CSF Adcmv-GMCSF-HSP-IL12 will establish constitutive
and IL-12 genes does not account for the regulation of expression of human GM-CSF and heat-inducible ex-
IL-12. Heat-based gene regulation is a ubiquitous stress pression of human IL-12. Large scale preparation of re-
responsetoheat shock in mammaliancells.Based onthis combinant Adcmv-GMCSF-HSP-IL12 was accomplished
feature, heat shock protein 70 promoter (hsp70B) has as previously described [17]. The control vector is an
been widely used in gene therapy to control gene expres- adenovirusexpressingGFPprotein(Figure 1).
sion [6]. The pharmacokinetics of GM-CSF and IL-12
productionaswellaspossibleinteractionsbetweenconsti- Invitroheatingexperiments
tutiveGM-CSFexpressionandheat-inducedIL-12expres- A549 and Hep3B cells were seeded in 24-well plates at a
sion should be investigated before clinical use. However, density of 6 × 104 cells/well. After cells were cultured
there is the dilemma that IL-12 has a restrict species- for 24 hrs, 100, 500, and 1000vp (viral particles) of
specificity.Forexample,humanIL-12showsnoactivityin Adcmv-hGMCSF-hsp-hIL12 virus were added into each
animalmodelsandmouseIL-12hasnoactivityinhuman. well. Twenty-four hours later, the culture medium was
Althoughthe efficacyand toxicityofsustainedhumanIL- replaced with 1 ml of fresh medium containing 2% FCS
12expressioncannotbeevaluatedinananimalmodel,the and cells were heated in a 45°C water bath for 45 min.
expression pattern of the adenoviral vector must be first Twenty-four hours later, the medium was collected for
testedinananimalmodelbeforeenteringclinicaltrials. hGM-CSF and hIL-12 measurement and replaced with
Currently, gene therapy with combined GM-CSF and 1 ml of fresh medium. Cells were heated again (45°C,
IL-12 has been established in several kinds of tumors 45 min) and the medium was collected 24 hrs post
using adenovirus to express constitutive GM-CSFand IL- heating.
12 levels. Although significant anti-tumor effect has been
observed, the level of GM-CSF or IL-12 is not regulatory Invivoheatingexperiments
[4,11]. In this study, we first constructed a novel adeno- Balb/C nude mice (BALB/c, nu/nu) weighing 20-22 g
viralvectorthatallowedconstitutiveexpressionof human were provided by the animal center of Shanghai Bio-
GM-CSF and heat-induced expression of human IL-12. logical Science Institution and housed in rooms under
The pharmacokinetics of gene expression triggered by standard lighting conditions and temperature. Water
hyperthermiawasthentestedincellcultureandinanani- and food were provided ad libitum. All animal experi-
malmodel.Ourstudyprovidedinsightsontumortherapy ments were conducted under an approved protocol from
bycombininggenetherapywithhyperthermia. Shanghai Jiaotong University and performed in accord-
ance with the animal care guidelines of the Chinese
Materials and methods Council. Hep3B tumors were introduced by subcutane-
Cellculture ousinjectionof1×107Hep3Bcellsin50μLofPBSinto
A549,ahumannon-smallcelllungcarcinomacellline,and the right hind limbs of mice. When tumor size reached
Hep3B, a human hepatoma cell line, were purchased from 1 cm in diameter, a total of 2 × 108 Adcmv-hGMCSF-
American Type Culture Collection. All cells were cultured hsp-hIL12 was injected into tumor. Mice were divided
in RPMI 1640 with 10% fetal bovine serum, 100 units/mL into 3 groups: non-heating group, one-time heating
penicillin,and100μg/mLstreptomycinat37°C,5%CO . group, and three-time heating group. In non-heating
2
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Figure1Aschematicdiagramofadenovirususedinthisstudy.HSP70-pro:heatshockprotein70genepromoter;hIL12:humaninterleukin
12;CMV-pro:CMVpromoter;hGMCSF:granulocyte-macrophagecolony-stimulating-factorgene;EGFP:enhancedGFP.
group, animals were sacrificed on day 1, 2, 3 and 4 post GM-CSF and human IL-12 ELISA kits (R&D Systems,
virus injection. In the one-time heating group, tumors Minneapolis,MN).
were heatedonce 24hrs post virusinjection andanimals
were sacrificed on day 1, 2, 3 and 4 post heat treatment. Results
In three-time heating group, tumors were heated on day hGM-CSFandhIL-12expressioninAdcmv-hGMCSF-hsp-
1, 3, and 5 post virus injection and animals were sacri- hIL12virusinfectedA549andHep3Bcells
ficed on day 4, 5, 6, 7 post first heat treatment. Tumors AsshowninFigure2,1000,500and100viralparticleper
were heated to 42°C in a water bath for 40 min by cell(vp)infectedcellsexhibitedsignificantincreasesinthe
immersing the tumor-bearing leg in the water bath [18]. production of hGM-CSF and hIL-12 in A549 after heat
Tumor tissues were homogenized for hGM-CSF and treatment(Figure2A, B). InHep3B cell medium, 1000vp
hIL-12detection. of virus infection significantly increased hIL-12 (p=0.001)
and hGM-CSF (p = 0.008) production 24 hrs after heat
DetectionofGM-CSFandIL-12levels treatment. 500 vp and 100 vp virus infected cells also
The hGM-CSF and hIL-12 levels in cell culture medium exhibited significant increases in the production of hGM-
andtumortissueshomogenateweredetectedwithhuman CSFand hIL-12 after heat treatment (Figure 2A, B). Heat
Figure2hGM-CSFandhIL-12expressioninheattreatedA549andHep3Bcells.A549andHep3Bcellsin24-wellplateswereinfectedwith
Adcmv-hGMCSF-hsp-hIL12virusfor24hrsandheatedat45°Cfor45min.Twenty-fourhourslate,mediumwascollectedforhGM-CSFandhIL-12
measurement.A)hIL-12expressionunderheatingandnoheatingtreatment.B)hGM-CSFexpressionunderheatingandnoheatingtreatment.
C)RelativehGM-CSFandhIL-12expressioninA549cells.D)RelativehGM-CSFandhIL-12expressioninHep3Bcells.HT:heatingtreatment.N=5
repeatedexperiments.
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treatment induced 8.79 ± 0.64 and 12.37 ± 2.41 fold actually lower than in non-heat treated A549 cells for 48
increases in hIL-12 production in 1000 vp and 500 vp hrs. In Hep3B cells, heat treatment for 24 hrs increased
virusinfectedA549cells(Figure2C).InHep3Bcells,heat hGM-CSF levels, but hGM-CSF levels were equal to or
treatment induced 6.13 ± 1.89 and 3.46 ± 0.36 fold higher than in non-heat treated Hep3B cells for 48 hrs.
increases in cells infected with 1000 vp and 500 vp virus These results suggest that hGM-CSF expression is time-
respectively, whereasheat treatment induced19.02 ± 4.95 dependentbut notheat-dependent.
fold increase in cells infected with 100 vp virus
(Figure2D).InbothA549andHep3Bcells,hGM-CSFex- TheeffectofheattreatmentoninvivohGM-CSFand
pression showed dependence on virus dosage. Although hIL12expression
hGM-CSF was driven by CMV promoter, hGM-CSF ex- As shown in Figure 4, virus infection produced consist-
pression was increased 1.48 ± 0.08 fold in A549 cells and ent hGM-CSF and hIL-12 expression under no heat
2.81±0.29foldinHepB3cellsafterheattreatment. treatment. hGM-CSF expression was significantly higher
than hIL-12, but both reached their peak at 24 hrs after
TheeffectofheattreatmentsonhGM-CSFandhIL-12 virusinfectionandbegantodeclineslowly at48hrspost
expression virus infection until day 7 of our observation. Under
AsshowninFigure3Ainnon-heatedA549cells,firstheat heat treatment, hIL-12 and hGM-CSF expressions were
treatment significantly increased hIL-12 levels in A549 significantly increased and reached a peak at 24 hrs after
cells infected with 100 vp 500 vp, 1000 vp virus, respec- eachheatingandbegantodecline 48hrsafterheating.
tively, while the second heat treatment was more efficient AsshowninFigure4A,intratumoralinjectionofadeno-
in increasing hIL-12 levels in A549 cells (p < 0.05 at all 3 viral vectors led to lower IL-12 expression. The first heat
viral dosages). In non-heat treated Hep3B cells, first heat treatment elevated hIL-12 level from 2500 ± 506 pg/ml
treatment significantly increased hIL-12 expressions in (noHT)to3966±661pg/ml(p=0.207),butsecondheat
Hep3B cells 24 hrs after first heat treatment. The second treatmentinduced 9.53 fold increase in hIL-12expression
heat treatment was also more efficient in increasing hIL- comparedtonoheattreatment(p=0.034)and4.1foldin-
12 levels in Hep3B (p < 0.05 at all 3 viral dosages). These creasecomparedtofirstheattreatment(HT1)(p=0.036).
resultssuggestthathIL-12expressionisheat-inducible.In Althoughthethirdheattreatment(HT3)waslesseffective
contrast,firstheattreatmentsignificantlyincreasedhGM- than the second heat treatment, hIL-12 level was still
CSF expression in A549 cells infected with 500 vp and higher in heat treated tumors than in non-heat treated
1000 vp virus in non-heat treated A549 cells shown in tumorsonday7sincefirsttreatment(p=0.039),suggest-
Figure 3B; however, second heat treatment did not signi- ing that multiple heat treatments could keep a constitu-
ficantly increase hGM-CSF expression in A549 cells tivelylowhIL-12expressionwithapeak-likeexpressionat
(p>0.05).Innon-heattreatedHep3Bcells,firstheattreat- 24hrsafterheating.
ment increased hGM-CSF levels in Hep3B cells but AsshowninFigure4B,theexpressionofhGM-CSFwas
showed no statistical difference (p > 0.05). After second controlledbyCMVpromoter;however,hGM-CSFexpres-
heat treatment, significant difference was observed in sion in tumor tissues increased 2.04 fold (p = 0.009) after
Hep3B cells infected with 1000 vp virus. These results first heat treatment compared to non-heat treated tumor
suggest that heat treatment can increase hGM-CSF ex- tissues(p=0.013).TheexpressionofhGM-CSFincreased
pression,buthGM-CSFexpressionisnotheat-dependent. intumortissueswithin24hoursafter2nd(p=0.002)and
We further compared the expression of hIL-12 third (p = 0.013)heat treatments.However, thepeakcon-
(Figure 3C) and hGM-CSF (Figure 3D) in A549 and centrations of hCM-GSF after heating were similar, and
Hep3B cells infected with the virus underlying heat no significant difference was observed between first, sec-
treatment for 24 hrs and no heat treatment for 24 and ond,andthirdheatingtreatments.
48hrs.Resultsshowed thattherewerenosignificantdif-
ferences in hIL-12 levels between 24 and 48 hrs in both Discussion
A549 and Hep3B cells infected with 3 different viral Combined gene delivery has been widely adopted in gene
doses underlying no heat treatment, but a significant in- therapy to increase therapeutic efficacy. However, some
crease in A549 and Hep3B cells was observed after 24 geneproductsareverytoxictonormaltissues,whichlimit
hrs of heat treatment. These results suggest that hIL-12 effective clinical application. To overcome this obstacle,
expression is heat-inducible, but not time-dependent. In the expression of one or more genes in the combined de-
contrast, significant differences in hGM-CSF levels were livery should be regulated. Gene therapy utilizing a com-
observed in A549 and Hep3B cells infected with 500 vp bination of IL-12 and GM-CSF has been previously
and 1000 vp virus underlying no heat treatment for 24 established [4,5]. In this strategy, GM-CSF is able to
and 48 hrs. In A549 cells, heat treatment for 24 hrs stimulate proliferation, maturation, and function of anti-
increased hGM-CSF levels, but hGM-CSF levels were gen representation cells, while IL-12 is able to enhance
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Figure3ThetimedependenceofhGM-CSFandhIL-12expressioninheattreatedA549andHep3Bcells.Cellswereinfectedandheated
asdescribedinFigure2.Mediumwascollectedat24and48hrsafterheatingtreatment.A)hIL-12expressioninA549andHep3Bcells.
B)hGM-CSFexpressioninA549andHep3bcells.C)ComparisonofhIL-12expressionbetweencellsheatedfor24hrsandcellswithoutheating
for24and48hrs.D)ComparisonofhGM-CSFexpressionbetweencellsheatedfor24hrsandcellswithoutheatingfor24and48hrs.N=5
repeatedexperiments.
T-helper1cell’simmunity,increase cytotoxicityofT-lym- via a heat-inducible promoter. After viral infection, heat
phocytes, and inhibit angiogenesis [5,8]. Another benefit stressinducedapulse-likeexpressionof hIL-12andahigh
of this strategy is that IL-12 can counteract the negative constitutiveexpressionofhGM-CSFinvitroandinvivo.
regulation of GM-CSF on Tc cells [7]. However, high Consistent with previous reports, constitutive hIL-12
toxicity was observed with this combination due to the expression was very low in both the A549 and Hep3B
consistently high IL-12 expression. To overcome the high cells under no heating. Heat stress induced 15 to 19 fold
toxicity, we constructed an adenovirus to constitutively increases in hIL-12 expression in cultured cells, while it
expresshumanGM-CSFwhilecontrollingIL-12expression induced a 16.9 fold increase in Hep3B tumor tissues
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producedrelativelyhigheramplificationrateinhIL-12ex-
pressiondue tothe existence oflowleak in hsp promoter
activity. This observation supports the idea that the virus
dose can be selected for clinical application. We acknow-
ledgethatwedidn’ttestthetemperature-dependenteffect
of IL-12 expression and that is a weakness to this study.
However, previous studies demonstrated a temperature-
dependent effect in hsp70 promoter controlled gene ex-
pression [19,20]. The second weakness is that the activity
andtoxicityofinduciblehumanIL-12cannotbetestedin
the animalmodelbecause human IL-12 shows noactivity
in animals and the nude mice used in this study are
immunodeficient.
In this study, the adenovirus was constructed with a
CMV-IEpromotertocontrolhumanGM-CSFexpression.
The CMV promoter should produce highly constitutive
hGM-CSF expression. However, heat treatment at 45°C
increased hGM-CSF expression by 1-1.5 folds in A549
cells and 2-3 folds in Hep3B cells. In addition, the in vivo
heating study revealed that although the first heat treat-
ment induced a 2-fold increase in hGM-CSF expression,
the second and third heat treatments didn’t increase
hGM-CSF expression as much as the first treatment. In
contrast, hGM-CSF expression was stable for 6 days after
first heat treatment and declined on day 7. This observa-
tionsuggeststhattheheat-induciblehGM-CSFexpression
isnotheat-dependentbuttime-dependent.Wealsonoted
thatheatinductionof hGM-CSFexpressionismoreobvi-
ousinHep3BcellsthaninA549cells,suggestingcelltype
dependence. Recently, the stimulating effect of heat stress
Figure4hGM-CSFandhIL-12expressioninHep3Btumor on CMV promoter activity has been studied [21,22]. Al-
tissues.Adcmv-GMCSF-hsp-hIL12wasintratumorlyinjected.Tumors thoughthepossiblemechanismsmightbecomplex,acon-
werenotheated,heatedfor1time,2time,and3timesat42°Cfor
siderable homology to the heat stress element core
40min.Animalsweresacrificedatdifferenttimepointandtumor
consensus(GA–TCC)within18bpelementsinIEenhan-
tissueswerehomogenizedforhGM-CSFandhIL12detection.A)hIL-12
expressionintumortissues.B)hGM-CSFexpressionintumortissues. cer might be the most reasonable explanation [21,23].
N=5micepergroup. Heat stress might regulate CMV-IE activity directly and
indirectly through heat-activated transcription factors.
Heat stress inducing various transcriptional factors, in-
after a second heat treatment. This suggests that hsp70 cluding those activating the CMV-IE promoter, has been
promoter is highly inducible with low background acti- reported [21,22]. Therefore, the cell type dependence
vity. Consistent with our previous findings, heat-induced might reflect the high specificity of the signaling pathway
hIL-12 expression peaked at 24 hrs and began to decline andtranscriptionfactors.
at 48 hrs post heat treatment [18]. This pattern can re- In this study, we established constitutive high expres-
ducethe consistentlyhighIL-12expression-inducedtox- sion of human GM-CSF and heat-induced expression of
icity. In addition, we found that the second heat human IL-12 with a single adenoviral vector. The heat-
treatment is more effective than the first heat treatment induced hIL-12 expression has a pulse like shape with a
in inducing hIL-12 expression, but the third heat treat- peak at 24 hrs post heat stress that is maintained for
ment is less effective than the second heat treatment. 24 hrs in tumor tissues. Repeated heat treatments are ef-
The lower efficacy of the third heat treatment in indu- fective but limited by the clearance of non-replicating
cing gene expression may suggest that one injection of adenovirus. Together with the low background activity
non-replicating adenovirus can only support a limited of hsp70 promoter, heat induced gene expression
number of heat treatments that induce gene expression. enables a fairly strict control of gene expression, which
Inaddition,highvirusdosecouldproducehighhIL-12ex- diminishes the cytotoxicity of toxic cytokines . We also
pression under heat stress. However, low dose infection observed that the CMV-IE promoter driven constitutive
Weietal.JournalofExperimental&ClinicalCancerResearch2013,32:5 Page7of7
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high expression of hGM-CSF could be stimulated by 18. HuangQ,HuJK,LohrF,ZhangL,BraunR,LanzenJ,LittleJB,DewhirstMW,
heat stress in a cell type dependent manner. However, LiCY:Heat-inducedgeneexpressionasanoveltargetedcancergene
therapystrategy.CancerRes2000,60:3435–3439.
the CMV-IE promoter activity cannot be regulated by
19. DammeyerP,JaramilloMC,PipesBL,BadowskiMS,TsangTC,HarrisDT:
heat stress. Our study provided solid evidence for the Heat-inducibleamplifiervectorforhigh-levelexpressionofgranulocyte-
feasibility of heat-induced regulation of gene expression macrophagecolony-stimulatingfactor.IntJHyperthermia2006,
22:407–419.
inacombinedgene deliveryvector.
20. SiddiquiF,LiCY,ZhangX,LarueSM,DewhirstMW,UllrichRL,AveryPR:
Characterizationofarecombinantadenovirusvectorencoding
Competinginterests
heat-induciblefelineinterleukin-12foruseinhyperthermia-induced
Allauthorsdeclarednoanyconflictofinterest. gene-therapy.IntJHyperthermia2006,22:117–134.
21. KobeltD,AumannJ,FichtnerI,SteinU,SchlagPM,WaltherW:Activation
Authors’contribution
oftheCMV-IEpromoterbyhyperthermiainvitroandinvivo:biphasic
FW:Conductexperiments,preparemanuscriptHW:performexperiment,data
heatinductionofcytosinedeaminasesuicidegeneexpression.
analysisJZ:performexperimentsXC:cellcultureCL:experimentdesign, MolBiotechnol2010,46:197–205.
manuscriptrevisionQH:experimentdesign,finalapprovalofmanuscript.All 22. PshenichkinS,SurinA,SurinaE,KlauzińskaM,GrajkowskaE,LuchenkoV,
authorsreadandapprovedthefinalmanuscript. DolińskaM,WroblewskaB,WroblewskiJT:HeatshockenhancesCMV-IE
promoter-drivenmetabotropicglutamatereceptorexpressionand
Acknowledgement toxicityintransfectedcells.Neuropharmacology2011,60:1292–1300.
ThisprojectwassupportedbygrantsfromNationalBasicResearchProgram
23. GeelenJL,BoomR,KlaverGP,MinnaarRP,FeltkampMC,vanMilligenFJ,SolCJ,
ofChina(2010CB529902).
vanderNoordaaJ:Transcriptionalactivationofthemajorimmediateearly
transcriptionunitofhumancytomegalovirusbyheat-shock,arseniteand
Received:16December2012Accepted:20January2013 proteinsynthesisinhibitors.JGenVirol1987,68:2925–2931.
Published:26January2013
doi:10.1186/1756-9966-32-5
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