Heat-activated Transgene Expression from Adenovirus Vectors Infected into Human Prostate Cancer...

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2001;61:1113-1121. Cancer Res Michael J. Borrelli, Diane M. Schoenherr, Alden Wong, et al. Infected into Human Prostate Cancer CellsHeat-activated Transgene Expression from Adenovirus Vectors

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[CANCER RESEARCH 61, 1113–1121, February 1, 2001]

Heat-activated Transgene Expression from Adenovirus Vectors Infected intoHuman Prostate Cancer Cells1

Michael J. Borrelli, 2 Diane M. Schoenherr, Alden Wong, Laura J. Bernock, and Peter M. CorryDepartment of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan 48073

ABSTRACT

Replication-deficient adenovirus expression vectors were used to intro-duce a recombinant DNA construct containing enhanced green fluores-cent protein (EGFP) under control of a truncated, human heat shockpromoter into human prostate cancer cells growing either exponentiallyor in plateau phase. This was done to measure controlled, heat shock-induced EGFP expression under conditions relevant to treating humancancers with heat-activated gene therapy. Both the temporal duration andmagnitude of EGFP expression increased proportionately with strongerheat shocks (time at temperature) up to maximum values that wereinduced by 4 h at 41.0°C or 2 h at 42.0°C. Longer heat shocks at eithertemperature yielded no additional EGFP expression and ultimately re-duced it. Maximal EGFP expression was induced in exponential culturesby heat shocks delivered 12–24 h after virus infection. Induction atprogressively later postinfection times induced increasingly lower, peakEGFP expression. Maximal EGFP expression could not be induced until48 h after infection of plateau phase cultures but could still be induced180 h after virus infection. However, peak EGFP levels in plateau cultureswere approximately 25–50% of those observed in identically inducedexponential cultures. Ostensibly, the differences in expression from theheat shock promoter observed in exponential and plateau cultures wereattributable to cell division diluting the vector within exponential culturesand the lower metabolic activity in serum-starved plateau cultures. For allexperimental conditions, EGFP expression induced from the heat shockpromoter was comparable with or higher than that from the constitutivelyactive cytomegalovirus promoter over any 24-h period.

The experimental results demonstrated that EGFP expression from theheat shock promoter was controllable in both exponential and plateauphase cultures and support the plausibility of using controlled heat shockactivation of this promoter as a means of regulating both the spatial andtemporal expression of therapeutic DNA constructs within human tissues.The ability to localize and regulate expression from the heat shock pro-moter may prove particularly advantageous for many cancer applications,especially if the therapeutic products are highly toxic,e.g.,proteotoxins orcytokines. However, the results of this study suggest that differentialgrowth conditions within tumors could markedly affect the expression ofrecombinant DNA under control of both inducible and constitutive pro-moters. Consequently, inducing schemes may need to be spatially adjustedto obtain the desired therapeutic results in all tumor domains usingheat-activated gene therapy.

INTRODUCTION

The heat shock promoter is one of several inducible promotersoffering the potential for controlled expression of therapeutic genesdelivered into diseased or normal tissues. A major advantage of theheat shock promoter would be the ability to use conformal heat shockto control and delimit its expression within the body even whenrecombinant DNA vectors are administered systemically. Microwave(1–3), ultrasound (4–6), or low frequency radiofrequency (7) devices

designed for localized clinical hyperthermia could be used to deliverconformal heat shocks to confine heat shock promoter activation,whereas the magnitude and temporal duration of the heat shock wouldregulate that of induced DNA expression (8–10).

Human tissues can be heated relatively quickly to temperaturescapable of activating the heat shock promoter,e.g.,40.5°C- 43.0°C,and heat diffuses rapidly from tissues once the heat source is removed.Consequently, the heat shock promoter can be activated over a widetemporal range, from minutes to hours. Promoters that are activatedby systemically delivered compounds,e.g.,the tetracycline promoter(11), are not necessarily subject to such strict temporal control unlessthe activating substance clears quickly from tissues and blood. How-ever, systemically delivered activation does lack the spatial control ofpromoter induction available with the heat shock promoter, which canbe activated in defined tissue volumes using conformal heating tech-niques.

Radiation-inducible promoters (12–14) could potentially providetight spatial and temporal control of induced promoter activity be-cause ionizing radiation doses can be delivered with great precision indefined tissue volumes. Unfortunately, radiation-inducible promotersexhibit substantial, constitutive expression, whereas that from thetruncated heat shock promoter is virtually undetectable. In somecircumstances, a baseline of constitutive expression from an induciblepromoter may not be a problem. However, if the therapeutic gene isextremely cytotoxic,e.g.,proteotoxins like diphtheria and Shiga tox-ins, even low levels of constitutive expression could prove very toxicto any normal tissues that inadvertently receives it.

Developing heat-activated gene therapy for treating human cancersis a logical approach to controlling gene therapy because hyperther-mia is already being investigated as an adjuvant modality for cancerradiation therapy (15–18). Consequently, the devices used to producelocal or conformal heating and the technical expertise to use them arealready available to many radiation oncology departments for testingthe feasibility of heat-activated gene therapy. If initial tests provepromising, heat-activated gene therapy could ultimately be adminis-tered concomitantly with hyperthermia and ionizing radiation treat-ments. The amount of administered, recombinant DNA would need tobe adjusted so that desired levels of therapeutic gene product weremaintained throughout the fractionated regimes typically used todeliver heat and ionizing radiation. Between-fraction boost heatingscould be used to adjust gene expression locally, should that provenecessary. Collaborations with other departments could then lead toexpanding heat-activated gene therapy to other medical applications.

Nonreplicating adenovirus vectors (19, 20) are particularly wellsuited for cancer therapy because the recombinant DNA they deliverinto cells is transiently resident and does not recombine into the hostcell genome. This is particularly important when the therapeutic geneproduct is very cytotoxic, which could lead to severe complicationsshould the recombinant DNA become permanently incorporated intothe genome of normal tissues.

One concern in using adenovirus vectors for gene therapy is theirinactivation by the host’s immune system (21, 22), which hasprompted attempts to circumvent this potential obstacle (23, 24).However, immune inactivation of adenovirus may not represent aproblem within tumors (25). This may result, in part, from the abnor-

Received 4/13/00; accepted 11/20/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant DAMD17-98-18496 (to M. J. B.) from the U.S.prostate cancer initiative and by William Beaumont Hospital Research Institute Grant98-11 (to M. J. B.).

2 To whom requests for reprints should be addressed, at Radiation Oncology ResearchLaboratory, Department of Radiation Oncology, William Beaumont Hospital, 3601 WestThirteen Mile Road, Royal Oak, MI 48073.

mal nature of tumor vascularization, which may restrict access ofantibodies and immuno-active cells to tumors (26) and/or reducestimulatory interactions between these cells and the tumor vascularendothelium (27–29). Consequently, immune inactivation of adeno-viruses in normal tissues may actually help focus expression ofadenovirus vectors in tumors. Adenovirus vectors delivered directlyinto the tumor would be capable of infecting cancerous cells withminimal inactivation by the immune response, whereas vectors escap-ing the tumor might be inactivated by a sensitized immune systemprior to their infecting normal tissues. Vector inactivation outside thetumor would prove most advantageous when the therapeutic DNAproduces a toxic product. For these and other reasons, adenovirusvectors were selected for delivering recombinant DNA into the cellsused in this study.

Many stresses other than heat shock elicit expression of HSPs,including oxidative stress (30) and suboptimal growth conditions (31,32). Because these two induction conditions can exist within tumors,it is plausible that they could result in uncontrolled expression fromthe heat shock promoter. Truncated forms of the heat shock promoterexhibit induced expression by a more limited range of stresses (33,34). As illustrated herein, a commercially available, truncated, humanheat shock promoter (StressGen) exhibited abundant and controllablegene expression in response to heat shock while being virtually silentat 37.0°C and unresponsive to the suboptimal growth conditions ofplateau-phase cultures. Fever still represents a potential for uncon-trolled expression from the truncated heat shock promoter. However,treatment with antibiotics and other fever-reducing measures couldpotentially be used until either the fever-inducing event passes or thetherapeutic gene construct is no longer functional,e.g.,as with tran-siently infecting adenovirus vectors.

MATERIALS AND METHODS

Cell Culture. Du-145 human prostate carcinoma cells were adapted togrowth in 10% iron-supplemented calf serum (Hyclone) in DMEM/F12, sup-plemented with MEM nonessential amino acids (Life Technologies, Inc.),MEM vitamin solution (0.53recommended concentration; Life Technologies,Inc.), and 1 mM L-glutamine. The adaptation process required 3 weeks, and theadapted cells were designated Dut-145 cells.

Dut-145 cells were grown as monolayers at 37.0°C in humidified incubatorswith 5% CO2 to maintain medium pH at 7.4. For experimentation, cells wereseeded into 25-, 75-, or 150-cm2 culture flasks to obtain cultures at 80–85%of confluency 48 h later.

Adenovirus Vector Construction. The plasmids and 293 cells used toproduce nonreplicating adenovirus vectors were obtained from Microbix, Inc.Adenoviral vectors expressing EGFP2 under control of the truncated heatshock promoter were produced by first cloning the truncated heat shockpromoter (StressGen, Inc.) into the adenovirus shuttle plasmids pDE1sp1A andpDE1sp1B (19), followed by cloning theEGFP gene sequence and poly(A)region from plasmid pEGFP-1 (Clontech, Inc.) downstream of the heat shockpromoter. Adenoviral vectors expressing EGFP under control of the CMVpromoter were produced by cloning theEGFPgene (from pEGFP-1) into themultiple cloning site of plasmid pCI-Neo (Promega, Inc.), after which theentire pCI-Neo expression cassette was excised and cloned into the adenovirusshuttle plasmids.

Adenovirus shuttle plasmids containing the EGFP expression cassettes werecotransfected (calcium phosphate) with plasmid PJM-17 into 293 cells (19).Recombination of the expression cassettes from the shuttle plasmids intoPJM-17 yielded viral DNA of packagable size that produced adenoviruscapable of replicating in 293 cells. Transfected 293 cells were overlaid withagarose to permit isolation of individual virus plaques.

Virus Clone Selection, Amplification, and Freezing.Virus clones wereselected as agarose plugs of individual plaques, and;10% of the virions from

each plaque were used to infect 293 cells in a 60-mm culture dish. The 293cells were harvested when they were cytopathic, pelleted, suspended in 5 ml ofDMEM/F12 containing 5% heat-inactivated horse serum and 10% glycerol,and then frozen at270°C. The cells were later thawed, and virions were freedby three freeze-thaw cycles using, alternately, liquid nitrogen and a 37.0°Cwater bath. Cell lysates were clarified by centrifugation for 10 min at14,0003 g and separation of the lysate from the pellet. The lysate was storedat 280°C as aliquots, one of which was later thawed to titer the viruspreparation (19). Each virus clone was then tested and selected for use inexperiments based upon their ability to infect Dut-145 cells and express EGFPunder control of either the CMV or truncated heat shock promoters.

Selected clones were further amplified to produce sufficient viruses forexperiments by infecting larger quantities of 293 cells (30–300; 100-mmculture dishes) at an MOI of 0.3. Cells were harvested when;80% of theculture exhibited cytopathic morphology, and freeze-thaw cell lysates werecentrifuged on cesium chloride step gradients at 60,0003 g for 2 h at 20.0°C(35) to separate viruses from defective particles and empty capsids. Recoveredvirus bands were dialyzed overnight into PBS. Glycerol was then added to10%, and aliquoted virus suspensions were frozen and stored at280°C. Again,one aliquot was thawed and used to titer the virus preparation.

Adenovirus vectors with [3H]thymidine-labeled DNA were produced in anidentical manner, except that the infected 293 cells were cultured in mediumcontaining 9.8mCi/ml [3H]thymidine (84 Ci/mmol).

Adenovirus Infection of Experimental Cells. Exponentially growingcells were trypsinized from monolayer cultures, resuspended in DMEM/F12containing 5% heat-inactivated horse serum (1.53 106 cells/ml), pelleted in 15ml of polypropylene centrifuge tubes (5 min at 1200 rpm), and resuspended inthe same medium at 13 107 cells/750ml. The determined amount of adeno-virus was added, the centrifuge tube was capped tightly and made watertightwith a paraffin film, and each tube was mounted horizontally into a racksubmerged in a 37.0°C water bath. The cell suspensions were agitated gentlyfor 2.0 h by attaching the rack to a wrist-action shaker. Afterward, the cellswere diluted appropriately into growth medium and then seeded into 35-mmculture dishes such that the cells would be at 80–85% of confluency whenassayed. For assay times longer than 72 h after infection, cells were maintainedin exponential growth by subculturing. Many different virus infection proce-dures were tested, and this suspension method produced the most efficient,reproducible, and uniform (cell-to-cell) infections.

Assaying EGFP Produced in Virus-infected Cells.The 35-mm disheswere rinsed twice with PBS and aspirated dry, and the cells were scraped into500ml of reporter lysis buffer (Promega, Inc.). The samples were collected intriplicate and then frozen at220°C for later fluorometric quantitation of EGFPcontent. For most experiments, triplicate samples were also collected foranalyses by Western blotting (36).

A standard curve was established by determining the fluorescence of meas-ured quantities of recombinant EGFP (Clontech, Inc.) using a Perkin-ElmerLS-50B spectrofluorimeter (excitation, 480 nm; emission, 510 nm). Frozenlysates of experimental cells were thawed, mixed, and then assayed with thespectrofluorimeter to determine EGFP fluorescence/volume. Each sample wasalso subjected to the BCA assay (Pierce, Inc.) to determine its protein con-centration, which was used to calculate the amount of EGFP/mg of totalcellular protein. This value was then converted to pg of EGFP/cell usingpredetermined values of the average protein content/cell.

Quantitative Western Blots. Quantitative Western Blots were performedas described previously (36), and measured quantities of recombinant EGFP(Promega, Inc.) were included in each gel to establish a standard curve fordetermining the EGFP content of experimental samples. The absorbance of allbands on the Western blot chemilumigraphs (ECL system; Amersham-Phar-macia) were measured with a laser densitometer.

RESULTS

Controlled, Induced Expression from the Heat ShockPromoter. TheEGFPgene was silent under control of the truncatedheat shock promoter in nonheated Dut-145 cells that were infected atan MOI of 40 or less. This is illustrated visually by both fluorescencemicrographs (Figs. 1Aand 2A) and Western blots (Fig. 1Band 2B).These figures also demonstrate that the magnitude of the heat shock

2 The abbreviations used are: EGFP, enhanced green fluorescent protein; CMV,cytomegalovirus; MOI, multiplicity of infection; HSP, heat shock protein.

HEAT-ACTIVATED TRANSGENE EXPRESSION

(time at temperature) at 41.0°C, 42.0°C, or 43.0°C, and adenoviralMOI determined the magnitude of EGFP expression. Heat shocks at40.5°C induced barely detectable EGFP expression, even after infec-tions at MOIs between 20 and 40 (data not shown). Induction attemperatures higher than 43.0°C produced progressively lower EGFPexpression, ostensibly because the harsher heat shocks markedlyreduced transcription and translation and caused significant cell kill-ing (10, 37).

Using MOIs.40 had several undesirable effects upon the infectedcells. The first was that EGFP expression could be detected in 2–5%of control, nonheated cells. Although the number of control cells

expressing EGFP increased with MOI, the experiment to experimentvariation was great and did not permit establishing a mathematicalrelationship for uninduced EGFP expression as a function of MOI.40. When the MOI was 120 or greater, all of the cells exhibitedsome level of uninduced EGFP expression, ceased dividing, had twicethe diameter (measured from cells spread on the substrate) of thecontrol cells, and exhibited elevated HSP levels (HSC-70, HSP-70,and HSP-90; data not shown). The latter indicated that infection withthe adenoviral vectors placed the cells in a state of stress, most likelycaused by overexpressing viral proteins, the genes of which are alsopresent within the adenoviral vectors. This phenomenon is different

Fig. 1.A, fluorescent micrographs showing heat-induced EGFP expression in Dut-145 cells infectedat MOI 10 with adenovirus vectors containing theEGFP gene under control of the truncated heatshock promoter. Cells were heated for the indicatedtimes at 41.0°C, 24 h after virus infection, and thenassayed 12 h after heat shock. The amount of EGFPexpressed increased with longer heat shocks. Thephase and fluorescent images of the same cultureregion demonstrate the silence of the heat shockpromoter at 37.0°C, and this was confirmed by theWestern blot data inB). The camera gain wasincreased for the fluorescent micrographs of thecontrol cells so that the cell and media autofluores-cence were detectable.B, Western blot data ofEGFP production in Dut-145 cells infected withadenovirus vectors (MOI 10) containing theEGFPgene under control of the truncated heat shockpromoter and heat shocked at 41.0°C, 42.0°C, or43.0°C for the indicated times. Samples were taken12 h after heat shock. The 41.0°C data were ob-tained from the cultures pictured inA. Longer heatshocks at each temperature produced more EGFP,and the lack of EGFP in the negative control sampleagain demonstrates the promoter’s silence at37.0°C. Actin was used as a housekeeping proteinto demonstrate equal sample loading.

from the stress induced by infection with replication-competent ade-novirus, which results when the cells enter a cytopathic state as thereplicating virus within cells distends them toward the point ofrupture.

Results similar to those reported for the Dut-145 cells were alsoobserved in three other cell lines,i.e.,PC3 human prostate carcinoma,A549 human lung carcinoma, and HeLa human cervical carcinoma(data not shown).

Temporal Profile of Heat-induced EGFP Expression.Priorstudies have demonstrated that the magnitude and temporal durationof stress-induced HSP synthesis, under control of their endogenous

heat shock promoters, is proportional to that of the inducing stress (8,9). The data in Figs. 1 and 2 depict induced EGFP expression at onlyone point after adenovirus infection and subsequent heat shock induc-tion. Establishing a quantitative relationship between induction stressand temporal expression of therapeutic, recombinant DNA would beessential for effective and safe application of heat-activated genetherapy. Such data would serve as a guide to selecting the heat shockrequired for producing desired, dosage profiles of therapeutic DNAexpression from the adenovirus vectors.

Fig. 3 shows that maximal EGFP expression from the truncatedheat shock promoter was observed when exponentially growing cells

Fig. 2. A, fluorescent micrographs showingheat-induced EGFP expression in Dut-145 cellsinfected at MOI 20 with adenovirus vectors con-taining theEGFP gene under control of the trun-cated heat shock promoter. Cells were heated forthe indicated times at 41.0°C, 24 h after virusinfection, and then assayed 12 h after heat shock.The amount of EGFP expressed increased withlonger heat shocks. The phase and fluorescent im-ages of the same culture region demonstrate thesilence of the heat shock promoter at 37.0°C, andthis was confirmed by the Western blot data inB).The camera gain was increased for the fluorescentmicrographs of the control cells so that the cell andmedia autofluorescence were detectable.B, West-ern blot data of EGFP production in Dut-145 cellsinfected with adenovirus vectors (MOI 20) contain-ing theEGFP gene under control of the truncatedheat shock promoter and heat shocked at 41.0°C,42.0°C, or 43.0°C for the indicated times. Sampleswere taken 12 h after heat shock. The 41.0°C datawere obtained from the cultures pictured in Fig.A.In general, longer heat shocks at each temperatureproduced more EGFP. However, a 2- and 4-h heatshock at 41.0°C produced essentially identical lev-els of EGFP (also seeA), suggesting that this wasthe maximal expression inducible from this vectorat 41.0°C, following an MOI 20 infection. The lackof EGFP in the negative control sample demon-strates the promoter’s silence at 37.0°C, even at thishigher MOI. Actin was used as a housekeeperprotein to demonstrate equal sample loading.

were heat shocked 12–24 h after infection with adenovirus vectors.Heat shocking cells at earlier or later postinfection times yieldedlower EGFP expression, with the ability to induce significant expres-sion being lost 90–100 h after infection. The majority of this loss wasattributed to cell division diluting the number ofEGFP gene copies/cell because the adenoviral DNA does not replicate in the Dut-145cells. This postulate of vector dilution by cell division was supportedby the ability to protract the period over which maximal EGFPexpression could be induced to.80 h after infection by treating cellswith the cell cycle inhibitor aphidicolin (3.0mM), which was admin-istered 12 h after infection and maintained in the culture mediumthereafter (data not shown).

Temporal profiles of heat shock-induced EGFP expression wereobtained for cells heated at 41.0°C (Fig. 4) or 42.0°C (Fig. 5) afterinfection at an MOI of 10 or 20. On the basis of the data in Fig. 3, heatshocks were administered 24 h after adenovirus infection to inducemaximal EGFP expression, and the EGFP content/cell was measuredperiodically thereafter.

Maximal EGFP expression occurred between 12 and 24 h after heatshock at 41.0°C, and this was not affected appreciably by increasingMOI. After identical heat shocks, the peak-induced EGFP levels forcells infected at MOI 20 were 2-fold or more greater than thoseinfected at MOI 10, and the decline from peak expression was slower.A 4-h heat shock yielded the maximum in both peak and temporalduration of EGFP expression after exposure to 41.0°C. Longer heatshocks at 41.0°C, up to 8 h, produced an EGFP expression profileidentical to that induced by a 4-h exposure (data not shown). Heatshocks longer than 8 h at 41.0°C produced proportionately lowerEGFP expression levels.

Compared with induction at 41.0°C, EGFP expression was delayedafter heat shocks at 42.0°C (Fig. 5), as illustrated by the ratio of EGFPexpression at 12 h to that at 24 h after heat shock. This ratio rangedbetween 0.85 and 1.1 following 41.0°C and 0.33 and 0.6 following42.0°C, with cells infected at MOI 10 exhibiting the lowest ratios afterinduction by 42.0°C. The maximal peak EGFP expression induced at42.0°C resulted from a 2-h heat shock. This maximum was essentiallyidentical to that induced by 41.0°C in cells infected at MOI 10 but was;40% lower when cells were infected at MOI 20. Using 42.0°C heatshocks longer than 2 h resulted in a progressive decrease in peakEGFP expression (Fig. 5). Interestingly, the duration over which

induced EGFP could be detected was relatively refractory to heatingtime or the use of 41.0°C or 42.0°C.

Spectrofluorimeter measurement of EGFP proved usable over awider range of cellular EGFP levels than measurement by Westernblots (data not shown), possibly because the linearity between EGFPlevels and fluorescence was more expansive than that for film densityfor exposed protein bands on chemilumigraphs. In addition, the spec-trofluorimeter measurements were easier to make (especially for nu-merous samples) and yielded greater reproducibility. Consequently,the spectrofluorimeter method of quantitating cellular EGFP was usedthroughout this study. Spot checks of the spectrofluorimetric data byquantitative Western blots showed good agreement between the twomethods (data not shown).

Depending upon the nature of the recombinant DNA being used fortherapy, it might be desirable to either re-induce or protract itsexpression under the heat shock promoter. These issues were ad-dressed by delivering 2-h, 41.0°C heat shock fractions at 24-h inter-vals, starting 24 h after virus infection. Fig. 6 shows that this frac-tionated heat shock regime extended EGFP expression such that it wasstill ;75% of peak 120 h after the initial heat shock (Fig. 6).Fractionated heat shocks at 24-h intervals may not have been optimalfor sustaining maximal EGFP expression; however, Fig. 6 shows clearlythat fractionated heat shocks markedly protracted high level EGFP ex-pression without requiring infection with additional virus vectors.

Expression from the CMV Promoter. To be effective, gene ther-apy vectors must produce sufficient recombinant product to treat the

Fig. 3. Exponentially growing Dut-145 cells were infected at an MOI of 10 withadenoviral vectors containing theEGFP gene under control of the truncated heat shockpromoter. Different culture dishes were then heat shocked at 41.0°C for 2 h at varyingtimes after viral infection, and samples were taken for analysis 24 h after the heat shock.Maximal EGFP expression was induced when the heat shock was administered 12–24 hafter infection, and the ability to induce EGFP expression was lost between 72 and 96 hafter infection.Bars,SD.

Fig. 4. Temporal EGFP expression profiles from the heat shock promoter in exponen-tially growing cultures, induced by 41.0°C for the indicated times. Cells were infectedwith viral vectors at an MOI of 10 (A) or 20 (B). Maximal expression was induced by heatshocks of 2–8 h (8-h data not shown).Bars,SD.

diseased state successfully. Many investigators have used strong,constitutive promoters,e.g., the CMV promoter, to maximize trans-gene expression to achieve this goal. Experiments were performedusing adenovirus vectors wherein EGFP was expressed under controlof the CMV promoter to determine how induced expression from thetruncated heat shock promoter compared with a strong constitutivepromoter that was already being used clinically.

Fig. 7 presents the temporal profile of EGFP expression from theCMV promoter in exponentially growing Dut-145 cells infected withadenovirus vectors at MOIs of 10, 20, or 30. For each MOI, cellularEGFP increased to a peak value that occurred 58–65 h after virusinfection; however, the peak for the MOI 10 infection is not obviousfrom this figure because EGFP expression was so low relative to thatfollowing the higher MOI infections. As with the heat shock pro-moter, the temporal expression peaks were asymmetric, with a distinctshoulder following the peak.

The peak EGFP levels achieved after the three different MOIs wereclearly not proportional to MOI (Fig. 7). EGFP expression after anMOI 10 infection was exceptionally low, being more than 15- and35-fold lower, respectively, than that observed after infections at MOI20 or 30. These were much higher than the factors of two and threethat were expected for MOI proportional expression. The 2.4-foldincrease in peak EGFP levels going from MOI 20 to MOI 30 wascloser to the expected increase but still significantly higher than theexpected 1.5-fold increase. Infections with virus containing radiola-beled DNA demonstrated that infection of viral DNA was propor-

tional to MOI (Fig. 8). Hence, the nonproportional EGFP expressionwas attributable to nonlinear CMV promoter activity.

A direct comparison shows that maximal expression from the heatshock promoter, after a 41.0°C, 2-h heat shock (Fig. 4), was 8- and1.3-fold greater than that from the CMV promoter for cells infected at,respectively, MOI 10 and MOI 20. Maximal expression from the heatshock promoter was still;8-fold greater after a 42.0°C heat shock tocells infected at MOI 10. However, in cells infected at MOI 20, peakexpression from the CMV promoter was 1.15-fold greater than fromthe heat shock promoter when expression from the latter was inducedby 42.0°C.

This peak production comparison demonstrates that the truncatedheat shock promoter was either more effective than or equally effec-tive as the CMV promoter, depending upon the MOI and inductiontemperature used. In actuality, the heat shock promoter was moreproductive because it was not transcribing continuously after theinducing heat shock. Prior studies have shown that transcription fromthe heat shock promoter ceases 4–8 h after induction, depending uponthe intensity of the inducing stress (8–10, 30). Consequently, if onecompares the amount of EGFP produced from the CMV and heatshock promoters over any 6- or 12-h period, the heat shock promoteris always found to be more productive.

Fig. 5. Temporal EGFP expression profiles from the heat shock promoter in exponen-tially growing cultures, induced by 42.0°C for the indicated times. Cells were infectedwith viral vectors at a MOI of 10 (A) or 20 (B). The post-heating time for maximal EGFPwas delayed 4–6 hours compared with that observed after induction by 41.0°C (Fig. 4).Maximal expression was induced by a 2-h heat shock, with longer heat shocks yieldinglower expression.

Fig. 6. Exponentially growing cells were infected at MOI 10 with adenovirus vectorscontaining EGFP under control of the heat shock promoter. Cells were heat shocked for2 h at 41.0°C every 24 h (arrows), starting 24 h after infection (initial arrow), and cellularEGFP levels were measured periodically. These data demonstrate the ability to maintainhigher EGFP levels longer by using sequential, fractionated heat shocks.Bars,SD.

Fig. 7. EGFP expression in exponentially growing cells infected with adenovirus vectorscontaining theEGFPgene under control of the CMV promoter. Maximal EGFP levels wereobserved at 58 h after infection. Expression after infection at an MOI of 10 was very low, andthe maximal EGFP level achieved clearly did not increase linearly with MOI.Bars,SD.

Expression from the Heat Shock and CMV Promoters inPlateau Phase Cells.Human tumors consist of cells exhibitinggrowth rates ranging from plateau phase to exponential. Furthermore,most cells in normal tissues grow more slowly than those in theexponential cultures used for the experiments presented thus far.Consequently, it was relevant to investigate expression from the heatshock and promoters in plateau phase cultures.

Maximum EGFP expression from the heat shock promoter wasinduced when plateau phase cells were heated 48 h after infection withthe adenovirus vectors (Fig. 9A) rather than 24 h after infection, aswith exponential cells (Fig. 3). The maximal EGFP level induced wasapproximately one-third of that expressed in exponential cells (com-pare maximum levels in Figs. 4Band 9), but it still occurred 12–24 hafter the heat shock (Fig. 9B). The postinfection period over whichnear-maximal EGFP expression could be induced from the heat shockpromoter was markedly protracted in plateau phase cultures (Fig. 9A).Almost 70% of the peak expression level could still be induced96–120 h after virus infection, whereas,10% of maximal EGFPexpression could be induced 96 h after infection of exponential cells(Fig. 3). Furthermore, EGFP levels decayed more slowly from thepeak induced level after heat induction in plateau phase cells (Fig.9B). Fig. 9 also demonstrates that the plateau phase did not constitutea stress that induced significant expression from the heat shockpromoter without administering an inducing stress.

Fig. 10 clearly shows that EGFP expression under control of the CMVwas delayed in plateau phase cultures, reaching peak values 96–120 hafter infection instead of 55–65 h after infection, as in exponentialcultures (Fig. 7). Expression also never achieved the same peak levelsobserved in exponential cells. However, cellular EGFP remained at peaklevels longer, showing very little decrease even.200 h after virusinfection. It is notable that EGFP expression in plateau cells infected atMOI 10 was higher than in identically infected exponential cells (Fig.10). Conversely, the peak EGFP levels achieved after infections at MOI20 and MOI 30 were less than in exponential cultures, with the biggestdecrease noted in the MOI 30-infected cells.

DISCUSSION

This study demonstrated that the magnitude and temporal duration oftransgene expression induced from a truncated heat shock promoter canbe controlled in both exponential and plateau phase cultures of humantumor cells infected with adenovirus expression vectors carrying the

transgene construct. Expression from the heat shock promoter was vir-tually undetectable until a heat shock.40.0°C was delivered, even incultures that were maintained in plateau phase for.2 weeks. Once thethreshold temperature was exceeded, transgene product was expressed ina dose-responsive manner that was proportional to the temperature andduration of the inducing heat shock. Sapareto and Dewey (38) establisheda time-temperature relationship for hyperthermic cell killing, which, insimplistic terms, states that equivalent levels of cell killing can be attainedat different temperatures by decreasing the heating time by a factor of twofor each degree the temperature is raised. The dose response for heatshock-induced transgene expression did not follow this time-temperaturerelationship strictly; however, the conformity was sufficient for the rela-tionship to be used to estimate the relative amount of heat shock-inducedtransgene expression for different time-temperature combination heatshocks (Figs. 1 and 2). Unfortunately, the estimates were good over alimited time-temperature range and quickly became inaccurate when theheat shock was severe enough to induce significant inhibition of cellularmetabolism and/or cell killing.

The current paradigms for heat shock activation of the stressresponse and hyperthermic cell killing incorporate thermal denatur-ation of cellular proteins as a critical event. Denatured proteins puta-tively interact with HSPs bound to heat shock transcription factormonomers such that they are freed to trimerize into the active form ofthe transcription factor, whereas aggregation of heat-denatured pro-

Fig. 8. Exponentially growing cells were infected with adenovirus vectors containingthe EGFP gene under control of the CMV promoter. The viral DNA was labeled with[3H]thymidine, and relative viral uptake by the cells, following different MOI infections,was measured by their content of radiolabeled DNA. The amount of radiolabeled DNA/cell did increase linearly with MOI, whereas the amount of EGFP expressed subsequentlydid not (Fig. 7).Bars,SD.

Fig. 9. Plateau phase cells were infected at an MOI of 10 with adenovirus vectorscontaining theEGFP gene under control of the heat shock promoter.A, cells were heatshocked at 41.0°C for 2 h at varying times after infection, and EGFP levels were measured24 h after heat shock. Maximal EGFP levels were achieved when cells were heat shocked48 h after infection, and;80% of this maximal level could still be induced.120 h afterinfection.B, cells were heat shocked for 2 h at 41.0°C 48 h after infection. Maximal EGFPexpression still occurred 12–24 h after a heat shock to infected, plateau phase cells.Maximal EGFP levels were lower (35–50% lower) than those achieved in identicallytreated, exponentially growing cells.Bars,SD.

teins onto critical cellular structures purportedly inhibits essentialcellular functions and results in cell killing. When the time-tempera-ture relationship for cell killing established by Sapareto and Dewey(38) was subjected to a thermodynamic, Arrhenius analysis, the re-sultant activation energy for cell killing was essentially identical to theaverage activation energy for thermal denaturation of cellular pro-teins, implicating protein denaturation as a lethal event for cell killing.Because thermal protein denaturation is putatively a common factor inheat shock promoter activation and the Sapareto-Dewey time-temper-ature relationship for cell killing, it is logical that heat shock promoteractivation would follow the Sapareto-Dewey relationship, at least overa limited time-temperature range. The Sapareto-Dewey relationshipfailed as a prognosticator of relative gene expression from the heatshock promoter when the inducing heat shock either resulted insignificant heat killing (survival,70%) or significantly inhibitedcellular metabolism, especially transcription and translation.

The demonstrated controllability of the truncated heat shock pro-moter supports the concept of using it to administer conformal,heat-activated gene therapy for treating tumors and other diseasedtissues. Data presented in this study can be used as a first approxi-mation to direct and planin vivo experiments that will be required todevelop heat-activated gene therapy for clinical applications.

A single heat shock at 41.0°C was capable of inducing EGFP expres-sion that was greater than or equal to the maximum provided by the CMVpromoter in cells infected with equivalent MOIs of the respective ade-novirus vectors. Thus, gene expression comparable with that from theCMV promoter can be elicited from the heat shock promoter by heatshocks that can be achieved readily within tissues, using devices currentlyavailable for clinical hyperthermia. A major advantage that the heat shockpromoter has over the CMV promoter and other constitutive promoters isthe ability to conformally regulate the magnitude and temporal durationof its expression, from undetectable levels (no heat shock) to levelsgreater than those achievable with the CMV promoter. Normal tissuetoxicity would be low or nonexistent when the inducing heat shock isdelivered conformally because the promoter would not be turned on inthe nonheated normal tissues. If expression from the heat shock promotercould be monitored during the course of treatment,e.g.,by noninvasiveimaging techniques, transgene expression could then be modulated con-formally throughout the treatment course using sophisticated hyperther-mia devices to maintain effective transgene expression within all regions

of the target tissue. Conversely, once CMV expression vectors are deliv-ered, transgene expression is unregulatable and committed to a givenlevel of transgene product. Additional vectors could be administered ifexpression from the CMV vectors is too low to be effective, but optionswould be limited if expression becomes too high. If vectors containingthe CMV promoter infect normal tissues, there is no way to suppress itsexpression. If the transgene product is toxic or otherwise affects normaltissue function, the consequences of such inadvertent, unwanted trans-gene expression in normal tissues could be dire (39).

Growth conditions of the cells markedly affected transgene expres-sion from both the CMV and heat shock promoters. Relative toexponential cultures, a much longer postinfection time was required toachieve the maximum, cellular EGFP level from the CMV promoterin plateau phase cells, and the postinfection time at which maximalexpression could be induced from the heat shock promoter wasdelayed from 24 h in exponential cultures to 48 h. Once expressioncommenced or was induced in plateau phase cells, the maximal levelof EGFP attained by the CMV or heat shock promoter, respectively,was reduced significantly, but these maximum EGFP levels weresustained for a significantly longer period.

The observed attenuation of the maximal amount of transgene productgenerated per cell could prove problematic for gene therapy in cellsgrowing in suboptimal conditions,e.g., within the central regions oftumors. The therapy would fail if the attenuated maximum fell below thethreshold level of transgene product required to achieve clinical efficacy.If, however, the temporal integral of the transgene product determinedtreatment efficacy, attenuation of transgene expression might be offset tosome extent by the protracted period over which the maximum level ofgene product persists in plateau phase cells.

The protracted period over which maximal, cellular EGFP levelswere sustained in plateau phase cultures was greater for the CMVpromoter, ostensibly because it remained constitutively expressedwhile expression from the heat shock promoter decayed after theinducing heat shock. This difference between the two promoters couldbe significant in normal tissues, where cell proliferation is usuallymuch less than in exponential cell cultures, yet nutrients for optimalmetabolism are abundant. Consequently, it is likely that CMV-controlled expression in normal tissues would be at the high levelobserved in exponential cultures and for the more protracted periodobserved in plateau phase cultures. This could result in uncontrollable,adverse side effects should too much normal tissue become infectedwith therapeutic vectors using the CMV promoter. The lack of unin-duced expression in plateau cultures bodes well for the heat shockpromoter being silent in nonheated normal tissues, wherein cellsgenerally proliferate more slowly than in culture. If more protractedexpression from the heat shock promoter is desired within a targettissues whose cells are in plateau phase, this can be accomplishedusing multiple heat shocks to sustain transgene expression (Fig. 9A).

Experiments with radioactively labeled viruses demonstrated thatadenovirus infection efficiency and the time required for virus uptakein plateau phase cells were similar to those in exponential cultures.Consequently, it was postulated that the time required to process theadsorbed viruses in plateau phase cells and/or for viral DNA tomigrate to the nucleus were the rate-limiting steps that delayed CMVexpression and the postinfection time at which maximum expressioncould be induced from the heat shock promoter. Reduced metabolismresulting from nutrient-poor medium and intercell competition weremost likely responsible for the attenuated EGFP expression in theplateau phase cultures. The protracted time over which cells main-tained maximal EGFP levels was attributed to reduced cell prolifer-ation in plateau phase cultures, a conclusion that was supported byexperiments with cell cycle inhibitors.

Spatial control of heat shock promoter expression was not investi-

Fig. 10. Plateau phase cells were infected at an MOI of 10, 20, or 30 with adenovirusvectors containing theEGFPgene under control of the CMV promoter. Cellular EGFP levelswere then measured periodically thereafter. The peak EGFP levels achieved at MOI 10 werehigher than that observed in similarly infected cultures growing exponentially, whereas thosefor cells infected at MOIs of 20 and 30 were significantly less than their exponentially growingcounterparts. However, the temporal duration of the maximal EGFP levels was markedlyprotracted compared with that observed in exponential cultures.Bars,SD.

gated, but its potential use for conformal gene therapy can be envi-sioned, as long as temperatures do not exceed 40.0°C in tissues, whereexpression is not wanted. Strong conformal control of promoter in-duction would be particularly important when the therapeutic trans-gene is toxic,e.g., a proteotoxin. In this instance, serious localcomplications could arise if undesired expression occurred in normaltissues surrounding diseased tissues injected with adenoviral vectors,with the potential for more serious, global complications if vectors areadministered systemically. The controlled inducibility of the heatshock promoter demonstrated in this report leaves open the potentialfor systemic delivery of a wide range of gene therapy vectors, eventhose expressing cytotoxic products. Vector expression would occuronly in selected, heat shocked sites. A major caveat would be thedanger of fever activating the heat shock promoter and resulting inwidespread toxicity. The potential for such a disaster, and the possi-bility of averting it by using high doses of systemically administeredantibiotics to control fever, need to be tested in animal models.

The ability to target the delivery and/or expression of gene therapyvectors to diseased tissues would greatly reduce the risks of inadvert-ent expression in normal tissues. Although some promising data withtissue-specific promoters have been reported, considerable work re-mains before they are used routinely. Furthermore, there are manytissues for which specific promoters are not available, and wherein aninducible promoter such as the heat shock promoter remains a goodoption for conformal gene therapy.

The results of this study support the plausibility for developingconformal, heat-activated gene therapy and provide data that can beused to anticipate the response of the heat shock promoter inin vivoexperiments. Additionalin vitro andin vivo experiments are requiredto address the many concerns and questions that must be answeredbefore heat-activated gene therapy can be introduced into the clinic.Clinical devices for delivering the necessary, local hyperthermia in therange of 40.0°C to 42.0°C exist, and there should be little difficulty inadapting these instruments for this application. The more dauntingtasks will involve designing effective therapeutic vectors, deliveringeffective amounts of the vectors throughout diseased tissues, andunderstanding how the biology and physiology of different diseasedtissues will affect the application of conformal, heat-activated genetherapy and its therapeutic outcome.

REFERENCES

1. Venn, S. N., Hughes, S. W., Montgomery, B. S., and Timothy, A. Heating charac-teristics of a 434 MHz transurethral system for the treatment of BPH and interstitialthermometry. Int. J. Hyperthermia,12: 271–278, 1996.

2. Ansher, M. S., Samulski, T. V., Dodge, R., Prosnitz, L. R., and Dewhirst, M. W.Combined external beam irradiation and external regional hyperthermia for locallyadvanced adenocarcinoma of the prostate. Int. J. Radiat. Oncol. Biol. Phys.,37:1059–1065, 1997.

3. Lantis, J. C., Carr, K. L., Grabowy, R., Connolly, R. J., and Schwaitzberg, S. D.Microwave applications in clinical medicine. Surg. Endosc.,12: 170–176, 1998.

4. Diedrich, C. J., and Hynynen, K. Ultrasound technology for hyperthermia. UltrasoundMed. Biol., 25: 871–887, 1999.

5. Lee, R. J., Buchanan, M., Kleine, L. J., and Hynynen, K. Arrays of multielementultrasound applicators for interstitial hyperthermia. IEEE Trans. Biomed.,46: 880–890, 1999.

6. Lizzi, F. L., Deng, C. X., Lee, P., Rosado, A., Silverman, R. H., and Coleman, D. J.A comparison of ultrasonic beams for thermal treatment of ocular tumors. Eur. J.Ultrasound,9: 71–78, 1999.

7. Corry, P. M., Martinez, A., Armour, E. P., and Edmundson, G. Simultaneoushyperthermia and brachytherapy with remote afterloading.In: A. Martinez, C. Orton,and R. Mould (eds.), Brachytherapy HDR and LDR: State of the Art, pp. 193–204.Columbia, MD: Nucletron, 1990.

8. Li, G. C., and Werb, Z. Correlation between synthesis of heat shock proteins anddevelopment of thermotolerance in Chinese hamster fibroblasts. Proc. Natl. Acad.Sci. USA,79: 3219–3222, 1982.

9. Landry, J., Bernier, D., Chretien, P., Nicole, L. M., Tanguay, R. M., and Marceau, N.Synthesis and degradation of heat shock proteins during development and decay ofthermotolerance. Cancer Res.,42: 2457–2461, 1982.

10. Lee, Y. J., and Dewey, W. C. Thermotolerance induced by heat, sodium arsenite, orpuromycin: its inhibition and differences between 43oC and 45oC. J. Cell. Physiol.,135: 397–406, 1988.

11. Gossen, M., and Bujard, H. Tight control of gene expression in mammalian cells bytetracycline responsive promoters. Proc. Natl. Acad. Sci. USA,89: 5547–5551, 1992.

12. Weichselbaum, R. R., Hallahan, D. E., Beckett, M. A., Mauceri, H. J., Lee, H.,Sukhatme, V. P., and Kufe, D. W. Gene therapy targeted by radiation preferentiallyradiosensitizes tumor cells. Cancer Res.,54: 4266–4269, 1994.

13. Advani, S. J., Chmura, S. J., and Weichselbaum, R. R. Radiogenic therapy: on theinteraction of viral therapy and ionizing radiation for improving local control oftumors. Semin. Oncol.,24: 633–638, 1997.

14. Manome, Y., Kunieda, T., Wen, P. Y., Koga, T., Kufe, D. W., and Ohno, T.Transgene expression in malignant glioma using a replication-defective adenoviralvector containing the Egr-1 promoter: activation by ionizing radiation or uptake ofiododeoxyuridine. Hum. Gene Ther.,9: 1409–1417, 1998.

15. Dewey, W. C., and Freeman, M. L. Rationale for use of hyperthermia in cancertherapy. Ann. NY Acad. Sci.,335: 372–378, 1980.

16. Bornstein, B. A., Zouranjian, P. S., Hansen, J. L., Fraser, S. M., Gelwan, L. A.,Teicher, B. A., and Svensson, G. K. Local hyperthermia, radiation therapy, andchemotherapy in patients with local-regional recurrence of breast carcinoma. Int. J.Radiat. Oncol. Biol. Phys.,25: 79–85, 1993.

17. Overgaard, J., Gonzalez Gonzalez, D., Hulshof, M. C., Arcangeli, G., Dahl, O., Mella,O., and Bentzen, S. M. Randomised trial of hyperthermia as adjuvant to radiotherapyfor recurrent or metastatic malignant melanoma. European Society for HyperthermicOncology. Lancet,345: 540–543, 1995.

18. de Wit, R., van der Zee, J., van der Berg, M. E., Kruit, W. H., Logmans, A., vanRhoon, G. C., and Verweij, J. A Phase I/II study of combined weekly systemiccisplatin and locoregional hyperthermia in patients with previously irradiated recur-rent carcinoma of the uterine cervix. Br. J. Cancer,80: 1387–1391, 1999.

19. Graham, F. L., and Prevec, L. Manipulation of adenovirus vectors.In: E. J. Murray (ed.),Methods in Molecular Biology, pp. 109–128. Clifton, NJ: The Humana Press, 1991.

20. Wilson, J. M. Adenoviruses as gene-delivery vehicles. Mol. Med.,334:1185–1187, 1996.21. Worgall, S., Wolff, G., Falck-Pedersen, E., and Crystal, R. G. Innate immune

mechanisms dominate elimination of adenoviral vectors followingin vivo adminis-tration. Hum. Gene Ther.,8: 37–44, 1997.

22. Chirmule, N., Propert, K., Magosin, S., Qian, Y., Qian, R., and Wilson, J. Immuneresponses to adenovirus and adeno-associated virus in humans. Gene Ther.,6:1574–1583, 1999.

23. Jooss, K., Turka, L. A., and Wilson. M. Blunting of immune responses to adenoviralvectors in mouse liver and lung with CTLA4Ig. Gene Ther.,5: 309–319, 1998.

24. Wold, W. S., Doronin, K., Toth, K., Kuppuswamy, M., Lichtenstein, D. L., andTollefson, A. E. Immune responses to adenoviruses: viral evasion mechanisms andtheir implications for the clinic. Curr. Opin. Immunol.,11: 380–386, 1999.

25. Li, Z., Rakkar, A., Katayose, Y., Kim, M., Shanmugam, N., Srivastava, S., Moul,J. W., McLeod, D. G., Cowan, K. H., and Seth, P. Efficacy of multiple administra-tions of a recombinant adenovirus expressing wild-type p53 in an immune-competentmouse tumor model. Gene Ther.,5: 605–613, 1998.

26. Jain, R. K. Physiological barriers to delivery of monoclonal antibodies and othermacromolecules in tumors. Cancer Res.,50: 814s–819s, 1990.

27. Wu, N. Z., Klitzman, B., Dodge, R., and Dewhirst, M. W. Diminished leukocyte-endothelium interaction in tumor microvessels. Cancer Res.,52: 4265–4268, 1992.

28. Jain, R. K., Koenig, G. C., Dellian, M., Fukumura, D., Munn, L. L., and Melder, R. J.Leukocyte-endothelial adhesion and angiogenesis in tumors. Cancer Metastasis Rev.,15: 195–204, 1996.

29. Melder, R. J., Koenig, G. C., Witwer, B. P., Safabahsh, N., Munn, L. L., and Jain,R. K. During angiogenesis, vascular endothelial growth factor and basic fibroblastgrowth factor regulate natural killer cell adhesion to tumor endothelium. Nat. Med.,2: 992–997, 1996.

30. Freeman, M. L., Borrelli, M. J., Syed, K., Senisterra, G., Stafford, D., and Lepock,J. R. Characterization of a signal generated by oxidation of protein thiols that activatesthe heat shock transcription factor. J. Cell. Physiol.,164: 356–366, 1995.

31. Ritossa, F. M. A new puffing pattern induced by a temperature shock and DNP inDrosophila. Experimentia,18: 571–573, 1962.

32. van Dongen, G., Geilenkirchen, W. L., van Rijn, J., and van Wijk, R. Increase ofthermoresistance after growth stimulation of resting Reuber H35 hepatoma cells.Alteration of nuclear characteristics, non-histone chromosomal protein phosphoryla-tion and basal heat shock protein synthesis. Exp. Cell Res.,166: 427–441, 1986.

33. Voellmy, R., Ahmed, A., Schiller, P., Bromley, P., and Rungger, D. Isolation andfunctional analysis of a human 70,000-dalton heat shock protein gene segment. Proc.Natl. Acad. Sci. USA,82: 4949–4953, 1985.

34. Schiller, P., Amin, J., Ananthan, J., Brown, M. E., Scott, W. A., and Voellmy, R.cis-acting elements involved in the regulated expression of a humanHSP70gene.J. Mol. Biol., 203: 97–105, 1988.

35. Gerard, R. D., and Meidell, R. S. Adenoviral vectors.In: B. D. Hames and D. M.Glover (eds.), DNA Cloning: A Practical Approach, pp. 285–306. Oxford: OxfordUniversity Press, 1995.

36. Borrelli, M. J., Stafford, D. M., Karczewski, L. A., Rausch, C. M., Lee, Y. J., andCorry, P. M. Thermotolerance expression in mitotic cells without increased transla-tion of heat shock proteins. J. Cell. Physiol.,169: 420–428, 1996.

37. Borrelli, M. J., Stafford, D. M., Rausch, C. M., Lee, Y. J., and Corry, P. M. The effectof thermotolerance on heat-induced excess nuclear associated proteins. J. Cell.Physiol.,156: 171–181, 1993.

38. Sapareto, S. A., and Dewey, W. C. Thermal dose determination in cancer therapy. Int.J. Radiat. Oncol. Biol. Phys.,10: 787–800, 1984.

39. Hollon, T. Researchers and regulators reflect on first gene therapy death. Nat. Med.6: 6, 2000.

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