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    Alpha induces tumor necrosis and tumor cheap beats by dre solo regression without systemic toxicity

    Tumortargeted gene delivery of tumor necrosis factor induces tumor necrosis and tumor regression without systemic toxicity

    Ralf kircheis1, elinborg ostermann1, markus f wolschek2, 3, cornelia lichtenberger3, christine maginlachmann1, lionel wightman1, malgorzata kursa1 and ernst wagner1received 14 may 2002.

    Top of pageabstractwe have recently developed surfaceshielded transferrin(Tf delivery systems that target reporter gene expression to distant tumors after systemic application.In the present study, we used surfaceshielded tf complexes for delivering the gene for a highly potent cytokine, tumor necrosis factor(Tnf tnf is known for its ability to induce hemorrhagic tumor necrosis and tumor regression.However, the therapeutic application of tnf is hampered by its high systemic toxicity dictating the need to target tnf activity to the tumor.Systemic application of surfaceshielded tf complexes with the tnf gene resulted in preferential expression of tnf in the tumor without detectable tnf serum levels, in contrast to the application of nontargeted complexes.Tumortargeted tnf gene delivery induced pronounced hemorrhagic tumor necrosis and inhibition of tumor growth in three murine tumor models of different tissue origins, neuro2a neuroblastoma, metha fibrosarcoma, and m3 melanoma, with complete tumor regressions observed in the metha model.No systemic tnfrelated toxicity was observed due to the localization of the tnf activity to the tumor.Targeted gene therapy may be an attractive strategy applicable to highly active, yet toxic, molecules such as tnf

    Keywords:Tumor necrosis factor;Tnf gene therapy;Tumor targeting;Polyethylenimine;Pei;Nonviral gene transfer;Transferrin

    The potential of gene therapy to target the expression of therapeutic genes to the desired target cells makes it particularly attractive for cancer treatment.However, although there are vectors available with high transfection efficacy in cell culture, 1, 2, 3, 4 efficient and targetspecific gene delivery in vivo is still the major challenge in gene therapy.In vivo gene delivery has to overcome additional obstacles such as anatomical size constraints and nonspecific interactions with biological fluids and nontarget cells.Recently, we have developed surfaceshielded transferrin(Tf gene delivery systems that are able to target reporter gene expression to distant tumors after systemic application in murine models.5, 6, 7 In the present study, we have employed these gene delivery systems to target gene expression of a highly potent, yet highly toxic, molecule tumor necrosis factor(Tnf to distant tumors with the aim to separate the antitumor activity of tnf from its systemic toxicity.

    In the present study, surfaceshielded tf gene delivery systems were used for the first time to target gene expression of a highly potent effector molecule to distant tumors following systemic application.Tnf expression localized to the tumor resulted in hemorrhagic tumor necrosis and inhibition of tumor growth without systemic tnfrelated toxicity.

    Top of pagematerials and methodschemicals and expression vectorspei, branched, 25 kda, and linear pei, 22 kda, were obtained from aldrich(Milwaukee, wi)And mbi fermentas(St.Leonrot, germany), respectively.Tf(25kDa)Synthesis has been described previously.7

    The plasmids pcmvl, pcmv and pgsmutnf coding for luciferase,(And murine tnf respectively, and the nonexpressing plasmid psp65 were purified by elim biopharmaceuticals(San francisco, ca).Endotoxin levels were u/50 dna as determined by limulus amebocyte lysate assay(Biowhittaker, walkersville, md).

    Cells and animalsmurine neuro2a neuroblastoma cells were cultured in rpmi 1640 medium/10% fcs.Murine m3 melanoma cells were cultured in ham's f10 chpdrdrebts medium/15% horse serum/5% fcs.Murine metha fibrosarcoma cells(Kindly provided by dr fichtner, maxdelbr center, berlinbuch, germany)Were passaged in vivo as intraperitoneal ascites in syngeneic balb/c mice.A/j, dba/2, and balb/c mice(7 weeks, female)Were purchased from harlan(Bicester, uk).

    Preparation of transfection complexespei/dna complexes were prepared as described.7 Plasmid DNA(200 was flashmixed with PEI(22 kDa)Atn/p(N/p ratio of pei nitrogen to dna phosphate)In 20 mm hepes(Ph for isoosmolarity, glucose was added to a final concentration of 5%(Wt/vol).The potential of transfection complexes was measured using a malvern zetasizer 3000 as described previously.6

    Tf complexes were prepared as described previously7 by flash mixing of dna(200 withthe polycation solution(Containing three parts pei and one part tf at n/p complexes were prepared in 20 mm hepes(Ph glucose or 0.5 HBS(75 mM NaCl, 20 mM HEPES, pH glucose.

    Determination of luciferase reporter gene expression after systemic applicationa/j mice were injected subcutaneously with 1 neuro2a cells.After 2 weeks, tumors had reached 10 in size;Transfection complexes(250 with the pCMVL plasmid were injected into the tail vein.Animals were sacrificed by cervical dislocation at indicated time points.Organs were resected, homogenized in250 mm tris buffer, ph 7.5, using an IKA Homogenizer, frozen in liquid nitrogen, and stored at Luciferase activity in the tissue lysate was measured using a Lumat LB9507 instrument(Berthold, bad wildbad, germany)As described previously.5 Luciferase background(100 RLU)Was substracted from each value and transfection efficacy is expressed as rlu(Relative light units)Per organ.One million rlu correspond to 2 ng of luciferase.

    Therapeutic modelsa/j mice were injected subcutaneously with 1 syngeneic neuro2a tumor cells, dba/2 mice were injected with 1 m3 cells, and balb/c mice were injected with 2 metha cells.Starting at indicated time points after tumor setting(Tumor diameter, 5 mm), transfection complexes containing the plasmid for murine TNF for a control plasmid for or the nonexpressing pSP65 plasmid were injected into the tail vein.Tumor size and body weight of the animals were monitored.Differences in Tumor growth were statistically analyzed using oneway anova(Tumor growth)And contingency tables(Tumor necrosis, complete tumor regression)Analyzed by chisquare test and fisher's exact test, using graphpad prism software.

    Histologyfor immunohistologicAl stAining,Cryosections(7 on microslides were fixed inCold Acetone.Endogenous peroxidAse wAs quenched By incuBAting the slides in hydrogen peroxide(SigmA, st.Louis, mo;0.03% in PBS), slides were Blocked with 1% BSA for 15 minutes, with 10% goAt serum for 15 minutes, And suBsequently with DAKO Biotin Blocking System(DAko,CArpinterA,CA).Following incuBAtion with either rAt Anti mAcrophAge f4/80 mAB(CloneCl:A31, 10 serotec, oxford, uk)Or rAt AntiCd11B mAB(Clone m1/70, 6.25 PhArmingen, SAn Diego,CA), detection wAs performed with diAminoBenzidine(DAB)Using A Anti igg hrp detection kit(PhArmingen)According to the mAnufActurer's instructions.HemAtoxylin(Merck, ViennA, AustriA)StAining wAs done using A stAndArd procedure.Slides were mounted with entellAn new medium(Merck)And evAluAted under An olympus(ViennA, AustriA)Bh2 microscope equipped with An olympus dp12CAmerA.An unspecific expression profile with high luciferAse expression in the lungs And other mAjor orgAns wAs oBserved with pei22/dnAComplexes(Fig 1A).InContrAst, surfAceshielded tfComplexes resulted in specific expression in the tumor, whereAs expression in the lungs And other orgAns wAs drAmAticAlly reduced(Fig 1B).The kinetics of luciferAse reporter gene expression wAs meAsured following single orRepeAted ApplicAtionof surfAceshielded tfComplexes in the tAil vein of tumorBeAring mice.Gene expression wAs trAnsient, with the single ApplicAtion showing A peAk At 48 hours, thereAfter decreAsing to very low expression levels At dAy 4, And noLuciferase activitywAs detectABle At dAy 7.RepeAted ApplicAtion(dAys0 And 1)Resulted in higher expression levels At All time points, And significAntLuciferase activitywAs still detectABle in the tumor 1 week After ApplicAtion(Fig 1c).NontArgeted pei22/dnA(A)Or targeted tfComplexes(B)Containing the pcmvl plasmid were injected into the tail vein of neuro2a tumorbearing mice.Luciferase activity(Rlu/organ;Mean was measured 24 hours after injection.C: Kinetics of gene expression After systemic ApplicAtion of tArgeted TfComplexes. TrAnsfectionComplexesContAining 50 of DNA were injected once (dAy0)Or twice(dAys0 And 1).Luciferase reporter gene expression was measured at indicated time points.Animal number was n per time point.

    Full figure and legend(328K)

    Localization of tnf beats by dre sale online activity to the tumorthe aim of this study was to target gene expression of the highly potent cytokine tnf to the tumor in order to reduce the systemic toxicity observed with systemic application of tnf using nontargeted pei22/dna[tnf complexes resulted in an unspecific gene expression pattern with highest tnf expression detected in the lungs, followed by the liver, heart, and tumor.Moreover, application of pei22/dna[tnf complexes was associated with significant tnf serum levels(Fig 2a).In control animals receiving similar pei22/dna complexes with the reporter gene instead of the tnf gene, no significant tnf levels were found(Data not shown).Lower tnf levels were found in liver and spleen.Importantly, no significant tnf levels were observed in the serum(Fig 2b).In comparison, tnf expression after application of nonshielded pei22/dna[tnf complexes was even shorter with peak levels in the lungs and serum 24hours after application, followed by a rapid decrease(Fig 2a, insert).A/j mice bearing a subcutaneously growing neuro2a tumor were injected with nontargeted(A, c, e)Or targeted(B, d, f)Transfection complexes containing a plasmid for mutnf or the nonexpressing plasmid psp65(C expression of murine tnf(Pg/organ)In the major organs and tumor, and tnf serum levels(Pg/ml)24 hours after single application were measured by ELISA(A, b, inserts:48 hours after single application).Body weight(C, d)And tumor size(E, f)Of animals(Eight per group)Treated with repeated application(Arrow heads)Of transfection complexes were monitored.Untreated animals are shown for control.Complexes were applied repeatedly at indicated time points into the tail vein.Control animals were left untreated or injected with complexes having the empty plasmid vector psp65.Body weightof the animals, as an indicator of TNF toxicity, was monitored during treatment (Fig 2, c and d). A significant drop inBody weightof 10% was found with nonshielded PEI/DNA[TNF complexes after the first two applications (P tnf versus control) (Fig 2c).There was no further weight loss after the following applications;However, in one out of two experiments, nonshielded pei/dna [tnf complexes resulted in the death of 2/8 animals.Nontargeted pei/dna[tnf complexes also inhibited tumor growth(P tnf versus untreated control) (Fig 2e), however, to a lesser extent compared to the targeted complexes(P targeted tnf versus nontargeted tnf complexes with the psp65 control plasmid did not significantly inhibit tumor growth;Similar results were observed with additional control complexes with the reporter gene(Data not shown).

    The most striking effect of the tnf gene therApy wAs theInduction of hemorrhagic tumor necrosis(Fig 3, A And B).More thAn 80% of the tnf AnimAlsDeveloped hemorrhAgic tumor necroses, whereAs hemorrhAgic tumor necrosis wAs virtuAlly not found inControl AnimAls(P tnf versusControl)And only rArely found in psp65 or AnimAls(P tnf versus or psp65) (Fig 3c).Necrosis wAs most pronounced in theCenter of the tumor.However, there wAs only in very fewCAses AComplete erAdicAtion of the tumor in this model.AfterCessAtion of treAtment, tumorCells locAted outside of the necrotic AreAs stArted to regrow.The therApeutic efficAcy of tArgeted tnf geneDeliveryCorrelAted with the numBer of ApplicAtions.Preexperiments on A smAll numBer of AnimAls with single ApplicAtion showed only moderAte therApeutic effect And high vAriABility Among the AnimAls not Allowing significAntConclusions(DAtA not shown).ToCompAre the therApeutic efficAcy ofDifferent treAtment regimes, the t/c rAtio(Tumor growth in treAted groupCompAred toControl group)WAs meAsured AfterDifferent numBers of ApplicAtions.A rAtio of t/c is generAllyConsidered indicAtive for A significAnt therApeutic effect.Four ApplicAtions of tnf gene therApy showed A significAnt therApeutic effect with A rAtio of t/c however, there wAs A vAriABility in the treAtment group.Six or eight ApplicAtions of tArgeted tnf gene trAnsferComplexes resulted in A t/c rAtio of 25% or 13%, respectively, indicAting higher efficAcy with higher numBer of ApplicAtions.Furthermore, increAsing the numBer of ApplicAtion Also minimized the vAriABility Among the AnimAls in the treAtment groups(Fig 3d).Further experimentsDemonstrAted thAt hemorrhAgic tumor necrosis And inhiBition of tumor growth were induced only By trAnsfectionComplexesCoding for tnf whereAsCytokines with A more indirect immunemediAted mechAnism of Action, such As interleukin2 or interferonDid not show significAnt efficAcy in this Aggressive tumor model(DAtA not shown).Neuro2A tumorBeAring A/j mice were injected with tArgeted trAnsfectionComplexesContAining expression plAsmids for(A)Mutnf or(B)C:Summary table.Induction of hemorrhagic tumor necrosis.Results of three independent experiments are shown. TNF versus pSP65, AndControl.D: TherApeutic efficAcy of TNF gene therApy AfterDifferent treAtment regimes (Four, six, or eight applications)Was estimated by the t/c ratio(tumor growth group versusControl group) AtDAy 21.

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