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Mouse mammary tumor virus promoter-containing retroviral promoter conversion vectors for gene-directed enzyme prodrug therapy are functional in vitro and in vivo.

Klein R, Ruttkowski B, Schwab S, Peterbauer T, Salmons B, Günzburg WH, Hohenadl C - J. Biomed. Biotechnol. (2008)

Bottom Line: The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals.The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA.Determination of the respective IC(50) values demonstrated that the effective IFA dose was reduced by sixteen folds.

View Article: PubMed Central - PubMed

Affiliation: Austrianova Biotechnology GmbH, Veterinärplatz 1, A-1210 Vienna, Austria. reinhard.klein@ccri.at

ABSTRACT
Gene directed-enzyme prodrug therapy (GDEPT) is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1) metabolizes the prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon) vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC(50) values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

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In vitro infection of T-47D/DsRed and CRFK/DsRed cells with pPCEMm1. (a)FACS analysis of CRFK/DsRed and T-47D/DsRed cells cocultivated with 2GP19Talf cells stably transfectedwith pPCEMm1 over a time period of five weeks. The numbers of red, green, andred/green fluorescent cells of a representative experiment are shown. As negative controls, 2GP19Talf, T-47D, and CRFK cells were used. Aspositive controls, pure 2GP19Talf/pPCEMm1, T-47D/DsRed, and CRFK/DsRed cellswere used. (b) Confocal laser-scanning microscopy of CRFK/DsRedcells cocultivated with 293/pPCEMm1 cells over a time period of five weeks.Upper left (UL), green fluorescence; upper right (UR), transmission; lower left (LL), redfluorescence; lower right (LR), UL + LL. Magnification 400 x. (c) Confocal laser-scanningmicroscopy of CRFK/DsRed cells cocultivated with 2GP19Talf/pPCEMm1cells over atime period of five weeks. Upper left (UL), green fluorescence; upper right (UR), red fluorescence; lower left (LL), transmission;lower right (LR), UL + UR. Magnification 400 x.
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fig2: In vitro infection of T-47D/DsRed and CRFK/DsRed cells with pPCEMm1. (a)FACS analysis of CRFK/DsRed and T-47D/DsRed cells cocultivated with 2GP19Talf cells stably transfectedwith pPCEMm1 over a time period of five weeks. The numbers of red, green, andred/green fluorescent cells of a representative experiment are shown. As negative controls, 2GP19Talf, T-47D, and CRFK cells were used. Aspositive controls, pure 2GP19Talf/pPCEMm1, T-47D/DsRed, and CRFK/DsRed cellswere used. (b) Confocal laser-scanning microscopy of CRFK/DsRedcells cocultivated with 293/pPCEMm1 cells over a time period of five weeks.Upper left (UL), green fluorescence; upper right (UR), transmission; lower left (LL), redfluorescence; lower right (LR), UL + LL. Magnification 400 x. (c) Confocal laser-scanningmicroscopy of CRFK/DsRed cells cocultivated with 2GP19Talf/pPCEMm1cells over atime period of five weeks. Upper left (UL), green fluorescence; upper right (UR), red fluorescence; lower left (LL), transmission;lower right (LR), UL + UR. Magnification 400 x.

Mentions: For in vitro infection studies, 2GP19Talf virus-producing cells that had stably been transfected with pPCEMm1were mixed in ratios of 1 : 10 or 1 : 5 with red fluorescent human T-47D breastcancer cells and as a control with red fluorescent feline CRFK kidney cellsthat are known to effectively being infected by retroviruses. Red fluorescence of target cells wasachieved by stably transfecting the cells with the DsRed protein-encodingvector pCMV-dsRed-Express and enrichment of red fluorescentcells was performed by cell sorting. Mixed cells werecocultivated for five weeks and the numbers of green, red, and green/red cellswere monitored by FACS (see Figure 2(a)). After five weeks, about 74% of redfluorescent T-47-D cells were also green fluorescent, indicating that they hadbeen infected with pPCEMm1. The initial ratio between virus-producing cells and target cells did notplay a role in this setting. The red fluorescent CRFKcells were infected to an extent of about 19% (ratio 1 : 10 between virus-producingcells and target cells) and 40% (ratio 1 : 5 between virus-producing cells andtarget cells). In contrast, when green fluorescent nonvirus-producing 293 cells stably infected with pPCEMm1 wheremixed with red fluorescent T-47D or CRFK cells, only a very low number (<1%) of false-positive red/green cells were detected by FACS after six weeks ofcocultivation (data not shown). The obtained results were also verified by confocal laser-scanningmicroscopy: no red/green double fluorescent cells could be detected asexemplified in Figure 2(b) for a mix of 293 cells stably infected with pPCEMm1and CRFK/DsRed cells. In contrast, when the cell mixesof cocultivated red fluorescent T-47D or CRFK cells and virus-producing greenfluorescent cells were analyzed, clearly a number of truly red/green doublefluorescent cells could be detected as demonstrated for a mix of CRFK/DsRed and2GP19Talf/pPCEMm1cells (see Figure 2(c)). Double fluorescence was not due tored autofluorescence of 2GP19Talf/pPCEMm1 or green autofluorescence ofCRFK/pPCEMm1 and T-47D/pPCEMm1 cells since no red fluorescent cells could bedetected in samples only consisting of green fluorescent 2GP19Talf/pPCEMm1cells and no green fluorescence could be detected in samples of red fluorescentCRFK/DsRed and T-47D/DsRed cells (data not shown).


Mouse mammary tumor virus promoter-containing retroviral promoter conversion vectors for gene-directed enzyme prodrug therapy are functional in vitro and in vivo.

Klein R, Ruttkowski B, Schwab S, Peterbauer T, Salmons B, Günzburg WH, Hohenadl C - J. Biomed. Biotechnol. (2008)

In vitro infection of T-47D/DsRed and CRFK/DsRed cells with pPCEMm1. (a)FACS analysis of CRFK/DsRed and T-47D/DsRed cells cocultivated with 2GP19Talf cells stably transfectedwith pPCEMm1 over a time period of five weeks. The numbers of red, green, andred/green fluorescent cells of a representative experiment are shown. As negative controls, 2GP19Talf, T-47D, and CRFK cells were used. Aspositive controls, pure 2GP19Talf/pPCEMm1, T-47D/DsRed, and CRFK/DsRed cellswere used. (b) Confocal laser-scanning microscopy of CRFK/DsRedcells cocultivated with 293/pPCEMm1 cells over a time period of five weeks.Upper left (UL), green fluorescence; upper right (UR), transmission; lower left (LL), redfluorescence; lower right (LR), UL + LL. Magnification 400 x. (c) Confocal laser-scanningmicroscopy of CRFK/DsRed cells cocultivated with 2GP19Talf/pPCEMm1cells over atime period of five weeks. Upper left (UL), green fluorescence; upper right (UR), red fluorescence; lower left (LL), transmission;lower right (LR), UL + UR. Magnification 400 x.
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fig2: In vitro infection of T-47D/DsRed and CRFK/DsRed cells with pPCEMm1. (a)FACS analysis of CRFK/DsRed and T-47D/DsRed cells cocultivated with 2GP19Talf cells stably transfectedwith pPCEMm1 over a time period of five weeks. The numbers of red, green, andred/green fluorescent cells of a representative experiment are shown. As negative controls, 2GP19Talf, T-47D, and CRFK cells were used. Aspositive controls, pure 2GP19Talf/pPCEMm1, T-47D/DsRed, and CRFK/DsRed cellswere used. (b) Confocal laser-scanning microscopy of CRFK/DsRedcells cocultivated with 293/pPCEMm1 cells over a time period of five weeks.Upper left (UL), green fluorescence; upper right (UR), transmission; lower left (LL), redfluorescence; lower right (LR), UL + LL. Magnification 400 x. (c) Confocal laser-scanningmicroscopy of CRFK/DsRed cells cocultivated with 2GP19Talf/pPCEMm1cells over atime period of five weeks. Upper left (UL), green fluorescence; upper right (UR), red fluorescence; lower left (LL), transmission;lower right (LR), UL + UR. Magnification 400 x.
Mentions: For in vitro infection studies, 2GP19Talf virus-producing cells that had stably been transfected with pPCEMm1were mixed in ratios of 1 : 10 or 1 : 5 with red fluorescent human T-47D breastcancer cells and as a control with red fluorescent feline CRFK kidney cellsthat are known to effectively being infected by retroviruses. Red fluorescence of target cells wasachieved by stably transfecting the cells with the DsRed protein-encodingvector pCMV-dsRed-Express and enrichment of red fluorescentcells was performed by cell sorting. Mixed cells werecocultivated for five weeks and the numbers of green, red, and green/red cellswere monitored by FACS (see Figure 2(a)). After five weeks, about 74% of redfluorescent T-47-D cells were also green fluorescent, indicating that they hadbeen infected with pPCEMm1. The initial ratio between virus-producing cells and target cells did notplay a role in this setting. The red fluorescent CRFKcells were infected to an extent of about 19% (ratio 1 : 10 between virus-producingcells and target cells) and 40% (ratio 1 : 5 between virus-producing cells andtarget cells). In contrast, when green fluorescent nonvirus-producing 293 cells stably infected with pPCEMm1 wheremixed with red fluorescent T-47D or CRFK cells, only a very low number (<1%) of false-positive red/green cells were detected by FACS after six weeks ofcocultivation (data not shown). The obtained results were also verified by confocal laser-scanningmicroscopy: no red/green double fluorescent cells could be detected asexemplified in Figure 2(b) for a mix of 293 cells stably infected with pPCEMm1and CRFK/DsRed cells. In contrast, when the cell mixesof cocultivated red fluorescent T-47D or CRFK cells and virus-producing greenfluorescent cells were analyzed, clearly a number of truly red/green doublefluorescent cells could be detected as demonstrated for a mix of CRFK/DsRed and2GP19Talf/pPCEMm1cells (see Figure 2(c)). Double fluorescence was not due tored autofluorescence of 2GP19Talf/pPCEMm1 or green autofluorescence ofCRFK/pPCEMm1 and T-47D/pPCEMm1 cells since no red fluorescent cells could bedetected in samples only consisting of green fluorescent 2GP19Talf/pPCEMm1cells and no green fluorescence could be detected in samples of red fluorescentCRFK/DsRed and T-47D/DsRed cells (data not shown).

Bottom Line: The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals.The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA.Determination of the respective IC(50) values demonstrated that the effective IFA dose was reduced by sixteen folds.

View Article: PubMed Central - PubMed

Affiliation: Austrianova Biotechnology GmbH, Veterinärplatz 1, A-1210 Vienna, Austria. reinhard.klein@ccri.at

ABSTRACT
Gene directed-enzyme prodrug therapy (GDEPT) is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1) metabolizes the prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon) vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC(50) values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

Show MeSH
Related in: MedlinePlus