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Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent.

Kirscher L, Deán-Ben XL, Scadeng M, Zaremba A, Zhang Q, Kober C, Fehm TF, Razansky D, Ntziachristos V, Stritzker J, Szalay AA - Theranostics (2015)

Bottom Line: In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models.Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography.At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

View Article: PubMed Central - PubMed

Affiliation: 1. University of Würzburg, Department of Biochemistry, Am Hubland, 97074 Würzburg, Germany.

ABSTRACT
We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

No MeSH data available.


Related in: MedlinePlus

rVACV mediated expression of proteins in A549 cancer cells. A) Map of relevant insertions into the genome of the different rVACV strains. B) Western blot analysis of viral marker (TetR, RFP, GusA, Ruc-GFP, mTyr) expression in rVACV infected A549 lung carcinoma cells in presence or absence of dox.
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Figure 1: rVACV mediated expression of proteins in A549 cancer cells. A) Map of relevant insertions into the genome of the different rVACV strains. B) Western blot analysis of viral marker (TetR, RFP, GusA, Ruc-GFP, mTyr) expression in rVACV infected A549 lung carcinoma cells in presence or absence of dox.

Mentions: The vaccinia virus strains GLV-1h312, GLV-1h460 and GLV-1h462 (Fig. 1A) were tested for expression of the inserted marker genes. For this, A549 lung carcinoma cells were infected with these different rVACVs using an MOI of 0.1. Protein samples were collected after an incubation time of 24h in presence or absence of doxycycline (1 µg/ml) and analyzed by Western blot (Fig. 1B). Human beta-actin which served as a loading control was detected in each lysate. As expected, expression of the 25 kDa Tet-repressor could be detected in GLV-1h312 and GLV-1h462 infected cells, in the presence or absence of doxycycline (dox), while Ruc-GFP (64 kDa) was found only in GLV-1h460 infected A549 cells (Fig. 1B). The functionality of the tet-system was confirmed as well. Expression of regulated genes could only be detected in the presence of dox, when the conformational change of TetR resulted in dissociation from the tet-operator allowing transcription of the viral promoter regulated expression cassette by the viral RNA-polymerase. The mRFP1 molecule (30 kDa) inserted into the TK locus of GLV-1h312, was detected solely in GLV-1h312 infected A549 cancer cells in presence of dox. GLV-1h462 infected A549 cells showed mTyr (70 kDa) expression in presence of dox but not when the inducer was absent. Lung carcinoma cells infected with GLV-1h460, which does not encode tetR, produced mTyr both in the absence or presence of dox. All three rVACV strains lead to expression of GusA, the E. coli beta-glucuronidase (60 kDa), inserted in the viral HA locus (Fig. 1A) in presence or absence of dox (Fig. 1B). Mock infected A549 cells served as negative control for the virus encoded marker proteins.


Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent.

Kirscher L, Deán-Ben XL, Scadeng M, Zaremba A, Zhang Q, Kober C, Fehm TF, Razansky D, Ntziachristos V, Stritzker J, Szalay AA - Theranostics (2015)

rVACV mediated expression of proteins in A549 cancer cells. A) Map of relevant insertions into the genome of the different rVACV strains. B) Western blot analysis of viral marker (TetR, RFP, GusA, Ruc-GFP, mTyr) expression in rVACV infected A549 lung carcinoma cells in presence or absence of dox.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4508495&req=5

Figure 1: rVACV mediated expression of proteins in A549 cancer cells. A) Map of relevant insertions into the genome of the different rVACV strains. B) Western blot analysis of viral marker (TetR, RFP, GusA, Ruc-GFP, mTyr) expression in rVACV infected A549 lung carcinoma cells in presence or absence of dox.
Mentions: The vaccinia virus strains GLV-1h312, GLV-1h460 and GLV-1h462 (Fig. 1A) were tested for expression of the inserted marker genes. For this, A549 lung carcinoma cells were infected with these different rVACVs using an MOI of 0.1. Protein samples were collected after an incubation time of 24h in presence or absence of doxycycline (1 µg/ml) and analyzed by Western blot (Fig. 1B). Human beta-actin which served as a loading control was detected in each lysate. As expected, expression of the 25 kDa Tet-repressor could be detected in GLV-1h312 and GLV-1h462 infected cells, in the presence or absence of doxycycline (dox), while Ruc-GFP (64 kDa) was found only in GLV-1h460 infected A549 cells (Fig. 1B). The functionality of the tet-system was confirmed as well. Expression of regulated genes could only be detected in the presence of dox, when the conformational change of TetR resulted in dissociation from the tet-operator allowing transcription of the viral promoter regulated expression cassette by the viral RNA-polymerase. The mRFP1 molecule (30 kDa) inserted into the TK locus of GLV-1h312, was detected solely in GLV-1h312 infected A549 cancer cells in presence of dox. GLV-1h462 infected A549 cells showed mTyr (70 kDa) expression in presence of dox but not when the inducer was absent. Lung carcinoma cells infected with GLV-1h460, which does not encode tetR, produced mTyr both in the absence or presence of dox. All three rVACV strains lead to expression of GusA, the E. coli beta-glucuronidase (60 kDa), inserted in the viral HA locus (Fig. 1A) in presence or absence of dox (Fig. 1B). Mock infected A549 cells served as negative control for the virus encoded marker proteins.

Bottom Line: In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models.Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography.At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

View Article: PubMed Central - PubMed

Affiliation: 1. University of Würzburg, Department of Biochemistry, Am Hubland, 97074 Würzburg, Germany.

ABSTRACT
We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

No MeSH data available.


Related in: MedlinePlus