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. 2000 Sep;106(6):763-71.
doi: 10.1172/JCI9180.

Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen

T Kurihara  1 , D E Brough, I Kovesdi, D W Kufe
Affiliations

Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen

T Kurihara et al. J Clin Invest. 2000 Sep.

Abstract

The DF3/MUC1 gene is aberrantly overexpressed in human breast and other carcinomas. Previous studies have demonstrated that the DF3/MUC1 promoter/enhancer confers selective expression of diverse transgenes in MUC1-positive breast cancer cells. In this study, we show that an adenoviral vector (Ad.DF3-E1) in which the DF3/MUC1 promoter drives expression of E1A selectively replicates in MUC1-positive breast cancer cells. We also show that Ad.DF3-E1 infection of human breast tumor xenografts in nude mice is associated with inhibition of tumor growth. In contrast to a replication-incompetent adenoviral vector that infects along the injection track, Ad.DF3-E1 infection was detectable throughout the tumor xenografts. To generate an Ad.DF3-E1 vector with the capacity for incorporating therapeutic products, we inserted the cytomegalovirus (CMV) promoter upstream of the TNF cDNA. Infection with Ad.DF3-E1/CMV-TNF was associated with selective replication and production of TNF in cells that express MUC1. Moreover, treatment of MUC1-positive, but not MUC1-negative, xenografts with a single injection of Ad.DF3-E1/CMV-TNF was effective in inducing stable tumor regression. These findings demonstrate that the DF3/MUC1 promoter confers competence for selective replication of Ad.DF3-E1 in MUC1-positive breast tumor cells, and that the antitumor activity of this vector is potentiated by integration of the TNF cDNA.

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Figures

Figure 1
Figure 1
Structure of Ad.DF3-E1. ITR, inverted terminal repeat; m.u., map unit.
Figure 2
Figure 2
Selective expression of E1A in MUC1-positive cells infected with Ad.DF3-E1. (a) The indicated cells were incubated with either mAb DF3 (open area) or an isotype-identical control Ab (shaded area) and then subjected to flow cytometric analysis. (b) Cells were infected with Ad.DF3-E1, Ad.CMV–β-gal, or wild-type Ad5. Lysates from control (noninfected) and infected cells were subjected to immunoblot analysis with anti-E1A Ab. (c) Lysates from Ad.DF3-E1–infected cells were subjected to immunoblot analysis with anti-E1B.
Figure 3
Figure 3
Replication efficiency of Ad.DF3-E1 (open circles), Ad.DF3-E1/CMV (open triangles), Ad.DF3-E1/CMV-TNF (filled diamonds), and wild-type Ad5 (open squares) in human cell lines. Monolayers in 24-well plates were infected at an moi of 1.0 pfu per cell. Virus production was assessed by plaque assays.
Figure 4
Figure 4
Cytopathic effects associated with Ad.DF3-E1 infection. MCF-7, PA-1, and Hs578Bst cells were infected with either Ad.DF3-E1 or wild-type Ad5 at the indicated moi. Photomicrographs were obtained at the indicated times after infection. ×200.
Figure 5
Figure 5
Effects of Ad.DF3-E1 on growth of MCF-7 and MDA-MB-231 tumor xenografts in nude mice. MCF-7 (a) or MDA-MB-231 (b) tumor xenografts were grown subcutaneously to volumes of 150–200 mm3. Groups of mice (n = 5) were treated with 2 × 108 pfu of Ad.DF3-E1 (filled diamonds) or Ad.DF3–β-gal (filled squares) by intratumoral injection on day 0. An equal volume of PBS was injected as a control (open squares). Tumors were measured weekly. The results are expressed as fractional tumor volume (V/V0). MCF-7 tumors infected with Ad.DF3-E1 were significantly smaller than those treated with PBS or Ad.DF3–β-gal at day 21 (P < 0.001), day 28 (P < 0.001), and day 35 (P < 0.01).
Figure 6
Figure 6
Characterization and intratumoral distribution of Ad.DF3-E1/CMV-GFP. (a) Structure of Ad.DF3-E1/CMV-GFP. (b) MCF-7 cells and PA-1 cells were infected with Ad.DF3-E1/CMV-GFP at an moi of 10. GFP expression was assessed by photomicrographic examination at 36 hours and 72 hours after infection. (c) MCF-7 tumor xenografts (150 mm3) were injected with 2 × 108 pfu of Ad.DF3–β-gal (upper panel) or Ad.DF3-E1/CMV-GFP (lower panel). At 21 days after injection, the tumors were removed, embedded in OCT (Tissue-Tek; Sakura Finetek USAInc., Torrance, California, USA), and frozen on dry ice. They were then cryosectioned with a microtome. Sections were fixed in 0.5% glutaraldehyde and stained with X-gal (upper panel; ×200). Sections shown in lower panel were visualized by STORM (Molecular Dynamics, Sunnyvale, California, USA).
Figure 7
Figure 7
Characterization of Ad.DF3-E1/CMV-TNF. (a) Structure of Ad.DF3-E1/CMV-TNF. (b) Cells were infected with Ad.DF3-E1 or Ad.DF3-E1/CMV-TNF. Lysates were subjected to immunoblot analysis with anti-E1A.
Figure 8
Figure 8
Antitumor effects of Ad.DF3-E1/CMV-TNF. MDA-MB-231 and MCF-7 tumor xenografts were grown subcutaneously in nude mice to volumes of 150–200 mm3. (a) Groups of mice (n = 5) bearing MDA-MB-231 tumors were injected intratumorally with PBS (open squares) or 108 pfu of Ad.DF3-E1/CMV-TNF (filled diamonds) on day 0. (b) Groups of mice (n = 5) bearing MCF-7 tumors were injected intratumorally with PBS (open squares), 108 pfu of Ad.CMV-TNF (filled squares), 108 pfu of Ad.DF3-E1/CMV-TNF (filled diamonds), or 108 pfu of Ad.DF3-E1/CMV-GFP (open triangles) on day 0. (c) Mice bearing MCF-7 tumors injected with Ad.DF3-E1/CMV-TNF were followed for more than 56 days. One mouse died on day 52 without any tumors. Of the remaining four mice, one exhibited tumor regrowth (open circles). This mouse was reinjected with 108 pfu of Ad.DF3-E1/CMV-TNF on day 105 (arrow). The results are expressed as fractional tumor volume (V/V0). Differences among the MDA-MB-231 treatment groups were not significant. MCF-7 tumors infected with Ad.DF3-E1/CMV-TNF were significantly smaller at day 35 than were those treated with Ad.CMV-TNF (P < 0.001) or Ad.DF3-E1/CMV-GFP (P < 0.01).
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References

    1. Haj-Ahmad Y, Graham F. Characterization of an adenovirus type 5 mutant carrying embedded inverted terminal repeats. Virology. 1986;153:22–34. - PubMed
    1. Bett A, Haddara W, Prevec L, Graham F. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci USA. 1994;91:8802–8806. - PMC - PubMed
    1. Heise C, Kirn DH. Replication-selective adenoviruses as oncolytic agents. J Clin Invest. 2000;105:847–851. - PMC - PubMed
    1. Rodriguez R, et al. Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res. 1997;57:2559–2563. - PubMed
    1. Miyatake S-I, et al. Hepatoma-specific antitumor activity of an albumin enhancer/promoter regulated herpes simplex virus in vivo. Gene Ther. 1999;6:564–572. - PubMed

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