Gene therapy for sarcoma utilizing adenoviral transfer of the beta-glucuronidase and bax genes and an anthracyline prodrug.
BACKGROUND
When acted on by beta-glucuronidase (BG), HMR1826 is metabolized to doxorubicin. Use of this prodrug with adenoviral transfer of beta-glucuronidase (AdBG) is limited by the drug's inability to enter cells and intracellular retention of BG after transduction. We evaluated a system combining AdBG, transfer of the proapoptotic gene bax (AdBax) at a low multiplicity of infection, and HMR1826 administration.


METHODS
Fibrosarcoma cells were treated with AdBG alone, AdBG plus HMR1826, AdBG followed by beta-galactosidase (AdLacZ) plus HMR1826, and AdBG followed by AdBax with no prodrug. In the experimental group, cells were transfected with AdBG, followed by AdBax plus HMR1826. Viability was measured 24 h after transfection and prodrug administration. Western blots for BG were performed on cell lysates and supernatants.


RESULTS
Minimal cellular killing was noted in the AdBG alone, AdBG plus HMR 1826, or AdBG:AdLacZ plus HMR 1826 groups, and Western blot did not demonstrate BG in the supernatant even though all AdBG-transfected cell lysates were positive. Cell killing was noted in the AdBG:AdBax group, but less than in the AdBG:AdBax plus HMR 1826 group (without prodrug versus with prodrug: 1:1 to 55.5% versus 75.9%, 5:1 to 10.0% versus 75.9%, 10:1 7.6% versus 49.0%, 20:1 4.6% versus 24.9%, P = 0.037). Western blot demonstrated BG in the supernatant of the AdBG:AdBax groups.


CONCLUSIONS
We have devised a novel enzyme prodrug method of killing tumor cells and engendering a bystander effect. AdBax leads to BG release from dying cells after AdBG transduction and conversion of HMR1826 to an active anthracycline.
Miller
Steven D
SD
Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Mohiuddin
Imran
I
Cao
Xiaobo X
XX
Ozvaran
Mustafa K
MK
Daniel
Jonathan C
JC
Roy Smythe
W
W
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D018943:Anthracyclines / Q000627:therapeutic use; D000903:Antibiotics, Antineoplastic / Q000627:therapeutic use; D017209:Apoptosis / Q000235:genetics; D045744:Cell Line, Tumor; D003131:Combined Modality Therapy; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000627:therapeutic use*; D004354:Drug Screening Assays, Antitumor; D005354:Fibrosarcoma / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D005965:Glucuronates / Q000627:therapeutic use*; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D011355:Prodrugs / Q000627:therapeutic use*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D051381:Rats; D011916:Rats, Inbred F344; D051028:bcl-2-Associated X Protein
D018943:Anthracyclines; D000903:Antibiotics, Antineoplastic; C494828:Bax protein, rat; D005965:Glucuronates; C114198:N-(4-glucuronyl-3-nitrobenzyloxycarbonyl)doxorubicin; D011355:Prodrugs; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D004317:Doxorubicin; D005966:Glucuronidase