Rat adenocarcinoma cell line infected with an adenovirus carrying a novel herpes-simplex virus-thymidine kinase suicide gene construct dies by apoptosis upon treatment with ganciclovir.
BACKGROUND
Eukaryotic initiation factor 4E (eIF4E) facilitates the translation of mRNAs with long 5' untranslated regions and thus regulates protein synthesis. This protein has been found in elevated quantities in breast, colon, and head and neck cancers. To exploit this dysfunction, the 619 base pair 5' untranslated regions of fibroblast growth factor-2 was spliced upstream of the herpes simplex virus thymidine kinase gene in an adenovirus vector (Ad-HSV-UTK), with the expectation that TK will be expressed in cells that overexpress eIF4E and, thus, render these cells susceptible to ganciclovir. In this study, we investigated the in vitro activity of this suicide gene therapy against the rat Mat BIII breast adenocarcinoma cell line, and assessed whether apoptosis was the responsible mechanism of cell killing.


METHODS
Mat BIII cells were infected with Ad-HSV-UTK, and optimal multiplicity of infection was determined using green fluorescent protein tagged adenovirus. Western blot analysis was used to detect eIF4E and TK expression. Cell viability was assessed by the MTT assay. Induction of apoptosis was determined using annexin V-FITC and propidium iodine detection kit and a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling in situ cell death detection kit.


RESULTS
Western Blot analysis confirmed successful infection of the cell line. Marked cytotoxicity was noted by the MTT assay in the infected group with a 100-fold less concentration of ganciclovir compared with the control groups. Annexin V-FITC/propidium iodide revealed apoptosis in infected cells following treatment with ganciclovir.


CONCLUSIONS
Suicide gene therapy targeting the overexpression of eIF4E induces apoptosis and cell death in rat Mat BIII mammary adenocarcinoma cells.
Chu
Quyen D
QD
Department of Surgery, Louisiana State University Health Sciences Center and Feist-Weiller Cancer Center, Shreveport, Louisiana, USA. qchu@lsuhsc.edu
Sun
Lisa
L
Li
Jie
J
Byrnes
Kerry
K
Chervenak
Deborah
D
DeBenedetti
Arrigo
A
Mathis
J Michael
JM
Li
Benjamin D L
BD
D000230:Adenocarcinoma / Q000378:metabolism / Q000628:therapy*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D015774:Ganciclovir / Q000494:pharmacology* / Q000627:therapeutic use; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods; D006561:Herpes Simplex / Q000188:drug therapy; D015674:Mammary Neoplasms, Animal / Q000378:metabolism / Q000628:therapy*; D051381:Rats; D018139:Simplexvirus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism
D000998:Antiviral Agents; D039561:Eukaryotic Initiation Factor-4E; D005456:Fluorescent Dyes; D013937:Thymidine Kinase; D016650:Fluorescein-5-isothiocyanate; D015774:Ganciclovir