Selective killing of cancer cells through translational regulation of the HTK/GCV gene therapy system

RJ DeFatta, PhDa, RP Chervenak, PhD b, Y Li, MS a,and A De Benedetti, PhD a

a Department of Biochemistry & Molecular Biology and the Feist-Weiller Cancer Center, b Department of Microbiology and Immunology, Louisiana State University Health Sciences Center. 1501 Kings Highway. Shreveport, LA 71130-3932, USA

AIM: Design a protocol permitting primary tumor and distant metastases killing while sparing normal cells by exploiting elevated levels of translation initiation factor/protooncogene eIF4E in cancer cells. METHODS: Herpes thymidine kinase (TK) cDNA was cloned into the BK-Shuttle vector (BK-TK). A hairpin structure that inhibits translation in low eIF4E-expressing cells (normal) was cloned upstream of TK (BK-UTK vector). eIF4E was overexpressed from a cDNA cloned into pREP7 (eIF4E). Vectors were stabily transfected into normal and tumorigenic mouse mammary cell lines. Western blots were performed to determine if altering eIF4E expression impacted translation of UTK mRNA, and thus, gancyclovir (GCV) sensitivity. Tumor regression was determined following systemic GCV administration in mice injected subcutaneously with BK-TK- or BK-UTK-transfected cells. Alternatively, DNA vectors were injected directly into preformed tumors. Effects on metastatic tumor burden, survival, and vector toxicity following GCV treatment were also determined. RESULTS: Efficient TK expression was seen in normal cells transfected with the BK-TK vector but only minimal expression was seen from the BK-UTK vector. Co-expressing eIF4E greatly enhanced the synthesis of TK protein from BK-UTK. BK-TK- and BK-UTK-transfected cancer cells showed similar, robust TK protein expression. Consistent with the TK expression pattern, cancer cells expressing BK-UTK were killed at 2,000-fold lower GCV concentration than normal cells expressing BK-UTK. The decreased sensitivity of normal cells was overcome by increasing the eIF4E expression level. While both vectors were equally effective in reducing subcutaneous tumors and lung metastases following GCV administration, systemic BK-TK delivery in mice was toxic, resulting in severe weight loss, organ degeneration, and early death. BK-UTK statistically increased mean survival without toxicity. CONCLUSIONS: These results provide compelling evidence that translational regulation of suicide gene expression may constitute a viable experimental approach to cancer gene therapy.

KEY WORDS: Gene therapy, suicide gene regulation, and translational control.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Gene Therapy, Part 2.