Experimental gene therapy of human colon cancer

Abstract

Background: Gastrin regulates growth of human colon cancer cells by activation of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA). Gastrin and 8-Br-cAMP, a membrane-permeable cAMP analog, inhibit growth of HCT116 cells; both stimulate growth of LoVo cells. This dual effect on growth may be explained by relative amounts of the regulatory subunit (RI alpha or RII beta) of PKA within the cancer cells. Antisense oligodeoxynucleotides (ASO) to either RI alpha or RII beta inhibit protein translation of the target mRNA by sequence-specific binding; subsequently, cellular PKA content and the cAMP-mediated growth may be altered. We determined whether ASO to either the RI alpha or RII beta subunit altered the cAMP-mediated growth of HCT116 and LoVo human colon cancer cells.

Methods: HCT116 cells were treated with RII beta ASO (15 mumol/L, 4 days) and then treated with 8-Br-cAMP (25 mumol/L); tritiated thymidine incorporation was measured after 24 hours, and the cell number was determined on alternate days. Protein and mRNA levels of the RII beta subunit were determined by Western and Northern blotting, respectively. Similar studies with an ASO against the RI alpha subunit were performed on LoVo cells.

Results: RII beta ASO reversed the cAMP-mediated inhibition of growth of HCT116 cells, and RII beta ASO decreased the protein level of the RII beta subunit. RII beta ASO did not alter the basal growth of HCT116 cells. RI alpha ASO reversed the cAMP-mediated stimulation of growth of LoVo cells.

Conclusions: The regulatory subunits of PKA are potential targets to alter growth of human colon cancer cells. Gene therapy directed to alter specific steps in signal transduction pathways may provide new therapeutic strategies.