Gene targeting approach to selectively kill colon cancer cells, with hyperactive K-Ras pathway.
BACKGROUND
Ras mutations are present in approximately 50% of human colorectal tumors. We have previously shown that transfection of a non-tumorigenic rat intestinal epithelial cell line, IEC18, by the K-Ras oncogene (R1 cells), resulted in malignant cell transformation. Utilizing the constantly active Ras signaling pathway to selectively target transformed but not normal cells is a plausible goal.


OBJECTIVE
To selectively kill Ras transformed cells by over expressing a lethal gene using a Ras-responsive promoter.


METHODS
IEC18, R1 and a number of colon cancer cell lines were transfected with luciferase (Luc) reporter gene under the control of different Ras-responsive elements. The Ras-responsive promoter Py2 contains two copies of adjacent Ets and AP I binding sites followed by a minimal promoter. Apoptotic genes (bax, caspase-8 and PKG) were cloned into the Py2 plasmids. RI cells co-transfected with expression constructs and a selected vector and then grown for 3 weeks under selection.


RESULTS
R1, SW480 and HCT116 with mutated c-K-Ras expressed high level of Luc activity following transfection with the Py2 element. IEC18 cell lines that do not contain this mutation expressed negligible low Luc activity. Following transfection of SW480 and R1 cells with Py2-bax, caspase-8 and PKG, there was a significant reduction in the number of colony formation.


CONCLUSIONS
1. Selective over-expression of pro-apoptotic genes, inhibits the growth of Ras transformed cells, and not normal cells. 2. This gene approach therapy may become a useful, effective and safe to target Ras mutated tumor cells with sparing of the normal cells.
Dvory-Sobol
H
H
Department of Cancer Prevention, Integrated Cancer Prevention Center, Tel Aviv Medical Center, Israel.
Kazanov
D
D
Arber
N
N
D017209:Apoptosis / Q000235:genetics; D053181:Caspase 8; D020169:Caspases / Q000235:genetics; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000628:therapy*; D006152:Cyclic GMP / Q000502:physiology; D018390:Gene Targeting; D017930:Genes, Reporter / Q000235:genetics; D011905:Genes, ras / Q000235:genetics*; D015316:Genetic Therapy; D006801:Humans; D008156:Luciferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection; D014410:Tumor Stem Cell Assay; D051028:bcl-2-Associated X Protein / Q000235:genetics
D051028:bcl-2-Associated X Protein; D008156:Luciferases; C505530:CASP8 protein, human; C505528:Casp8 protein, rat; D053181:Caspase 8; D020169:Caspases; D006152:Cyclic GMP