LACK OF BYSTANDER KILLING IN HERPES-SIMPLEX VIRUS THYMIDINE KINASE-TRANSDUCED COLON CELL-LINES DUE TO DEFICIENT CONNEXIN43 GAP JUNCTION FORMATION

The efficacy of herpes simplex virus thymidine kinase (HSV-TK) gene th erapy for colorectal carcinoma has been investigated in an in vitro sy stem, The magnitude and the mechanism of the HSV-TK bystander effect w as determined in three human colon tumor cell lines: HCT-116, HCT-8, a nd HT-29. Each HSV-TK(+) cell line was generated by stable transductio n with a bicistronic retroviral vector containing the HSV-TK and neomy cin resistance (neo) genes; each exhibited an IC50 for GCV of less tha n or equal to 4 mu M, When GCV was added to HSV-TK(+) cells mixed with parental cells or known bystander-positive cell lines, no bystander k illing was evident in the HT-29 or HCT-8 cells. Western blots detected the expression of the gap junction protein connexin43 (Cx43) in HCT-8 and HT-29 cells; however, immunolocalization studies indicated predom inantly cytoplasmic staining of Cx43 and no cell surface staining in t hese cell lines, Stable transfection of HCT-8 and MT-29 cells with Cx4 3 resulted in increased levels of Cx43 expression with the same subcel lular distribution as before, yet there was again no apparent bystande r killing, Tn contrast, Cx43 expression was localized to the cell surf ace in the bystander-positive colon tumor cell line HCT-116. These res ults demonstrate that expression and proper surface localization of Cx 43 gap junctions are necessary components of the bystander effect in h uman colon tumor cells, They also indicate that future combination gen e therapy approaches using coexpression of HSV-TK and Cx43 genes may n ot be applicable to all tumor systems.