INHIBITION OF COLON-TUMOR CELL-GROWTH BY 8-CHLORO-CAMP IS DEPENDENT UPON ITS CONVERSION TO 8-CHLORO-ADENOSINE

Recent interest in site-selective cAMP analogs has focused on the role of 8-chloro-adenosine (8-Cl-adenosine) in the inhibition of tumor cell growth by 8-chloro-cAMP (8-Cl-cAMP) (Van Lookeren Campagne, et al. Cancer Res 1991; 51: 1600-5). We have evaluated 8-Cl-cAMP and 8-Cl-adenosine for their growth inhibitory activity against two human colon adenocarcinoma cell lines, HCT116 and FET. Because these cell lines have been adapted to grow in chemically defined medium we were able to evaluate the effect of serum on 8-Cl-cAMP's growth inhibitory activity. in addition, cells grown in serum-free medium were tested for their sensitivity to 8-Cl-cAMP, serum-activated 8-Cl-cAMP and 8-Cl-adenosine. IC50 values, determined by measuring cell growth using a MTT colorimetric assay, showed that 'serum activation' of 8-Cl-cAMP was required to achieve inhibition of HCT116 (IC50 = 1.3 +/- 0.1 muM) and FET (IC50 = 2.0 +/- 0.1 muM) cell growth. IC50 values were not reached at the highest concentrations tested (IC50 > 500 muM) in the absence of serum, permitting us to conclude that 8-Cl-cAMP does not have growth inhibitory activity between 1.0 and 500 muM doses. HCT116 and FET cells grown in media containing serum and in the presence of 8-Cl-adenosine had IC50 values of 0.6 +/- 0.1 and 0.9 +/- 0.2 muM, respectively. HCT1 16 and FET cells grown in chemically defined medium containing 8-Cl-adenosine exhibited IC50 values of 1.0 +/- 0.1 and 3.1 muM, respectively. Reversed-phase HPLC analysis showed an 11.4 +/- 0.7% conversion of 8-Cl-cAMP to 8-Cl-adenosine in 1 h at 37-degrees-C in the presence of 10% fetal bovine serum (FBS). Analysis of the continued conversion ot 8-Cl-cAMP after 72 h in media containing 10% FBS revealed that 69.5 +/- 0.7% of the 8-Cl-cAMP was converted to 8-Cl-adenosine. These results strongly support the conclusion that enzymatic conversion of 8-Cl-cAMP to 8-Cl-adenosine occurs in the presence of serum and that 8-Cl-adenosine is the active inhibitory compound.