Reduced cytocidal efficacy for adriamycin in cultured human carcinoma cells depleted of polyamines by difluoromethylornithine treatment.

We have studied the effect of pretreatment with difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, on the cytocidal responses of four human adenocarcinoma cell lines to Adriamycin (ADR). The cell lines utilized included HuTu-80 (duodenum), HT-29 (colon), ME-180 (cervix), and A-427 (lung). A 48-h DFMO pretreatment reduced putrescine and spermidine content to less than 10 and less than 1% of control levels and decreased spermine to between 70 and 30% of controls. Plating efficiency assays were used to generate ADR dose-response survival curves for DFMO-treated and control cultures. The DFMO pretreatment significantly protected human adenocarcinoma cells from the lethal effects of ADR. Addition of exogenous putrescine to the DFMO-treated cultures 24 h before treatment with ADR restored their cytocidal response to ADR to near control levels. Putrescine had no effect on cell survival in cultures that were not pretreated with DFMO. These observations suggest that DFMO-induced protection from ADR may be a specific consequence of DFMO-induced inhibition of polyamine biosynthesis. Alternatively, since ADR efficacy varies directly with cellular growth rates and DFMO inhibits proliferation, the protection may have resulted from DFMO-induced growth inhibition. Comparison of ADR uptake in DFMO-pretreated and control cells showed that the protection did not result from decreased intracellular accumulation of ADR.

Altered cell cycle phase distributions in cultured human carcinoma cells partially depleted of polyamines by treatment with difluoromethylornithine.

We have studied the effects of partial polyamine depletion, induced by treatment with alpha-difluoromethylornithine (DFMO) on cell cycle phase distributions in five cultured human carcinoma cell lines. We used flow cytometry of cells stained with chromomycin-A3 and computer analysis to measure phase distributions of treated and control cultures. All five lines respond to 1-5 mM DFMO treatment with a total absence of measurable putrescine, a loss of greater than 90% of spermidine, and a 30-40% decline in spermine by 48 h after DFMO addition. The proliferation of all five lines is inhibited as well. Nonetheless, only four of the cell lines (HuTu-80, HT-29, MCF-7, and A-427) show a marked increase in the G1-phase fraction and decrease in the S-phase fraction as a consequence of DFMO treatment. Small, but significant, decreases in the G2-M populations of these cell lines also occurred after DFMO treatment. Exogenous putrescine (5-50 microM) reversed both the polyamine depletion and the perturbed phase distributions of DFMO-treated cultures but was without effect on phase distributions of cultures not treated with DFMO. The fifth cell line (ME-180) showed no effect of polyamine depletion on cell cycle phase distributions in DFMO-treated cultures and also no effect of exogenous putrescine on phase fractions of either control or DFMO-treated cells. These observations indicate that some human tumor cell lines are dependent upon adequate intracellular polyamine content for maintenance of cell cycle traverse. They also imply that human tumor cell lines are heterogeneous with regard to their cell cycle response to DFMO-induced polyamine deficiency.

Effects of DFMO-induced polyamine depletion on human tumor cell sensitivity to antineoplastic DNA-crosslinking drugs.

We investigated the effect of pretreatment with difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, on the cytocidal responses of four human adenocarcinoma cell lines to two alkylating and crosslinking agents: chlorambucil and N,N',N"-triethylenethiophosphoramide (thiotepa). The cell lines studied included HuTu-80 (duodenum), HT-29 (colon), ME-180 (cervix), and A-427 (lung). A 48- to 72-h pretreatment with DFMO reduced intracellular putrescine and spermidine contents to less than 10% and less than 1% of control levels. This treatment also caused a 30%-70% decline in spermine content. Survival of control and DFMO-pretreated cells after treatment with chlorambucil or thiotepa was measured by a plating efficiency assay. For three of the four lines studied, the DFMO-induced partial polyamine depletion significantly protected cells from the lethal effects of chlorambucil. In ME-180 cultures alone, DFMO pretreatment did not alter the cytocidal efficacy of chlorambucil. Addition of exogenous putrescine to cultures of HuTu-80, HT-29, or A-427 24 h after DFMO addition but 24 h before treatment with chlorambucil reversed the polyamine depletion and its protective effects on chlorambucil-induced cell kill. In contrast to the above observations, DFMO and partial polyamine depletion had no effect on cell survival after thiotepa treatment for any of the cell lines investigated.

Chemosensitization of cultured human carcinoma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea by difluoromethylornithine-induced polyamine depletion.

We have investigated the effect of pretreatment with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) on the cytocidal efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a series of five cultured human adenocarcinoma cell lines. Plating efficiency assays were used to generate BCNU dose-response survival curves for DFMO-treated and control cells. The cell lines varied in their sensitivity to BCNU, with A-427 (lung) and HuTu-80 (duodenum) cells being most sensitive, HT-29 (colon) and ME-180 (cervix) most resistant, and MCF-7 (breast) showing intermediate sensitivity. For all five cell lines, a 48-h pretreatment with 5 mM DFMO reduced intracellular putrescine and spermidine content to less than 10% of control levels and decreased spermine content to between 60 and 70% of controls. This pretreatment resulted in a shift of the BCNU survival curves for each of the five cell lines downward and to the left, indicating that the cells were sensitized to the lethal effects of BCNU. Dose enhancement ratios for DFMO-induced chemosensitization ranged from 1.2 (HuTu-80 cells at the 1% survival level) to 1.9 (HT-29 cells at the 10% survival level). The cell lines most resistant to BCNU appeared to give the greatest degree of potentiation by DFMO pretreatment. For four of the five cell lines, addition of 50 to 100 microM exogenous putrescine to DFMO-pretreated cultures 12 to 24 h before BCNU addition reversed the chemosensitization. ME-180 cells were the sole exception. Exogenous putrescine did not increase the surviving fraction after BCNU of any cells not pretreated with DFMO. These results suggest that DFMO-induced chemosensitization to BCNU in the four cell lines other than ME-180 is a specific consequence of the inhibition of ornithine decarboxylase by DFMO and the resulting depletion of intracellular polyamine content.