Combination chemotherapy with Clostridium perfringens phospholipase C and cytosine antimetabolites: complementary inhibition directed at membrane lipids.

Abstract

Tumor cell membranes were susceptible to the action of Clostridium perfringens phospholipase C, and this was reflected by inhibition of cellular replication in culture. The differential susceptibility of two neoplastic cell lines to this enzyme was studied in detail. The growth of sarcoma 180 cells cultured in Fischer's medium was markedly inhibited by phospholipase C; whereas, in contrast, cultured L1210 leukemia cells were relatively resistant to the cytotoxic effects of this enzyme. The differential sensitivity of these two neoplastic cell lines to phospholipase C was corroborated by dye-exclusion tests. Thus, leukemia L1210 cells exposed to a concentration of 0.2 mg of phospholipase C per ml of Fischer's medium for 30 min at 37 degrees C were able to exclude Trypan Blue; whereas, only about 21% of sarcoma 180 cells treated under identical conditions were able to exclude the dye. That the cytotoxicity of phospholipase C to sarcoma 180 was the result of hydrolysis of phospholipids of the plasma membrane was supported by measurements of the rate of hydrolysis of radioactivity from the phospholipid of neoplastic cells prelabeled with [3H]choline. Eighty-two percent of incorporated radioactive choline was released from sarcoma 180 cells treated with phospholipase C in Fischer's medium, whereas, only 20% of the label from [3H]choline was solubilized from L1210 leukemia cells treated with the enzyme under similar conditions. Scanning electron microscopy revealed significant damage to sarcoma 180 cells exposed to phospholipase C in Fischer's medium, which was characterized by alterations in size and shape of cells, disappearance of microvilli, and appearance of fistulas in cell membranes; relatively resistant L1210 leukemic cells did not appear to be markedly damaged by comparable enzyme treatment. Exposure of leukemia L1210 cells to phospholipase C in Puck's saline A increased the sensitivity of these cells to enzymatic action. Under these conditions, a comparable amount of phospholipid was hydrolyzed from surface membranes of sarcoma 180 and leukemia L1210 cells, and the degree of membrane damage appeared to be similar, as measured by the capacity of the tumor cell lines to exclude Trypan Blue and by scanning electron microscopy. The extensive damage to membranes by hydrolysis of phospholipids was not accompanied by a change in the degree of specific binding of [3H]concanavalin A(ConA).(ABSTRACT TRUNCATED AT 400 WORDS)