Leakage of lactate dehydrogenase and staining by the vital dye trypan blue were investigated in adult rat hepatocytes at the time of isolation, in suspensions up to 3 h and in primary monolayer cultures up to 3 d. These two parameters of plasma membrane integrity were found to correlate closely in hepatocyte suspensions, but to a lesser degree in monolayer cultures. Functional activity was demonstrated in culture by glucose consumption and lactic acid production. There was a balance of total lactate dehydrogenase (LDH) activity over time for both hepatocyte suspensions and cultures. Loss of LDH activity in the cell fraction was accompanied by a corresponding increase in enzyme activity in the media fraction. Lactate dehydrogenase activity per dye-excluding hepatocyte was calculated to be 9.2 +/- 1.5 X 10(-6) IU assayed at 37 degrees C for 25 preparations of isolated hepatocytes. The results suggest that leakage of cytoplasmic enzyme and vital dye staining are of comparable sensitivity in evaluating hepatocyte preparations. Measurement of LDH leakage offers a less subjective alternative to cell counting procedures and is applicable to both attached and suspended cells.
Interference with growth factor-receptor interactions may have particular relevance in efforts to intervene clinically in both autocrine and paracrine aspects of malignancy. Suramin is a synthetic anticancer agent that works, in part, by blocking the binding of growth factors to their receptors. While initial clinical trials have been encouraging, its use in clinical applications is associated with significant toxicities. Suradista is a novel sulfonated distamycin derivative that is also effective at complexing and inactivating growth factors and cytokines while remaining relatively nontoxic. The goal of this study was to compare the antineoplastic properties of suramin and Suradista. To achieve this, the effects of these compounds on growth factor induced mitogenesis in normal mouse fibroblasts and human umbilical vein endothelial cells were examined, as well as their ability to inhibit the growth of NIH/3T3 cells that had been transformed by the introduction of a fibroblast growth factor (FGF) 1 coding region (residues 1–154) fused to the signal peptide of the hst/KS3 gene (sp-hst/ KS3:FGF1–154). In each case, Suradista was more effective than suramin in inhibiting mitogenesis in normal cells, as well as the growth of the transformed cells. Furthermore, Suradista was also shown to be as effective as suramin at inhibiting the growth of sp-hst/KS3: FGF1–154-transformed NIH/3T3 xenografts grown in athymic nude mice when given at only 50% the dosage used for suramin (50 mg/kg for Suradista versus 100 mg/kg for suramin). In summary, these results indicate that novel compounds acting like suramin may be developed as effective antineoplastic agents and may also prove to be of clinical benefit.
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