Nuclear DNA polymerases of human carcinomas.

Comparisons have been made of the DNA polymerases of normal human lung and cecum, primary carcinomas of human lung, breast, and cecum, and resting and regeneration rat liver. The picture for the normal human tissues is similar to the one for unstimulated rat liver, that for the human carcinomas resembles regenerating rat liver. The human tissues contain two polymerases with sedimentation coefficients of about 3 and 7 S, the enzymes are restricted to the nucleus, and the specific activities of the 7 S polymerase, but not of the 3 S enzyme, are elevated in the cancers. Just as with the regenerating rat liver polymerases, the 3 S activity of a bronchogenic carcinoma is unaffected by cytosine arabinoside 5'-triphosphate and only little reduced by novobiocin, whereas DNA synthesis by the 7 S enzyme is abolished by both compounds. A variety of other inhibitory agents have similar effects on the 7 S polymerases of the human carcinomas and regenerating rat liver.

The 7.1 S nuclear DNA polymerase and DNA replication in intact liver.

Just as after 70% hepatectomy, the activity of the 7.1 S DNA polymerase, but not the 3.2 S polymerase, is elevated in liver nuclei from unoperated animals in which hepatic DNA replication has been induced with a mixture of biochemicals or by a dietary manipulation. Again as with regenerating liver, the stimulated intact livers show a relationship between the increases in the enzyme activity and thymidine incorporation in vivo over a wide range of hepatic responses. These observations are consistent with a role for the 7.1 S activity in nuclear DNA replication. Cytosine arabinoside 5'-triphosphate and novobiocin can be used to distinguish between the 3.2 S and 7.1 S polymerases from nuclei of stimulated intact liver as well as of regenerating liver.

Nuclear deoxyribonucleic acid polymerases of liver.

Hepatic nuclei that are isolated in aquenous solutions of low ionic strength or glycerol contain all or nearly all the nonmitochondrial DNA polymerase activity of the cell. The presence of polymerase activity in the cytoplasm is due to extraction of nuclear enzymes by buffer and inorganic salts. Even with low ionic strength solutions, some leaching of nuclear enzymes occurs if the concentration of liver in the homogenizing medium is greater than 10%. As defined by sucrose gradient analysis, the normal adult rat liver nucleus contains mainly or entirely a single species of DNA polymerase (3.2 S) whereas the regenerating nucleus after 70% hepatectomy has an additional enzyme (7.1 S). The total activity of regenerating nuclei is about twice the normal value. The increase resides in the 7.1 S activity. The 7.1 S DNA polymerase had been purified partially from regenerating liver nuclei (isolated in low ionic strength solutions) and cytosol (prepared under conditions of nuclear enzyme extraction). The properties of the activity from the two sources are indistinguishable. A mixture of albumin and spermidine enhances by several-fold the activities of the 3.2 S and 7.1 S DNA polymerases. In the presence of spermidine, but not in its absence, the activity of the 7.1 S DNA polymerase is strictly proportional to the amount of the enzyme preparation.

Erythrocytes as carriers of chemotherapeutic agents for targeting the reticuloendothelial system.

The object of this work was to define a model using hypotonically loaded erythrocytes as a vehicle to target drugs to the reticuloendothelial system (RES). The optimum hemolytic event was found to occur at 100 mOsm/kg using a 0.5-min exposure at 0 degrees C. Approximately one third of the total volume of the cells could be replaced with hypotonic drug solutions under these conditions. Although cytosine-beta-D-arabinofuranoside, ara C, is membrane permeable and could not be entrapped in the erythrocytes, phosphorylation of this nucleoside antimetabolite enabled it to be loaded efficiently. Actinomycin D could be loaded and retained within the cells at 0 degrees C, but 90% of this loaded drug leaked out of the erythrocytes in 1 min at 37 degrees C. Actinomycin D-DNA complexes, however, could be loaded and retained for longer periods. In this case, 50% of the DNA-bound drug was retained in the cells for one hour at 37 degrees C. It was found that the glycopeptide antitumor antibiotic, bleomycin, could be entrapped and retained in the cells without appreciable leakage. It was possible to load a human therapeutic dose of this drug in 1-2 ml of packed cells. Furthermore, it was demonstrated that bleomycin entrapped in erythrocytes was significantly more effective than the same dose of free drug in suppressing the phagocytic function of the RES in Balb/C and C3H mice. The rationale is discussed for the possible use of these drugs, entrapped in erythrocytes, for the production of RES blockade in the treatment of disorders in man.

The use of erythrocytes for delivery of chemotherapeutic agents to the reticuloendothelial system.

The object of our work is to define the possible role of hypotonically loaded erythrocytes as carriers to target drugs to the reticuloendothelial system. We have examined choices of drugs for loading into the erythrocytes and have considered methods of altering potentially useful agents so that they will load. We have demonstrated that the delivery of bleomycin to the reticuloendothelial system of mice, inside erythrocyte carriers, potentiates the effect of this drug on phagocytosis. We speculate, that this targeted delivery of bleomycin to phagocytes could be beneficial in the treatment of diseases characterized by an important phagocytic component.