The application of microanalytical techniques was used to elucidate the enzyme profiles of adenylate cyclase and cAMP-dependent protein kinase within a series of malignant and benign human mammary tumors. The methods allow for determination of enzyme activity in histologically-confirmed neoplastic areas within individual tumors which permits comparisons to be made with particle and cytosolic fractions prepared from mammary tumors. The potential for cAMP formation by adenylate cyclase and mediation of cAMP effects by cAMP-dependent protein kinase were studied to provide information on the levels of enzyme activity and the extent and type(s) of regulation of these enzymes in a cell-free system.

Adenylate cyclase (AC) activity levels were higher in benign than malignant tumor tissue on a dry wt basis. A 2 to 3-fold activation by Gpp(NH)p and a 4 to 5-fold stimulation by fluoride was demonstrated. Calmodulin activation of tumor-associated AC was not observed. Membrane-associated AC could be stimulated by PGE₁, prolactin and TSH. Thermal stability studies suggested that AC from malignant tissue was more labile than that from benign tissue.

Cyclic AMP-dependent protein kinase associated with human mammary tumors was primarily type II kinase based upon DEAE-Sephacel chromatography and demonstrated a preference for histone VS as a substrate. Cyclic AMP-dependency was supported by enzyme activation by cAMP, by cAMP binding, and inhibition by protein kinase inhibitors and regulatory subunit. The activity ratio was slightly higher in benign than malignant tissue. Histone kinase activity was higher in benign than malignant tumor microsections. Cyclic AMP-binding activity was higher in malignant than benign tumor cytosolic fractions, although cAMP affinity was similar. Catalytic subunit was prepared from tumor histone kinase by cAMP affinity chromatography. Reassociation of tumor catalytic subunit and heterologous regulatory subunit was shown. A cAMP-independent casein kinase was also partially characterized in human mammary tumors. Endogenous phosphorylation in tumor cytosolic fractions was examined and polypeptides identified which showed phosphorylation differences in the presence of cAMP.