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Characterization of the Cyclic Adenosine 3':5'-Monophosphate Effector System in Hormone-dependent and Hormone-independent Rat Mammary Carcinomas¹

Cell Biology Research Group, Institute of Anatomy [D. Ø., R. E., O. V., S. O. D.] and Biochemistry [J. H.], University of Bergen, Årstadveien 19, N-5000 Bergen, Norway, and Cellular Biochemistry Section, Laboratory of Tumor Immunology and Biology [Y. S. C-C.], NIH, National Cancer Institute, Bethesda, Maryland 20205

We have compared the properties of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases I and II in hormone-dependent/cAMP-sensitive (DMBA tumor) and hormone-independent/cAMP-resistant (DMBA 1 tumor) rat mammary carcinomas.

cAMP-resistance was not due to (a) less total kinase in the hormone-independent tumor, (b) grossly altered distribution between soluble and particulate forms of the kinase (80% soluble in either tumor), (c) alteration in the relative proportion of isozymes I and II of the protein kinase (the soluble and the particulate fraction from both tumors contained about 50% of either isozyme), or (d) a decreased sensitivity towards cAMP (both isozymes had affinities for cAMP and its derivatives that corresponded closely with those of isozymes from normal tissues). Furthermore, the sensitivity of the enzymes towards thermal denaturation was identical for samples from the two tumor types.

Subtle differences did, however, exist between the regulatory moieties [regulatory subunit of cAMP-dependent protein kinase II (RII)] of isozyme II from the two tumors: (a) autophosphorylated RII from the hormone-independent tumor migrated as a doublet corresponding to M_rs 54,000 and 52,000 on sodium dodecyl sulfate-polyacrylamide gels, against M_rs 53,000 and 52,000 for RII from the hormone-dependent tumor; (b) RII from the two tumors showed different elution profiles upon DEAE-cellulose chromatography; (c) a considerable proportion of the soluble RII in the hormone-independent tumor formed supramolecular aggregates as judged by size-exclusion chromatography.

No such microheterogeneity was noted for isozyme I. This study thus shows that the lack of cAMP-responsiveness of one tumor is related either to a defect distal to the cAMP-dependent protein kinases or to the appearance of the new subtype of RII in the resistant tumor. If the latter explanation is correct, it means that the part of the RII molecule responsible for interaction with other proteins rather than that responsible for cAMP-binding and control of protein kinase activity modulates the growth-inhibiting response to cAMP.

¹ This work was supported by the Norwegian Cancer Society (LMK).

² To whom requests for reprints should be addressed.

Received 8/14/86. Revised 1/ 8/87. Accepted 2/11/87.