8-Chloro-cAMP inhibits transforming growth factor alpha transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation.

F Ciardiello,G Tortora,N Kim,T Clair,S Ally,D S Salomon,Y S Cho-Chung

doi:10.1016/s0021-9258(19)40152-x

F Ciardiello, G Tortora + Show 5 more

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https://doi.org/10.1016/s0021-9258(19)40152-x

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Abstract

Differential regulation of the regulatory subunits of cAMP-dependent protein kinase isozymes correlates with the growth inhibitory effect of site-selective 8-Cl-cAMP demonstrated in cancer cell lines (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., Ogreid, D., Døskeland, S.O., Jahnsen, T., and Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6319-6322). Such selective modulation of protein kinase isozyme regulatory subunits was also found in the 8-Cl-cAMP-induced inhibition of both transformation and transforming growth factor alpha (TGF alpha) production in Ki-ras-transformed rat kidney fibroblasts (Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and Cho-Chung, Y. S. (1989) FEBS Lett. 242, 363-367). In this work, we have demonstrated that 8-Cl-cAMP antagonizes the TGF alpha effect in TGF alpha-transformed mouse mammary epithelial cells (NOG-8TFC17) at the level of gene expression for cAMP receptor protein isoforms, RI and RII (the regulatory subunits of protein kinase isozymes). Northern blot analysis demonstrated that in the transformed NOG-8TFC17 cells, compared with the nontransformed counterpart NOG-8 cells, the mRNA levels for the RI alpha cAMP receptor protein markedly increased, whereas the mRNA levels for the RII alpha and RII beta cAMP receptor proteins decreased. 8-Cl-cAMP, which induced growth inhibition and phenotypic reversion in NOG-8TFC17 cells, caused an inverse change in the mRNA patterns of the cAMP receptor proteins; RI alpha cAMP receptor mRNA sharply decreased to levels comparable with that of the nontransformed NOG-8 cells, whereas RII beta mRNA increased to a level even greater than that in the NOG-8 cells. In addition, one mRNA species of RII alpha increased, whereas the other RII alpha mRNA species decreased during the treatment. The mRNA level for the catalytic subunit of protein kinase, however, did not change during 8-Cl-cAMP treatment. In addition, 8-Cl-cAMP brought about a reduction in both TGF alpha mRNA and protein levels. These coordinated changes in the expression of the cAMP receptor proteins and TGF alpha were not observed during cis-hydroxyprolineor TGF beta-induced growth inhibition of the NOG-8TFC17 cells. Thus, the antagonistic effect of 8-Cl-cAMP toward TGF alpha-induced transformation involves modulation of the expression of a specific set of cellular genes.

Highlights

Our study presents the first evidence of the CAMP antagonism for TGFa at the level of cellular gene expression
The data presented in this study show that there is an antagonistic interaction between TGFa and 8-Cl-CAMP in the regulation of mammary epithelial cell proliferation
The transformation of mammary epithelial cells with TGFa resulted in a marked increase in the RI, CAMP receptor mRNA level along with a decrease in the RII, and RIIB CAMP receptor mRNA levels

Summary

EXPERIMENTAL PROCEDURES

The cells (2 X lo cells/well) were plated in multiwell cluster dishes (Costar) in 2 ml of DMEM containing 10% FBS. Starting at day 0, 8-Cl-CAMP, CHP, or TGFp was added when the medium was replenished. Agar/medium base layer in 35-mm dishes (Costar) and treated with various concentrations of 8-Cl-CAMP, CHP, or TGFP. 60% confluency in 10% FBS containing DMEM in T-150 flasks (Costar), washed twice in serum-free medium, and cultured in PC-l serum-free medium (Ventrex) for an additional 48 h in the absence or presence of various concentrations of 8-Cl-CAMP, CHP, TGFP. TGFa antiserum that is specific for TGFa and fails to recognize mouse or human epidermal growth factor (EGF) as previously described [22, 23]. The gels were transferred to Biotrans nylon membranes (ICN Biomedicals) by the method of Thomas [33] and hybridized to the following “P-labeled nick-translated cDNA probes: a 406-bp EcoRI-ApaI restriction fragment derived from a human TGFol cDNA clone; pTGF-Cl [34]; a 600-bp I’stI restriction fragment of the mouse CAMP-dependent progein kinase type I regulatory subunit, RI, cDNA clone [35]; a 1600-bp BamHI restriction fragment of the human CAMP-dependent protein kinase type II regulatory subunit, RIIB cDNA clone [36]; a 1650-bp EcoRI restriction fragment of the human CAMP-dependent protein kinase type II regulatory subunit, RII, cDNA clone [37]; and 600-bp EcoRI restriction fragment of the mouse CAMP-dependent protein kinase catalytic subunit, C, cDNA clone [38]

RESULTS

25 PM 5w’M

DISCUSSION