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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.

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.

Such selective modulation of protein kinase isozyme regulatory subunits was also found in the S-Cl-CAMP-induced inhibition of both transformation and transforming growth factor (Y (TGFa) 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 Two different catalytic subunits (C, (10) and Cp (11, 12)) have also been identified; however, preferential coexpression of either one of these catalytic subunits with either the type I or II protein kinase regulatory subunit has not been found (12).
Recently, we discovered that site selective CAMP analogs, which show a preference for binding to type II rather than type I protein kinase of purified preparations in vitro (13,14), provoke potent growth inhibition, differentiation, and reverse transformation in a broad spectrum of human and rodent cancer cell lines (15)(16)(17). We have also demonstrated that the antineoplastic effects of site-selective 8-Cl-CAMP are associated with an antagonistic activity on the effects of exogenous transforming growth factor (Y (TGFol) in normal rat kidney fibroblasts and on the production of TGFol in Ki-ras-transformed rat kidney cells (18).
TGFa is a potent mitogen for fibroblasts and epithelial cells (19) and has been implicated in the autocrine growth of rodent and human tumor cells. In fact, enhanced expression of TGFa has been detected in a number of rodent and human breast cancer cell lines and in a majority of primary rodent and human breast carcinomas (20-24). In addition, the ability of estrogens to stimulate the growth of estrogen-dependent breast cancer cells may be related in part to an increased '  production of TGFa in response to estrogen stimulus (20,21,24). Also, experimentally, overexpression of the human TGFa gene in an expression vector plasmid successfully converts in vitro (25) an immortalized nontransformed mouse mammary epithelial cell clone (NOG-8) to a transformed phenotype (NOG-8TFC17) that produces tumors in viuo (25). NOG-8 TFC17 cells, therefore, represent a novel in vitro system to investigate the regulatory mechanisms underlying cell proliferation and transformation and to eventually identify the antitransforming factor that has the ability to interfere with the production and/or action of TGFcL In this work, we have examined the effect of 8-Cl-CAMP on TGFLu-induced transformation of NOG-8TFC17 cells in comparison with that of two other known growth inhibitors, cis-hydroxyproline (26, 27) and TGFP (28,29). The results are correlated with the expression of CAMP-dependent protein kinase isozymes and TGFa production in NOG-8TFC17 cells.

RESULTS
Effect on Cell Growth-The effect of 8-Cl-CAMP on the growth of the transformed NOG-8TFC17 cells and nontransformed counterpart NOG-8 cells is shown in Fig. 1. 8-Cl-CAMP inhibited the monolayer growth of the NOG-8TFC17 cells in a concentration-dependent manner. At 10 and 25 pM, the analog produced 50 and 70% growth inhibition, respectively, when compared with that in the untreated control cells (Fig. IA). In contrast, up to a 50 pM concentration of 8-Cl-CAMP produced no growth inhibition in the nontransformed NOG-8 cells. A greater degree in the growth inhibitory effect of 8-Cl-CAMP was shown when the transformed NOG-8TFC17 cells were grown in soft agar. At 5 pM, the analog produced over 90% inhibition of colony formation compared with that in the untreated control cells (Fig. 1B). Fig. 1 also shows the effect of CHP on the growth of the NOG-8 and NOG-8TFC17 cells. The proline analog CHP has been shown to inhibit the growth of rat mammary tumors in uiuo (26) and of virally transformed rodent cell lines in vitro (27). CHP exhibited growth inhibition in both the NOG-8 and NOG-8TFC17 cells, although it produced a more potent effect on the transformed cells than on the nontransformed cells ( Fig.  1C). At 25 pg/ml, CHP inhibited over 90% of the colony formation of the NOG-8TFC17 cells in soft agar (Fig. 1D). TGF/3 at l-5 rig/ml produced a weak growth inhibitory effect (-30% inhibition) on both NOG-8 (in monolayer culture) and NOG-8TFC17 (in monolayer culture or in soft agar) cells. Time courses of the growth inhibition produced by 8-Cl-CAMP (25 pM) and CHP (30 pg/ml) in the NOG-8TFC17 cells in monolayer culture are shown in Fig. 2. The growth inhibition by these analogs required two to three population doublings. On day 3, both analogs produced a 30-50% growth inhibition; and by day 4, a 60-70% growth inhibition was achieved compared with that in the untreated control cells.
The growth inhibition in NOG-8TFC17 cells brought about by 8-Cl-CAMP or CHP accompanied distinct morphological changes. The growth-arrested cells exhibited a reverted morphology that resembled that of nontransformed NOG-8 cells (data not shown).
Effect of TGFcr Production-We examined whether the growth inhibitory effects of 8-U-CAMP, CHP, and TGFP on the NOG-8TFC17 cells are associated with a specific interference by these agents on TGFa production.
The conditioned media (see "Experimental Procedures") were collected from the culture of the NOG-8TFC17 cells, and the levels of the immunoreactive and biologically active TGFo( were measured by RIA and RRA, respectively. As shown in Table I Table  I). The results in Table I indicate that the growth inhibitory effect of 8-Cl-CAMP is associated with inhibition of TGFa production, whereas that of CHP and TGF/? does not involve interference of TGFL~ production. mRNAs of CAMP Receptor Proteins and TGFor-The effects of 8-Cl-CAMP, CHP, and TGFP on the mRNA levels of the regulatory (RI,, RII,,, and RI&,) and catalytic (C,) subunits of CAMP-dependent protein kinases and TGFcv were determined in the NOG-8TFC17 cells. The RNA filters prepared were probed with '"P-labeled cDNAs of RII,,, RII,,, RI,,, and TGFcu; Northern blot analysis is shown in Fig. 3. The results are expressed in relation to the mRNA levels in the nontransformed NOG-8 cells (Fig. 3, lower). The human RII, cDNA probe detected 6.0-and 2.2-kb mRNA species in the NOG-8 cells (Fig. 3,  in total quantity, RII, mRNA decreased to 30% of that in the untreated transformed cells (Fig. 3). CHP and TGFP treatment brought about little or no effect on the levels of the 6.0-and 2.2-kb RII, mRNA species (Fig. 3). The human RIIB cDNA detected a 1.9-kb mRNA species. The RII, mRNA level decreased by 50% in the transformed NOG8TFC17 cells (lane C) compared with that in the nontransformed cells (lane NOG-8). 8-Cl-CAMP treatment of NOG-8TFC17 cells brought about a 16-fold increase in the RIIB mRNA level over that in the untreated control cells (Fig. 3). CHP caused little or no effect on the RIIp mRNA level, but TGFp caused a substantial increase (6-fold) in the RIIp mRNA level (Fig. 3). The mouse RI, cDNA probe detected a 3.2-and 1.7-kb mRNA species. Both the 3.2-and 1.7-kb mRNA species markedly increased (-IO-fold) in the transformed NOG-8TFC17 cells (lane C) compared with the nontransformed cells (lane NOG-8). 8-Cl-CAMP caused a marked decrease in both the 3.2-and 1.7-kb mRNA levels comparable to that in the nontransformed cells (Fig. 3). CHP also decreased the RI, mRNA level by 60%, but TGFP caused little or no effect on RI, mRNA (Fig, 3). The mouse C, cDNA probe detected a 2.4-kb mRNA species in the transformed and nontransformed cells in a similar quantity, and treatment with 8-Cl-CAMP, CHP, or TGFp caused no change in the mRNA levels (data not shown).
The human TGFa cDNA probe detected a 2.3-kb mRNA species in the transformed NOG-8TFC17 cells (Fig. 3, upper, lane C), but not in the nontransformed cells (lane NOG-8). 8-Cl-CAMP treatment brought about a 90% reduction in the TGFa mRNA level of NOG-8TFC17 cells (Fig. 3), whereas CHP decreased the TGFa mRNA level only by 20%, and TGF@ caused a slight increase in the TGFa mRNA level (Fig.   3).
In previous work, we demonstrated that site l-and 2selective CAMP analogs, which in combination produce synergistic enhancement of binding to type II rather than type I CAMP-dependent protein kinase (13,14), also produces a synergistic growth inhibitory effect (l&17,39).
We examined whether such synergism exists between 8-Cl-CAMP (site lselective) and NG-benzyl-CAMP (site 2-selective) in changing levels of mRNAs for CAMP receptor proteins and TGFa in NOG-8 TFCl7 cells. When cells were treated with 5 pM 8-Cl-CAMP, which alone gave a 20% growth inhibition (Fig. l), the mRNA levels of CAMP receptors and TGFL~ were not significantly different from those in the untreated control cells (Table II) 15 PM NG-benzyl-CAMP brought about changes in these mRNA levels to an extent similar to that produced by higher concentrations of ~-U-CAMP (25 pM) alone (Table II). In addition, 15 FM NG-benzyl-CAMP alone neither exerted growth inhibition or changed these mRNA levels (Table II). However, 50 pM NG-benzyl-CAMP, which demonstrated a moderate growth inhibitory effect, brought about changes in these mRNA levels to a lesser extent than that produced by 25 pM 8-Cl-CAMP (Table II).

DISCUSSION
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.
TGFa induces transformation in an immortalized population of mouse mammary epithelial cells (25), whereas 8-Cl-CAMP, a potent growth inhibitor for a spectrum of human cancer cell lines (15-17), can reverse such transformation, This antagonism between TGFa and 8-Cl-CAMP is expressed in the modulation of the genes for different species of the CAMP receptor proteins. 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. In the reverse transformation by 8-Cl-CAMP, the RI, mRNA level decreased, whereas one species of RII, mRNA and RIIB mRNA levels sharply increased, converting the RII/RI CAMP receptor mRNA ratio to a value similar to that in nontransformed mammary epithelial cells. Thus, transformation by TGFa brought about a disruption in the normal mRNA patterns of CAMP receptor species, and 8-Cl-CAMP restored the normal CAMP receptor mRNA patterns in the mammary epithelial cells. The changing patterns of CAMP receptor mRNAs brought about by 8-Cl-CAMP are the specific effects of the analog rather than a nonspecific event related to growth inhibition in general since two other known growth inhibitors, namely CHP (a proline analog) and TGFP, inhibited the growth of the transformed mammary epithelial cells without reverting the abnormal CAMP receptor mRNA patterns to the normal patterns.
That these effects of 8-Cl-CAMP are due to the interaction of this CAMP analog with type II CAMP-dependent protein kinase is supported by the synergistic effect observed with 8-Cl-CAMP and NG-benzyl-CAMP in combination (Table II). Such synergism between site l-and 2-selective CAMP analogs has been previously demonstrated with purified preparations of protein kinase in vitro (13,14) and in the growth inhibition and differentiation induced in cancer cells by these CAMP analogs (15)(16)(17)39). Moreover, in the growth inhibition induced by 8-Cl-CAMP of LS-174T human cancer cells, there was rapid translocation of RIIB CAMP receptor protein from the cytoplasm to the nucleus that preceded increased transcription of the RIIB gene with decreased transcription of the RI, gene (40). Based on these previous findings, we interpret our present results of the changing levels of mRNAs for CAMP receptor protein to be due to changes in transcription rather than to effects on mRNA stability.
Concomitantly, with these changes in mRNAs for the CAMP receptor proteins, 8-Cl-CAMP also brought about a reduction in the TGFa mRNA level. In these cells, TGFa production is driven by the SV40 promoter. Whether the inhibitory effects of 8-Cl-CAMP were directed against the SV40 promoter is not known at present. A CAMP-and phorbol ester-responsive element (activator protein P)-binding sequence has been identified in the transcription enhancer of