Cyclic AMP-binding protein and estrogen receptor: antagonism during nuclear translocation in a hormone-dependent mammary tumor

Incubation of nuclei from hormone-dependent rat mammary tumors with its cytosol activated with 5 nM 17β-estradiol resulted in a 4-fold increase of nuclear estrogen binding activity over the control nuclei. The presence of 100 nM cAMP in the activated cytosol inhibited this nuclear uptake of estrogen receptor by 50%. Conversely, incubation of the nuclei with cytosol activated with 100 nM cAMP increased nuclear cAMP binding and cAMP-dependent protein kinase activity 4-fold, while the presence of 5 nM 17β-estradiol in the activated cytosol inhibited the nuclear cAMP binding and the protein kinase activity by 50%. No competition was found between estrogen and cAMP for each other's cytoplasmic binding proteins or the nuclear acceptor sites. These data suggest that a mutual antagonism exists between the cAMP-binding protein and estrogen receptor during their nuclear translocation.     

Increase of cyclic AMP-dependent protein kinase type II as an early event in hormone-dependent mammary tumor regression.

Primary, 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary carcinoma in the rat contains cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent and -independent forms of protein kinase. When growth of DMBA-induced tumors was arrested by either ovariectomy or N⁶,O²′-dibutyryl cAMP treatment of the host, the activity of cAMP-dependent protein kinase type II markedly increased in the tumor cytosol, as shown by DEAE-cellulose chromatography and autophosphorylation. The increase in activity of cAMP-dependent protein kinase was also demonstrable in the tumor cytosol and nuclei following $\text{in}$$\text{vitro}$ incubation of tumor slices with cAMP. These results suggest that protein kinase type II is involved in the regression of hormone-dependent mammary tumors.     

Prostaglandin synthesis and binding is increased in regressing NMU mammary carcinoma

Mammary tumors induced in Buffalo rats by treatment with nitrosomethyl urea will regress after oophorectomy. Their ability to synthesize and bind prostaglandins E and F₂α was studied in the growing and regressing states. Prostaglandins present in suspensions of 100,000 xg tumor membranes after 2 hr incubation at 37°C ± 5×10⁻⁴M indomethacin were partially purified by silica gel column chromatography before assay by specific PG RIA. The amounts of PGE and F₂α synthesized rose from 0.13 and 10.5 ng/mg protein in the growing tumors to a maximum of 1.2 and 26.5 ng/mg protein 5 days after oophorectomy. Specific binding of ³H-PGE₂ and ³H-PGF₂α to 15,000 xg tumor membranes was achieved during a 45 min incubation at 23°C ± excess unlabelled PG. Free and bound prostaglandins were separated by filtration. Binding reached equilibrium after 30 min, was saturable and reversible. Scatchard analysis revealed high affinity binding of PGF₂α but only low affinity PGE₂ binding in membranes obtained from growing tumors. A 2–3-fold increase in specific binding of PGE₂ and PGF₂α was noted at 4 days after oophorectomy which represented an increase in the number of PGF₂α receptors. PGE₂ binding retained a low affinity character. The elevated PGF₂α synthesis rates observed in the regressing tumors coupled with a regression-associated increase in receptor number suggests that PGF₂α-plays a significant role in hormone-dependent mammary tumor regression.     

Dibutyryl cyclic AMP treatment mimics ovariectomy: new genomic regulation in mammary tumor regression

The $\text{in}\text{vitro}$ translated proteins from poly(A)RNAs differed when hormone-dependent mammary carcinomas were compared during their growth and regression. Within 6 hours post ovariectomy the concentration of one protein band increased and those of two protein bands decreased in the regressing as compared to the growing tumors. The translated protein patterns of the regressing tumors were identical whether regression was induced by ovariectomy or dibutyryl cyclic AMP treatment. The results suggest that mammary tumor growth is subject to genomic regulation and that the same new genetic event occurs in dibytyryl cyclic AMP- and ovariectomy-induced regression.     

Antigenicity of UV radiation-induced murine tumors correlates positively with the level of adenosine deaminase activity

Summary The specific activities of adenosine deaminase (ADA) in 16 murine tumor cell lines derived from seven UV light-induced neoplasms (melanoma and fibrosarcoma) were determined. In each case, the specific activity of ADA correlated positively with the antigenicity of the tumor cells. Highly antigenic cell lines that regress upon introduction into syngeneic hosts had on average 4- to 6-fold higher ADA specific activities than cell lines of low antigenicity that grow progressively in syngeneic hosts. The antigenic differences are probably not related to intracellular cAMP levels, as the level of cAMP differed only 2-fold between the two groups of cell lines.     

Anticarcinogenic effect of retinoids on 7,12-dimethylbenz(a)anthracene-induced mammary tumor induction, and its relationship to cyclic AMP-dependent protein kinase

Administration of 13-cis retinoic acid and N-(4-hydroxyphenyl) retinamide daily in the diet to female Sprague-Dawley rats beginning one day after intubation with 7,12-dimethylbenz(a)anthracene (DMBA) prolonged the latency periods and inhibited the percentage incidence of mammary tumors. A significant reduction in the total number of tumors was also evident. The inhibition of mammary tumor growth by retinoids was associated with a significant increase (3-fold) in cytosolic cAMP-binding and histone kinase activities. The increase of histone kinase activity was almost totally in the cAMP-dependent protein kinase Type II. Retinoic acid increased the amount of the regulatory subunit (R11) rather than altering its cAMP binding affinity. These results suggest that cAMP-dependent protein kinase Type II may be involved in mediating the retinoid action in the inhibition of mammary tumor growth in vivo.     

Induction of mammary prolactin receptors and lactose synthesis after ovariectomy in the pregnant mouse.

The development of prolactin receptors in the mammary gland after ovariectomy was investigated in pregnant KA mice. Mice were ovariectomized on day 13 of pregnancy and used for the determination of the amount of specific binding of 125I-labelled prolactin to the mammary tissue, and the contents of lactose and nucleic acids in the mammary gland 0, 8, 24, and 72 hr after the operation. The specific binding of 125I-labelled prolactin, lactose and RNA contents in the mammary gland remained low until 8 hr, sharply increased 24 hr and decreased 72 hr after ovariectomy. When ovariectomized mice were treated with 0.2 mg progesterone, pregnancy was maintained and an increase (1.5-fold) in the amount of specific binding was observed with an increase of lactose content. Five mg progesterone completely inhibited lactose synthesis. Cortisol administered with progesterone did not show any specific change at the dose used (0.5 to 10 mg). Although the amount of specific binding was also increased after hysterectomy, this increase (2-fold) did not fully cover the increase after ovariectomy (3-fold). These results suggest that the recepter site for prolactin is induced before the initiation of lactose synthesis caused by ovariectomy during pregnancy.     

Cyclic AMP regulation of mammary tumor virus production.

Addition of dibutyryl cyclic AMP (cAMP) and agents (isoproterenol and epinephrine) that stimulate the activities of adenylate cyclase enhance the stimulation of mammary tumor virus (MTV) production two- to threefold by glucocorticoid in short-term primary cultures of mammary tumors. This cAMP potentiation seems to depend on the stimulated level of MTV production by glucocorticoid alone, which increases MTV production 5- to 10-fold over basal level but varies greatly in absolute terms. When the stimulated level by glucocorticoid alone is suboptimal, cAMP seems to restore the sensitivity of the cells to the stimulatory effect of glucocorticoid to its maximum.     

A study of cyclic nucleotide metabolism and the histology of rat liver during 3'-methyl-4-dimethylamino-azobenzene carcinogenesis. II. Cyclic AMP metabolism.

We have studied cAMP metabolism in rat livers undergoing carcinogenesis induced by dietary 3'-methyl-4-dimethylaminoazobenzene. A correlation between the biochemical and the histological changes described in the companion paper has been made. In this study, we saw 100% incidence of cholangiocarcinoma by 10 weeks. During weeks 1--10, the biochemistry of tumor-free areas of the livers only was studied; during weeks 11-13, the increased size of the tumors made possible a biochemical study of the tumor tissue as well as the non-tumor tissue, and a comparison between the two was made. Alterations in all parameters of cAMP metabolism were seen from the earliest stages of treatemnt. Most striking were those of adenylate cyclase activity which preceded and accompanied tumor formation, and were seen in both non-tumor and tumor tissue. In the first few weeks of treatment, small acidophilic glycogen-deficient hepatocytes appeared in the periportal areas of the liver lobules. During this time, there was an increase in maximal isoproterenol stimulation of adenylate cyclase and to a lesser extent in the basal activity of the enzyme; increases in phosphodiesterase activity were seen, and were greatest in weeks 1, 2; cAMP levels were diminished in weeks 1, 2 and slightly but not significantly elevated at week 3. From week 4 onwards an even smaller glycogen-deficient cell population appeared in perilobular areas amongst the acidophilic hepatocytes, and tumors began to appear elsewhere in the livers; at this time, there were further marked increases in the basal activity and isoproterenol responsiveness of adenylate cyclase, and the appearance of increased Gpp(NH)p responsiveness of the enzyme; the increase in phosphodiesterase activities seen at week 3 (smaller than that seen in weeks 1, 2) was sustained but did not further increase; cAMP levels were now significantly elevated also, but they did not rise steadily as did the activity of adenylate cyclase. There was a marked difference between the adenylate cyclase activities in non-tumor tissue from tumor-bearing and non-tumor-bearing livers in weeks 4--10, but there was no difference between the phosphodiesterase activities or cAMP levels in these two groups. Adenylate cyclase activity was extremely high in both non-tumor tissue of tumor-bearing livers from weeks 4--10 and tumors from weeks 11--13. Although phosphodiesterase activities were most elevated in the tumors, there were extremely high cyclic AMP levels in these tissues. The difference between the cAMP levels of tumor and non-tumor tissue was striking. Our findings are discussed with respect to the two-state model of carcinogenesis...     

Kinetics of glucocorticoid interaction with wild-type and variant receptors

Data concerning the short- and longterm effects of ovariectomy on the levels of estrogen binding sites in the rat uterus and liver are presented. The information increases the understanding of the regulation of estrogen receptor synthesis. The circulating estrogen level is suggested to affect receptor synthesis in the uterus and liver differently. Shortly after gonadectomy (2–20h), an elevation in the concentration of cytoplasmic binding sites in the uterus of 35% was observed, whereas no effect was seen in the liver cell. A longer period of time after ovariectomy (2–3 months) caused a reduction in the number of uterine receptor sites by 74%, whereas in the liver an increase of 84% was detected.     

Cholera toxin: mitogen for Harderian glands of golden hamsters

1. Within 2 days, a single injection of 10 micrograms cholera toxin induces a 6-20-fold increase in mitotic activity in the Harderian glands of male and female golden hamsters. 2. Neither hypophysectomy nor ovariectomy had any influence on this response. 3. The cellular proliferation does not appear to involve cAMP nor is it the result of stress, nor a release from an early mitotic block. 4. Nuclear polyploidy increases within a few hours after treatment. 5. DNA density per unit area increases within 24 hr and is maintained for at least 3 days.     

Expression of transforming growth factor alpha (TGF alpha) in differentiated rat mammary tumors: estrogen induction of TGF alpha production

Primary well-differentiated dimethylbenzene alpha-anthracene (DMBA)-or nitrosomethylurea (NMU)-induced rat mammary adenocarcinomas that are estrogen dependent possess biologically active and immunoreactive transforming growth factor alpha (TGF alpha), which can be detected in a sort agar growth-promoting assay and by a specific liquid-phase competitive RIA, respectively. In contrast, tissue extracts prepared from transplantable undifferentiated DMBA-I and NMU-II rat mammary carcinomas that are estrogen independent and metastatic exhibit low or undetectable levels of TGF alpha. In addition, the primary DMBA- and NMU-induced rat mammary adenocarcinomas express a specific 4.8-kilobase TGF alpha mRNA species, whereas little or no TGF alpha mRNA can be detected in the transplantable DMBA-I and NMU-II tumors. Primary tumors synthesize type IV basement membrane collagen, whereas the transplantable tumors elaborate very little type IV collagen. Either TGF alpha or estrogens can differentially enhance the synthesis of type IV collagen by 0.5- to 4-fold over total protein synthesis in primary cultures of normal mouse mammary epithelial cells or in primary NMU-induced tumor cells, respectively. Therefore, TGF alpha could function as an estrogen-inducible autocrine growth factor for well differentiated rat mammary tumor cells by its ability to selectively regulate type IV collagen synthesis. Estrogens can modulate TGF alpha production in vivo in primary DMBA-induced rat mammary tumors, because ovariectomy results in a rapid decline (within 6 h) of TGF alpha mRNA levels. This response to estrogens can also be observed in vitro. Primary DMBA- or NMU-induced rat mammary tumor cells cultured in the presence of 17 beta-estradiol (10(-8) M) for 4 days show an increase in the level of TGF alpha mRNA over cells not treated with estrogen. This increase in TGF alpha mRNA is paralleled by a 2- to 3-fold increase in the levels of immunoreactive TGF alpha that can be detected and in the conditioned medium from estrogen-treated cells. These results suggest that TGF alpha may be an adjunct marker for those mammary tumors that are well differentiated adenocarcinomas and estrogen dependent and that estrogen-independent tumors do not constitutively produce TGF alpha or express TGF alpha mRNA.     

Immunological responsiveness against tumors induced by avian sarcoma virus: reduced expression of pp60src kinase activity in regressing tumors.

Tumors which are induced in chickens by avian sarcoma virus frequently grow progressively for several weeks and then regress. We showed that tumor cells which are derived from the progressively growing phase of tumor growth produce large quantities of progeny-transforming virus, are reactive with antiviral antibody, and are susceptible to lysis in cell-mediated cytotoxicity assays by splenic lymphocytes of sensitized hosts. In contrast, tumor cells derived from regressing sarcomas are poor producers of progeny virus and are relatively unreactive with both antiviral antibody and sensitized lymphocytes. We further found that pp60src kinase activity was reduced by about 75% in regressing compared with progressively growing tumor cells. The half-lives of directly precipitable pp60src in tumor cells derived from progressively growing and regressing neoplasms were 6 and 1.5 h, respectively. Studies on each of three other cellular enzymes did not reveal any regression-associated decreases in enzyme activity. These data support the notion that expression of adequate levels of long-lived pp60src kinase activity is essential to progressive tumor growth.     

Hormone-regulated expression of cellular rasH oncogene in mammary carcinomas in rats

Differential gene expression has been observed in hormone-dependent rat mammary carcinomas during their growth and regression. A 22K MW protein, a prominent in vitro translation product of the growing as compared to the regressing tumor, was identified as the c-rasH-21,000-dalton transforming protein (p21) using a monoclonal antibody that reacts specifically with Harvey-related p21 species. The amount of p21-translated protein sharply decreased in the translation products of the regressing tumors within 6 hours post ovariectomy or dibutyryl cyclic AMP treatment. The results show that an enhanced expression of the c-rasH oncogene is associated with hormone-dependent growth of mammary carcinomas in vivo and that suppression of this oncogene precedes the tumor regression induced by either hormone withdrawal or dibutyryl cyclic AMP treatment.     

EFFECTS OF NOREPINEPHRINE ON THE MORPHOLOGY AND SOME ENZYME ACTIVITIES OF PRIMARY MONOLAYER CULTURES FROM RAT BRAIN

Addition of norepinephrine or isoproterenol to primary cultures started from the brains of 1-3 day old rats caused up to 200-fold increases in cAMP levels, which reached a maximum by 5-10 min and then declined. This effect was studied in detail for norepinephrine. The rise in cAMP levels was followed by morphological changes, in which up to 65% of the cells exhibited an astrocyte-like morphology, and 2-3 fold increases in carbonic anhydrase and (Na⁺-K⁺) ATPase activities. However, morphological transformation also occurred after much smaller increases in total cAMP levels. These effects on cell morphology and enzyme activities reached a maximum 1-2 h after addition of norepinephrine and then declined. Carbonic anhydrase activity was found both in the particulate and post 100,000 g supernatant fractions from homogenates of these cultured cells, and in the latter case the activity was activated 3-fold by addition of cAMP. The significance of these obscrvations on the cellular localization of, and functional role for similar increases in cAMP in brain tissue is discussed.     

The cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum. Binding characteristics of aggregation-competent cells and variation of binding levels during the life cycle.

Both cyclic guanosine 3':5'-monophosphate and dithiothreitol stimulate binding of cyclic adenosine 3':5'-monophosphate (cAMP) to aggregation-competent amoebae. Both compounds appear to function solely by preventing the hydrolysis of cAMP by the cell-bound phosphodiesterase. The dissociation constant for binding of cAMP is 36 nM. Both cAMP binding and membrane-bound phosphodiesterase activities increase dramatically as cells develop aggregation competence, reach a maximum at about 11 hours, and remain at high levels for up to 48 hours if cells are maintained in shaken suspension. When amoebae are allowed to aggregate and develop naturally, binding of cAMP increases during aggregation, decreases during tip formation, and disappears during culmination. Phosphodiesterase activity parallels binding activity except that the decreased level after tip formation is retained throughout culmination. Two N-6-modified cAMP derivatives compete with cAMP for binding sites. One derivative is fluorescent (1,N-6-etheno-cAMP); the other is photolyzable [N-6(ethyl-2-diazomalonyl)cAMP]. This result opens the possibilities of using fluorescence quenching for assay of in vitro binding and of affinity labeling of binding sites. Competition by the derivatives is only partial, indicating possible heterogeneity of binding sites. Both compounds inhibit hydrolysis of cAMP by the membrane-bound phosphodiesterase.     

Identification of tumor-infiltrating macrophages as the killers of tumor cells after immunization in a rat model system.

Immunization can prevent tumor growth, but the effector cells directly responsible for tumor cell killing in immunized hosts remain undetermined. The present study compares tumor grafts that progress in naive syngeneic rats with the same grafts that completely regress in hosts preimmunized with an immunogenic cell variant. The progressive tumors contain only a few macrophages that remain at the periphery of the tumor without direct contact with the cancer cells. These macrophages do not kill tumor cells in vitro. In contrast, tumors grafted in immunized hosts and examined at the beginning of tumor regression show a dramatic infiltration with mature macrophages, many of them in direct contact with the cancer cells. These macrophages are strongly cytotoxic for the tumor cells in vitro. In contrast to macrophages, tumor-associated lymphocytes are not directly cytotoxic to the tumor cells, even when obtained from tumor-immune rats. However, CD4(+) and CD8(+) T cells prepared from the regressing tumors induce tumoricidal activity in splenic macrophages from normal or tumor-bearing rats and in macrophages that infiltrate progressive tumors. These results strongly suggest that the main tumoricidal effector cells in preimmunized rats are macrophages that have been activated by adjacent tumor-immune lymphocytes.     

Effect of chronic estrogen treatment of Syrian hamsters on microsomal enzymes mediating formation of catecholestrogens and their redox cycling: implications for carcinogenesis

Estrogens have previously been shown to induce DNA damage in Syrian hamster kidney, a target organ of estrogen-induced cancer. The biochemical mechanism of DNA adduction has been postulated to involve free radicals generated by redox cycling of estrogens. As part of an examination of this postulate, we measured the effect of chronic estrogen treatment of hamsters on renal microsomal enzymes mediating catechol estrogen formation and free radical generation by redox cycling of catechol estrogens. In addition, the activities of the same enzymes were assayed in liver in which tumors do not develop under these conditions. At saturating substrate concentration, 2- and 4-hydroxyestradiol were formed in approximately equal amounts (26 and 28 pmol/mg protein/min, respectively), which is 1-2 orders of magnitude higher than reported previously. Estradiol treatment for 2 months decreased 2-hydroxylase activity per mg protein by 75% and 4-hydroxylase activity by 25%. Hepatic 2- and 4-hydroxylase activities were 1256 and 250 pmol/mg protein/min, respectively. Estrogen treatment decreased both activities by 40-60%. Basal peroxidatic activity of cytochrome P-450, the enzyme which oxidizes estrogen hydroquinones to quinones in the redox cycle, was 2.5-fold higher in liver than in kidney and did not change with estrogen treatment. However, when normalized for specific content of cytochrome P-450 the enzyme activity in kidney was 2.5-fold higher than in liver and increased further by 2-3-fold with chronic estrogen treatment. The activity of cytochrome P-450 reductase, which reduces quinones to hydroquinones in the estrogen redox cycle, was 6-fold higher in liver than in kidney of both control and estrogen-treated animals. When normalized for cytochrome P-450, the activity of this enzyme was similar in liver and kidney, but over 4-fold higher in kidney than liver after estrogen treatment. Basal concentrations of superoxide, a product of redox cycling, were 2-fold higher in liver than in kidney. Estrogen treatment did not affect this parameter in liver, but increased it in kidney by 40%. These data provide evidence for a preferential preservation of enzymes involved in estrogen activation.     

Influence of estrogen on glutathione levels and glutathione-metabolizing enzymes in uteri and R3230AC mammary tumors of rats

The glutathione content and the activities of several enzymes in its metabolism, glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase, were assayed in uteri obtained from estrogen-treated rats and in R3230AC mammary adenocarcinomas obtained from ovariectomized, intact and estrogen-treated hosts. Normal mammary glands, obtained 10–12 days post-partum, were also examined for these parameters.A daily pharmacological dose of 0.4 μg of estradiol-17β induced a maximal increase in uterine weight and in reduced glutathione (GSH); higher doses of estrogen did not significantly increase either of these parameters. Levels of oxidized glutathione (GSSG) were comparable in both estrogen-treated and untreated rats. The time course of the estrogen-induced uterotrophic response was associated with increases in glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase activities with the increased GSH level preceding the increase in uterine weight. Compared to neoplasms from intact or ovariectomized animals, tumors from estrogen-treated hosts exhibited significant decreases in levels of GSSG and GSH, as well as in glutathione reductase and glutathione peroxidase activities, but demonstrated a significant elevation of γ-glutamyl transpeptidase activity. Normal glands from lactating rats had decreased GSH levels, lower activities of glutathione reductase and glutathione peroxidase, but elevated γ-glutamyl transpeptidase activity versus tumors from intact rats. Tumors from estrogen-treated rats more closely resembled mammary glands during lactation. The divergent growth responses elicited by estrogen in the uterus and mammary tumor are correlated with the observed changes in GSH levels and enzymes involved in glutathione metabolism.     

Hormone-dependent changes of blood vessels in DMBA-induced rat mammary carcinoma and its regression studied by 3H-thymidine autoradiography

Using DMBA-induced rat breast cancer, the changes in the histology and proliferative activity underlying the phenomenon of tumor regression by hormone therapy were studied by 3H-thymidine autoradiography. The control tumor was found to essentially consist of two histologically different areas, medullary (A area) and tubular or cystic (B area). The cancer cells in the A area were homogeneously proliferating with a cell cycle time of 51h, and among those in the B area, 65% were proliferating with a cell cycle time of 81h while 35% were non-proliferating. Among the various-kinds of hormone therapies, ovariectomy plus male sex hormone administration was most effective in inducing tumor regression. In the regressed tumor, the A area was greatly diminished due to central necrosis and replaced by cystic B area. In the remaining A area, the cell cycle time was lengthened to 97h, and that for the proliferating cells in the B area was as long as 118h. The most striking histological change after ovariectomy plus male sex hormone administration was the diffuse necrosis of the capillary endothelial cell within 24h, followed by hemorrhage, central necrosis in the A area (1W), and final stage of fibrosis (2W). The tumor administered with female sex hormone after ovariectomy showed a rebound growth from the regression, due to the initial reactivation of the endothelial cell proliferation and following stimulation of cancer cell mitotic activity. From these observations, it is concluded that the capillary endothelial cells in DMBA-induced rat breast cancer are estrogen dependent, and that the tumor regression induced by decreased estrogen-level is attributable to the massive necrosis from capillary insufficiency and anoxia.