Influence of some dietary chemopreventive agents on the expression of functional differentiation of the mouse mammary gland in vitro

The purpose of the present studies was to determine the influence of the chemopreventive agents selenium, 4-(hydroxyphenyl)-retinamide (4-HPR) and beta-carotene, on functional differentiation (lactogenesis) of the mouse mammary gland cells. The hormone-induced expression of the milk-protein genes, beta-casein, epsilon-casein and the whey acidic protein (WAP) was used as molecular marker of differentiation of the mammary cells in organ culture medium containing insulin, prolactin, aldosterone and hydrocortisone. Quantitative determination of the cellular concentration of the respective mRNA was ascertained by molecular hybridization of RNA to the specific cloned cDNA probes using both the solution and the filter hybridization methods. Selenium at 100 nm concentration caused a pronounced inhibition of accumulation of the respective mRNAs. The retinoid, 4-HPR, also caused a dose-dependent inhibition of expression of these mRNA sequences. In contrast, concentrations of the 3 mRNAs in beta-carotene-treated glands remained similar to those observed in glands cultured in medium containing the hormones and hexane (the latter being the solvent for beta-carotene). The significant antagonistic action of selenium and 4-HPR, however, was reversible after removal of the chemicals from the culture medium. Thus, among the 3 chemicals tested, selenium and retinoid can cause an adverse effect on functional differentiation (lactogenesis) of the mammary cells. This inhibitory effect, however, was reversible. beta-Carotene, on the other hand, caused no apparent antagonistic effect on expression of the milk-protein genes in the isolated whole mammary organ in culture.     

Cell cycle-related hormone carcinogen interaction during chemical carcinogen induction of nodule-like mammary lesions in organ culture.

The immature mammary glands of BALB/c female mice were treated with 7,12-dimethylbenz[a]anthracene (DMBA), 2 micrograms/ml, or 3-methylcholanthrene (10 micrograms/ml) for a 24-hr period at different times during the inital six days of lobuloalveolar growth in hormone-supplemented organ culture. Nodule-like alveolar lesions (NLAL) were detectable in 80% of the glands treated with DMBA (40% in 3-methylcholanthrene-treated glands) in the presence of insulin + prolactin + aldosterone + cortisol in the medium. No NLAL were present in dimethyl sulfoxide-treated control glands cultivated with the same hormones. The hormone combination insulin + prolactin + cortisol was unfavorable for NLAL induction by DMBA, and the combination of aldosterone + insulin + prolactin was only moderately conducive. Thus, the presence of cortisol with insulin + prolactin + aldosterone enhances NLAL incidence of mammary cells by DMBA. The highest incidence was found in glands that were treated with DMBA for 24 hr between the third and fourth day of culture, the period corresponding to the onset of the second wave of DNA synthesis in the gland. Cytotoxicity of DMBA was pronounced between 24 and 48 hr, when a high frequency of cells were in DNA synthesis, and survival of the cells after the cytotoxic effect of DMBA appeared to play a role in NLAL incidence. This suggests that DMBA-induction of NLAL in mammary glands in organ culture involves a complex carcinogen-hormone-cell cycle interaction. We emphasize that, although NLAL morphologically resembles the hyperplastic alveolar nodules of mouse mammary gland in vivo, the abilitity of NLAL to produce typical hyperactive alveolar outgrowth and mammary tumor after transplantation iv vivo remains to be determined.     

Role of the progesterone receptor (PR) in susceptibility of mouse mammary gland to 7,12-dimethylbenz[a]anthracene-induced hormone-independent preneoplastic lesions in vitro

Glands of wild-type (WT) and progesterone receptor knockout (PRKO) mice were exposed to 7,12-dimethylbenz[a]anthracene (DMBA) while cultured in serum-free medium containing insulin, prolactin, aldosterone, and cortisol. Glands of WT but not PRKO mice responded to DMBA with epithelial hyperplasia after 10 days in this medium. After culture without prolactin and adrenocortical hormones for an additional 14 days, hyperplastic lesions were present only in glands of WT mice. We conclude that in the absence of PR, epithelial structures are resistant to the carcinogenic action of DMBA.     

Bowman-Birk protease inhibitor and its palmitic acid conjugate prevent 7,12-dimethylbenz[a]anthracene-induced transformation in cultured mouse mammary glands

The chemopreventive effects of Bowman-Birk protease inhibitor (BBI), a soybean polypeptide, and its palmitic acid conjugate, Pal-BBI, on 7,12-dimethylbenz[a]anthracene (DMBA)-induced transformation were investigated by using an in vitro whole organ culture system of mouse mammary glands. The transformation incidence was measured as the number of glands containing nodule-like alveolar lesions (NLAL). Both BBI and Pal-BBI were effective in preventing DMBA-induced transformation, especially when added to the medium during the developing period after the exposure of mammary glands to DMBA, i.e. 35.9 and 53.4% prevention, respectively. However, when only present in the medium before the promotion period, Pal-BBI was effective in decreasing the transformation incidence (32.2%) while BBI was not (10.3%). This difference could possibly be due to the high lipophilicity and tissue retention of Pal-BBI in mammary gland cultures.     

Long-term cultivation of a human breast cancer cell line, MCF-7, in a chemically defined medium. Effect of estradiol

The human breast cancer cell line, MCF-7, has been adapted to long-term growth in chemically defined medium without loss of estrogen and progesterone receptors or tumorigenicity in athymic mice. An estrogen reversible inhibition of cell proliferation is exerted by newborn calf serum (NCS) 10%, athymic mouse serum (AMS) 2% or tamoxifen 0.1 to 1.0 microM. The mamma-related hormones, hydrocortisone, progesterone, prolactin, and insulin could not mimic this growth inhibitory effect, and estradiol alone or combined with these hormones did not stimulate cell proliferation in chemically defined medium.     

Plasma hormone levels and the incidence of carcinogen-induced mammary tumours in two strains of rat.

The incidence of mammary tumours developing after administration of the carcinogen DMBA (at 50 days of age) has been determined in 2 strains of Sprague-Dawley rat. Untreated animals of each strain were exsanguinated in dioestrus at a time corresponding to the early post-carcinogen stage (at 70 days of age) and the plasma concentrations of prolactin, oestradiol-17B and progesterone were measured by radioimmunoassay. In an inbred strain of rats, tumour-induction rate was 6-4% and plasma prolactin concentration was 2-5 x lower than that found in a random-bred strain with a tumour-induction rate of 41-6%. No difference was found between the 2 strains in the level of either ovarian hormone. It is concluded that the difference between these strains in mammary gland susceptibility to DMBA may be related to plasma prolactin concentration, but it is unlikely to be determined by the ovarian hormones.     

Absence of an effect of dietary corn oil content on plasma prolactin, progesterone, and 17 beta-estradiol in female Sprague-Dawley rats

Enhancement of mammary tumorigenesis in rats by high-fat diets has been postulated to be due to altered hormonal status. Elevated serum prolactin and, in some cases, estrogen have been reported in rats fed diets high in corn oil or lard that increase 7,12-dimethylbenz(a)anthracene (DMBA) tumorigenesis. However, we have found no difference in plasma prolactin during the proestrus surge or at the other stages of the estrous cycle in rats fed a diet high (24%) in corn oil that augments DMBA-induced tumorigenesis or a control diet containing 5% corn oil. There was no effect of the same dietary treatment on plasma progesterone or serum estradiol in the same experiments. In addition, we found that DMBA administration did not change the blood concentration of any of the three hormones. Female Sprague-Dawley rats, bearing atrial cannulae and given a carcinogenic dose of DMBA at 8 weeks of age, were studied 2 to 5 weeks or 10 to 13 weeks after DMBA administration. Rats given only the oil vehicle for DMBA were studied at the same ages. Blood samples were taken from rats with 4-day estrous cycles at 3-hr intervals on the day of proestrus and at 6- to 12-hr intervals on the other days of the cycle. No effect of dietary corn oil content or of DMBA administration on plasma prolactin and progesterone was detected in either age group. On the afternoon of proestrus, rats were sampled more frequently to examine the hormonal patterns in detail; again, no dietary effect was detected. Serum estradiol was measured in rats that were fed the control or high-corn-oil diets, treated with DMBA or vehicle, and decapitated on one of the 4 days of the estrous cycle. There was no detectable effect of dietary corn oil content or DMBA treatment during any stage of the cycle. All three hormones showed qualitative and quantitative patterns identical to normal cycling rats at both ages studied. Our results indicate that earlier reports of an effect of dietary fat on blood prolactin and estrogen content may have been due either to diets low in essential fatty acids or to anesthesia-induced hormonal responses.     

Influence of underfeeding during the "critical period" or thereafter on carcinogen-induced mammary tumors in rats

Normal cycling virgin female Sprague-Dawley rats were divided into different treatment groups and subjected to half ad libitum feed intake for 2 or 4 weeks at consecutive periods of time before and/or after administration of 7,12-dimethylbenz(a)anthracene (DMBA) and then were returned to full-feed. All rats at the end of their respective under-feeding periods showed reductions in serum prolactin and cessation of estrous cycles. However, only rats underfed 1 week prior to and 1 week after DMBA administration showed significant reductions in mammary tumorigenesis or the entire 21 weeks of the experiment when compared to full-feed controls. Rat groups underfed during subsequent weeks after DMBA administration showed no alterations in mammary tumor development as compared to full-fed controls. These results demonstrate that inhibition and perhaps permanent suppression of mammary tumorigenesis occurred only in rats underfed 1 week before and 1 week after DMBA administration. The inhibitory effects produced by this under-feeding regimen on mammary tumorigenesis may be mediated by suppression of prolactin and estrogen secretion.     

Enhanced inhibition of mammary carcinogenesis by combined treatment with N-(4-hydroxyphenyl)retinamide and ovariectomy

Bilateral ovariectomy and dietary administration of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) are both effective inhibitors of chemical carcinogenesis in the rat mammary gland. The present study was designed to determine whether an enhanced inhibitory effect is obtained with combined ovariectomy and 4-HPR administration, compared to either treatment alone. In separate experiments, 50-day-old virgin female Sprague-Dawley rats received either a single i.v. injection of 50 mg N-methyl-N-nitrosourea per kg body weight or a single intragastric dose of 20 mg 7,12-dimethylbenz(a)anthracene. The experimental design was the same in both the N-methyl-N-nitrosourea and 7,12-dimethylbenz(a)anthracene experiments: Group 1, 25 intact rats, placebo diet; Group 2, 25 intact rats, supplement of 782 mg 4-HPR per kg diet; Group 3, 50 ovariectomized rats, placebo diet; Group 4, 50 ovariectomized rats, supplement of 782 mg 4-HPR per kg diet. Feeding of the 4-HPR supplement was begun 7 days after carcinogen administration; ovariectomy was performed 7 days post-7,12-dimethylbenz(a)anthracene or 14 days post-N-methyl-N-nitrosourea. In both experiments, combined ovariectomy plus 4-HPR was significantly more active in suppressing mammary cancer induction than was either manipulation alone. 4-HPR was a more effective inhibitor of carcinogenesis in ovariectomized rats than in intact animals. These data indicate that 4-HPR is highly effective in inhibiting ovarian hormone-independent cancers and suggest that retinoid inhibition of mammary carcinogenesis does not involve an influence on ovarian hormone action.     

An expression study of hormone receptors in spontaneously developed,carcinogen-induced and hormone-induced mammary tumors in female Noble rats

The Noble (Nb) rat model has been used in the study of hormonal carcinogenesis of mammary and prostate glands, as this rat strain is susceptible to tumor induction in these glands by hormonal treatments. Recently, we demonstrated that this rat strain can develop spontaneously mammary tumors at high incidence in aged animals and also show high sensitivity to chemical carcinogens (DMBA and MNU) and combined treatments with sex hormones in mammary tumor induction. In the present study, we examined and compared the expression of hormone receptors [including estrogen receptors (ERalpha and ERbeta), androgen receptor (AR), progesterone receptor (PR), prolactin receptor (PRLR)] and prolactin (PRL) by immunohistochemistry, Western blotting and RT-PCR in spontaneous mammary tumors, and mammary tumors induced by sex hormones (T+E2 and T+DES for 8-10 months) and DMBA in Nb rat model. Immunohistochemistry and Western blotting showed that both the spontaneously developed and hormone-induced carcinomas exhibited strong immunoreactivity of ERalpha, ERbeta, AR, PR and PRLR, while the spontaneous fibroadenomas showed weak to moderate immunoreactivity of ERalpha and PRLR, whereas the DMBA-induced carcinomas exhibited weak to moderate immunoreactivity of ERalpha, AR, PR and PRLR, and sporadic weak ERbeta immunoreactivity. RT-PCR analyses showed that mRNA expression pattern of these markers resembled that of proteins. In addition, weak mRNA expression of PRL was detected in spontaneous carcinomas and carcinomas induced by DMBA and hormones, suggesting that PRL could be produced locally within the tumors. The results showed that the expression status of hormone receptors and PRL was different in spontaneous mammary tumors and tumors induced by carcinogen or hormones, suggesting that the extent of involvement of steroid hormones and their receptors in the spontaneous, carcinogen- or hormone-induced mammary carcinogenesis might be different.     

Reduction by pituitary isograft of inhibitory effect of large dose of estrogen on incidence of mammary tumors induced by carcinogen in ovariectomized rats

Interaction of prolactin and estrogen on the incidence of mammary tumors induced by 7,12-dimethylbenz (a) anthracene (DMBA) in female Sprague-Dawley rats was studied. Rats were bilaterally ovariectomized at 45 days of age and then treated subcutaneously with 20μg of estradiol benzoate (EB) every 2 days beginning the next morning. They received single intravenous injections of 5mg DMBA at 52 days of age. Two months later they were divided into four groups: Group I received no pituitary grafting: group II was grafted with three isologous pituitaries under the right kidney capsule: groups III and IV received six pituitaries each. EB injections were continued throughout the experiment to every group except group IV. Serum prolactin levels were determined by radioimmunoassay just before pituitary grafting, 20 days, 2 and 3 months after grafting. At the end of 5 months after DMBA injection, the mammary tumor incidence and the number of palpable tumors per tumor-bearing rat apparently increased, and the percentage of completely regressed tumors decreased in groups II and III as compared to group I. Serum prolactin level was significantly higher in groups II and III than in group I at 3 months after pituitary grafting. Only one rat had palpable mammary tumors in group IV and serum prolactin levels were always significantly lower in this group than in the others. These results indicate that prolactin secreted by the grafted pituitaries overcomes the inhibitory action of a large dose of estrogen on the growth of DMBA-induced mammary tumors of the rat and results in an increased incidence of the tumors.     

Antagonistic action between cyclic AMP and estrogen in phosphorylation of mammary tumor nuclear proteins.

A new protein kinase-dependent phosphorylation occurs in the nuclei of hormone-dependent, 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced mammary carcinoma following preincubation of tumor slices with cyclic adenosine 3',5'-monophosphate (cAMP). The presence of 17beta-estradiol in the medium inhibits this effect. Both events have been observed in vivo in the nuclei of DMBA-induced tumors. The phosphorylation pattern of nuclei in hormone-independent mammary tumor, DMBA No. 1, however, is not affected by preincubation with either cAMP or estrogen. These findings suggest that the antagonistic effect of cAMP and estrogen in the growth control of mammary tumors is exerted through a specific action on nuclear protein phosphorylation and that these events correlate with the hormone-dependency of the tumors.     

N-Nitrosodiethylamine-induced nodule-like alveolar lesion and its prevention by a retinoid in BALB/c mouse mammary glands in the whole organ in culture

The ability of N-nitrosodiethylamine (DENA) to induce transformationof the mammary cells was studied in culture of the whole mammaryorgan from BALB/c female mice. Incidence of nodule-like alveolarlesions (NLAL) in the glands in vitro has been used as a measureof transformation. NLALs are analogous to the precancerous hyperplasticalveolar nodules (HAN) of mouse mammary gland in vivo. The mammaryglands were treated with graded concentrations (0.1–2.5µg/ml) of DENA during lobuloalveolar morphogenesis inmedium (Waymouth's MB752/1) containing insulin, prolactin, hydrocortisoneand aldosterone. DENA treatment caused a dose-related increasedoccurrence of NLAL in the glands in vitro and concentrationof 1.5 µg/ml produced the highest incidence of 85%. Thehigh incidence of NLAL was accompanied by a 3-fold increaseof DNA repair activity in the DENA treated glands. Incubationof the glands for 6 days after DENA treatment in medium containingN-(4-hydroxyphenyl)retinamide and the same hormone mixture caused61% inhibition of NLAL incidence. The results indicate thatDENA is capable of inducing a high level of transformation ofthe mammary epithelial cells in vitro and that this retinoidcan inhibit expression of the transformed cells acting at thepromotional level.     

4-(hydroxyphenyl)retinamide selectively inhibits the development and progression of ductal hyperplastic lesions and carcinoma in situ in mammary gland.

In most previous chemoprevention studies on inhibition of mammary carcinogenesis, the formation of palpable tumors has been used as an end-point. Little is known about whether chemopreventive agents may similarly or selectively suppress hyperplastic and premalignant stages of the neoplastic process. In this study, we evaluated the effect of 4-(hydroxyphenyl)retinamide (4-HPR) on the development and progression of hyperplastic lesions and carcinoma in situ (CIS) in the N-methyl-N-nitrosourea (MNU) mammary carcinogenesis model in rats. 4-HPR was used as the chemopreventive agent because of its proven inhibitory effect on both the early and late phases of mammary carcinogenesis. Treatment with 4-HPR (2.0 mM/kg diet), beginning 2 days after MNU administration and administered continuously for 10 weeks, suppressed all mammary gland lesions (hyperplasia, CIS and invasive carcinoma) in 35% of animals. In the remaining 65%, 4-HPR allowed the development of hyperplastic lesions, alone or combined with CIS, and/or invasive carcinomas (CA). 4-HPR also increased by 2-fold the ratio between CIS and CA (0.75 per animal in control versus 1.5 in 4-HPR-treated animals), suggesting that it may also suppress the transition of CIS into CA. 4-HPR, when administered beginning 4 weeks after MNU administration [when hyperplastic and premalignant (CIS) lesions are present in the mammary gland], inhibited the frequency of terminal end bud hyperplasia (TEBH) and CA but did not significantly suppress ductal hyperplasia, ductal alveolar hyperplasia, alveolar hyperplasia and CIS. In these animals, 4-HPR induced partial disintegration of mostly peripheral areas of lesions, including carcinomas. Taken together, our data indicate that 4-HPR selectively suppresses the development and progression of hyperplastic lesions and CIS in TEBs. Furthermore, it appears that, in addition to mammary carcinomas, TEBH and CIS could also be used as end-point biomarkers in breast cancer chemoprevention studies.     

Stimulation of mammary tumourigenesis and inhibition of uterine adenomyosis by suppressed progesterone effects in SHN mice

It has been shown in mice that progesterone stimulates both mammary tumourigenesis and uterine adenomyosis in intact animals in the presence of normal levels of prolactin and oestrogen. From the view-point of the high dependence of progesterone upon other hormones, the effects of suppressed progesterone action on mammary and uterine lesions were studied in this paper. Subcutaneous implantation of RU 486, an antiprogesterone, enhanced preneoplastic and neoplastic mammary gland growth in SHN virgin mice associated with elevated plasma prolactin levels and continued vaginal oestrous stage. This mammary gland growth stimulated by RU 486 was arrested by additional treatment with progesterone. On the contrary, RU 486 treatment markedly suppressed the development of uterine adenomyosis, which was counteracted only by progesterone supplement for a long period in old mice. The previous and the present findings conclude that progesterone stimulates mammary tumourigenesis by acting synergistically with prolactin and oestrogen when the latter hormones are at normal levels, but it prevents tumourigenesis by acting antagonistically with high levels of prolactin and oestrogen. They further confirmed the importance of progesterone in the development of adenomyosis, while its relationship with prolactin and oestrogen is not understood so well as it is in mammary tumourigenesis.     

Effects of high dietary fat on the growth and development of ovarian-independent carcinogen-induced mammary tumors in rats

This study examined the influence of high dietary fat intake on the development of ovarian-independent mammary tumors in both vehicle-treated controls and rats made deficient in estrogen and prolactin during tumor induction. The majority of 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors in rats are dependent on estrogen and prolactin for growth, and suppression of prolactin and estrogen at the time of tumor initiation causes a reduction in tumor incidence and increase in tumor latency. However, the majority of mammary tumors which do develop in these animals exhibit ovarian-independent growth. Sprague-Dawley rats were given 7.5 mg DMBA p.o. at 57 days of age. Starting 1 day prior to and continuing for 7 days after DMBA administration, rats were given daily injection of vehicle or the combination of tamoxifen (20 micrograms/rat) plus bromocryptine (5 mg/kg). At the end of drug treatment, rats in each treatment group were equally divided and placed on normal fat (5% corn oil) or high fat (20% corn oil) diets for the duration of the experiment. Vehicle-treated rats were ovariectomized 27 wk and drug-treated rats 47 wk after DMBA administration to determine tumor ovarian dependency. Vehicle-treated rats fed high fat diets showed significant increases in mammary tumor incidence and number as compared to similarly treated rats fed a normal fat diet, with approximately 80% of the tumors in each group being ovarian dependent. Likewise, tamoxifen-bromocryptine-treated rats fed a high fat diet showed a significant enhancement in mammary tumor number, although not incidence, as compared to similarly treated rats fed a normal diet. Tumors in these drug-treated groups displayed essentially the same incidence of ovarian dependence (23%). Tamoxifen-bromocryptine-treated groups displayed a 2-fold increase in latency of tumor appearance as compared to vehicle-treated controls; however, this long latency was not reduced when these rats were fed a high fat diet. These results demonstrate that high dietary fat stimulates ovarian-dependent and -independent mammary tumorigenesis in rats but does not influence the hormonal responsiveness of these tumors.     

Effects of progesterone on mammary carcinogenesis by DMBA applied directly to rat mammae.

The effects and site(s) of action of progesterone on DMBA mammary carcinogenesis in the rat, when a small dose of the carcinogen was applied directly to the inguinal mammary gland, were investigated. No reduction in tumour yield was apparent when progesterone was administered s.c. for 18 days before dusting DMBA. This finding contrasts with a previously reported inhibitory effect on carcinogenesis when hormone treatment was followed by intragastric administration of DMBA. When progesterone injections were begun either 2 days before or 2 days after direct application of DMBA, and were continued until the end of the experiment (135 or 195 days) an enhancement in carcinogenesis was observed similar to that previously demonstrated after gastric intubation of DMBA. These findings, together with previously reported observations, suggest that progesterone may exert its inhibitory effect on carcinogenesis by acting at a site outside the breast, perhaps on the liver. However, it is likely that the hormone acts directly on the mammary tissue to exert its enhancing effect on tumorigenesis.     

Influence of pituitary grafts or prolactin administrations on the hormone sensitivity of ovarian hormone-independent mouse mammary MXT tumors

The sensitivity of MXT mouse mammary tumors to ovarian hormones was assessed by the following parameters: growth in vivo; presence or absence of estrogen receptors and progesterone receptors; and histological differentiation. A spontaneous evolution from hormone sensitivity (HS) to hormone independence (HI) was observed when the tumors underwent monthly transplantation onto intact recipient mice, with the tumors fulfilling the criteria of HI tumors after the 12th transplantation. In contrast, the tumors recovered most of the criteria of hormone sensitivity when pituitary isografts were placed under the kidney capsules of HI tumor-bearing animals or when these animals received daily administrations of prolactin over several months. Sensitivity to 17 beta-estradiol, progesterone, or prolactin was further assessed by actinomycin binding on the nucleus and thymidine labeling index, both measured by autoradiography. These technical approaches revealed that 17 beta-estradiol and prolactin stimulated the thymidine labeling index of both HI (despite the lack of detectable estrogen receptors) and HS MXT tumors whereas progesterone influenced only that of HS cancers. The three hormones significantly stimulated [3H]actinomycin D binding within HS tumors but not within HI ones. However, such "HI" tumors were characterized by increased actinomycin binding and thymidine labeling index in comparison with HS neoplasms. Thus, all the data presently reported strongly suggest that prolactin is able to restore the hormone-sensitive phenotype within so-called MXT hormone-independent tumors.     

Retinoids as chemopreventive agents for breast cancer.

Although several retinoids have been evaluated for prevention of mammary carcinogenesis in rats and mice, retinyl acetate (RA) and N-(4-hydroxyphenyl)retinamide (4-HPR) proved most effective. In rats, dietary administration of the retinoids reduced the incidence and number, and increased the latency of N-methyl-N-nitrosourea (MNU)-induced mammary cancers. 4-HPR reduced the number of hyperplastic alveolar nodules (HAN) in MTV- mice and the number of tumors in MTV+ mice. Other studies indicate that the synergistic effect of retinoid administration and hormonal deprivation is more efficacious in prevention of MNU-induced mammary cancer than either modality alone. Furthermore, retinoids alone and the combination of retinoid and tamoxifen inhibit the appearance of mammary cancers following the surgical removal of the first cancer as well as inhibit the growth of established cancers. Again, the combined modality was the most effective. Retinoids also exert an antiproliferative effect upon the mammary epithelium in vivo, which is represented morphologically by a bare duct system with little branching, end buds, and few, if any, alveoli. In organ culture, retinoids inhibit mammary end bud differentiation and proliferation induced by insulin and prolactin or carcinogens.     

Effect of retinyl acetate and 4-hydroxyphenylretinamide on initiation of chemically-induced mammary tumors

The anti-promotional effect of retinoids on chemically-induced mammary carcinogenesis in the rat is well established. The present studies were performed to determine the effect of long-term feeding of retinyl acetate and 4-hydroxyphenylretinamide (4-HPR) on initiation of mammary tumors induced by MNU or DMBA. Retinyl acetate (328 mg/kg of diet) or 4-HPR (782 mg/kg of diet) was added to the diet of female Sprague-Dawley rats for two months prior to the administration of the carcinogens. In the MNU model, a 50% increase in the number of mammary adenocarcinomas was observed in rats pretreated with retinyl acetate, while pretreatment with 4-HPR resulted in a 93% increase in the number of cancers. Continued treatment with 4-HPR throughout the study, however, caused a reduction in cancer number. In the DMBA mode, pretreatment with these retinoids significantly increased the number of benign mammary tumors, but not mammary cancers. These data suggest that newly synthesized retinoids should be evaluated for chemopreventive activity against mammary cancer initiation as well as for their anti promotional activity.