Effect of progesterone on cell proliferation in the MXT mouse hormone-sensitive mammary neoplasm.

The transplantable MXT mouse mammary tumor is an experimental model for which the growth is modulated by ovarian hormones; because of scant knowledge about the effects of progesterone (Pg) alone on cancer cells, the present investigation was made. By measuring the percentage of cells with labeled nuclei after tritiated thymidine ([3H]dThd) injection 1 hour prior to sacrifice [dThd labeling index (TLI)], a study was made of the effect (in vivo) of a near physiologic dose of Pg (125 micrograms ip) on cell proliferation in uteri (as the control Pg target organ) and on tumors of spayed (C57BL female X DBA/2F male)F1 mice. Pg induced a significant and transient rise in TLI, which increased from the 12th to the 24th hour after its injection. This mitogenic effect on tumors was comparable to that elicited by 0.25 microgram 17 beta-estradiol injected under similar conditions. In vivo brief exposure of castrated mice to Pg induced a significant mitogenic effect on MXT tumors. These observations might have important consequences for a better understanding of the endocrine mechanisms involved in human hormone-dependent breast cancer.

Synergism or antagonism between estradiol and progesterone on the cell kinetic parameters of the MXT mouse mammary tumor.

With the use of an in vivo tritiated thymidine ([3H]dThd) nuclear labeling followed by autoradiography, the effects at different times before sacrifice of single or paired injections of 17 beta-estradiol (E2) and/or progesterone (Pg), at different concentrations, were studied in (C57BL x DBA/2f)F1 (B6D2F1) mice transplanted with the MXT hormone-sensitive mammary tumor. Uteri were chosen as controls for the methodology. With regard to MXT tumors, E2 (0.25, 2.50, or 25.00 micrograms/animal) or Pg (125, 600, or 5,000 micrograms/animal) exerts almost a similar mitogenic influence that is dose related to the former and nor for the later, at least within the range of concentrations studied here; and the mitogenic effect of Pg seems to appear earlier than that of E2. When these steroids are concomitantly given, no obvious synergistic or antagonistic effect can be observed. A second E2 administration seems to have a less pronounced effect on cell proliferation as compared to that exerted by the first injection performed 12 or 24 hours earlier. On the contrary, the repetition of a Pg treatment would rather exert an additive or even a synergistic effect on the tumoral dThd labeling indices with regard to the total duration of stimulation. When one of these two steroids is administered first, it blocks totally the mitogenic effect of the second, provided the latter is given 24 hours later. It is concluded that E2 and Pg have almost a similar mitogenic effect on the MXT tumor and that these hormones exert complex interactions that are probably very important for the growth regulation of this cancer. Further investigations are needed to better understand the precise biochemical mechanisms involved in the modulating actions of these steroids on the MXT mammary growth.

Effect of castration and 17 beta-estradiol pulse on cell proliferation in the uterus and the MXT mouse mammary tumor.

The effects of a single 17 beta-estradiol (E2) injection on cell proliferation were studied in 3 groups of 30 mice transplanted with the MXT ovary-dependent mammary tumor. In group A, all animals were castrated prior to tumor implantation; groups B and C had intact ovaries at the time of transplantation, but group B was left intact throughout the experiment, while group C underwent castration 4 weeks later. On day 40 in groups B and C and on day 80 in group A, in which tumor development was significantly delayed, the same procedure for testing the effects of E2 was applied: Ten controls received 0.1 ml saline ip and were killed on the next day; 4 lots of 5 mice received 0.25 micrograms E2 ip and were killed one by one at 12-hour intervals. Exactly 1 hour prior to sacrifice, each animal received 25 microCi [methyl-3H]thymidine ip. Histologic sections of tumors and uteri were processed for autoradiography, and nuclear thymidine (dThd) labeling indices (LI) were determined. All tumors of group A grafted under unfavorable hormonal conditions were poorly differentiated, and E2 injection induced no appreciable changes in their dThd LI. Tumors B and C were well-differentiated adenocarcinomas, in which E2 induced significant modifications of cell proliferation. In group B, complex changes in dThd LI occurred in tumors as well as in uteri, probably due to interferences with the ovarian hormonal production. In group C, E2 produced a marked rise in dThd LI in tumors, lasting from the 12th to the 36th hour after its injection. Stimulation was maximum at the 24th hour, representing a 2.8-fold increase over mean basal dThd LI. It is concluded that the presence of an intact ovarian function at the time of transplantation is critical for maintaining the properties of hormone dependence in MXT tumors. In mice castrated after tumor implantation, a single E2 injection induces a marked and partially synchronous proliferation of neoplastic cells. The hypothesis that such hormonal manipulation might amplify the killing effect of cell cycle- or phase-specific cytotoxic drugs could be adequately tested with this model.

Effects on insulin, prolactin, progesterone, and estradiol on DNA synthesis in organ culture of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

The effect of various hormones or hormone combinations on DNA synthesis was investigated in organ cultures of 20 dimethylbenz(a)anthracene-induced rat mammary tumors. Three tumors were insulin independent and were totally insensitive to all other hormones tested. Seventeen tumors were insulin dependent for DNA synthesis and, in the presence of insulin, displayed variable responses to the other hormones. Nine of 12 such tumors were significantly stimulated by the combination of prolactin and progesterone. Given alone, these hormones were effective in only 25% of the tumors tested. Estradiol used at 2 dose levels, 0.001 or 1.0 mug/ml, acted in a reverse manner to progesterone and proved inhibitory in combination with prolactin in 40% of cases. It was ineffective alone except in 1 of 10 cases in which a stimulatory effect was recorded. A comparison in 4 tumors between estimation of DNA synthesis ([3H]thymidine incorporation into DNA) and colchicine-blocked mitoses demonstrated a good concordance. These results are discussed in terms of variations in the degree of hormone responsiveness of individual tumors and of the known hormone-dependent properties of the 7,12-dimethylbenz(a)-anthracene tumors in vivo.

Estradiol-dependent collagenolytic enzyme activity in long-term organ culture of human breast cancer.

An organ culture method suitable for the maintenance of viable human breast cancer for at least 14 days has been described. This method was applied to a total of 94 breast cancer specimens. It allowed good survival of "soft" tumors of various histological types, with loose connective stroma even in hormone-free medium. In contrast, "scirrhous" cancers showed poor survival in hormone-free medium; viable cells were maintained only at the very periphery of the explants. Supplementation of the medium with insulin (10 mug/ml), ovine prolactin (5 mug/ml), and hydrocortisone (1 mug/ml) in various combinations seemed to induce enlargement of viable cancer cells and moderate loosening of the stroma in some cases. However, it did not improve the survival of central tumor cords in scirrhous explants. Further supplementation of the medium with 17 beta-estradiol (minimum effective dose, 0.1 to 10 ng/ml), although it did not affect soft tumors, markedly improved survival of the cancer cells of scirrhous tumors throughout the whole explants, with evidence of collagen digestion around the neoplastic cells. This was observed in 18 of 20 scirrhous cancers subjected to this treatment. Estradiol need not be present during the whole culture period; the results at 14 days were identical in explants treated with estradiol for the first 7 days only or for the entire period. Addition of purified collagenase during the first 24 or 48 hr of culture resulted in complete dissolution of the collage. After such treatment, culture under the usual conditions resulted in excellent survival of the explants without improvement from hormone supplementation; thus, while estradiol was necessary when collagen was present, it was not longer required after collagen digestion. It can be concluded that breast cancer cells in organ culture are only slightly, or not at all, hormone dependent for survival, provided that they are not restrained by a dense collagen barrier. The estrogen-induced changes allowing survival inside the scirrhous explants strongly suggest the presence of an estrogen-dependent collagenolytic enzyme system in the collagen-rich breast cancers. This system could represent an important component of the hormone dependency of human breast cancer growth.

Physiological and pharmacological effects of estrogens in breast cancer.

PIP: Focus here is on the mechanism of action of both estrogens and antiestrogens at the tumor cell levels in breast cancer. The interactions of estrogens and their antagonists are emphasized and analyzed in terms of current and potential clinical applications to breast cancer treatment. This review deals with these interrelationships at the molecular levels, not just with general aspects of endocrine interrelationships. The article is divided into 8 main parts: 1) an introduction, which reviews historical understanding of receptor technology and significances; 2) main properties of estrogens and estrogen receptors; 3) the influence of estrogens and antiestrogens on growth of experimental mammary tumor systems; 4) the suppression of or administration of estrogens for treatment of advanced human breast cancer; 5) estrogen receptivity of mammary tumors; 6) progress in treatment of advanced breast cancer derived from studies on the mode of action of estrogens; 7) the prognostic significance of estrogens and estrogenic receptivity (the estriol theory); and 8) concluding remarks on the future paths of receptor research.^ieng

Assessment of estrogenic recruitment before chemotherapy in advanced breast cancer: a double-blind randomized study. European Organization for Research and Treatment of Cancer Breast Cancer Cooperative Group.

PURPOSE: In the present phase III study, the specific effect of estrogenic recruitment was assessed by comparing two groups of patients with advanced breast cancer receiving either ethinylestradiol (EE2) or placebo (PL) before chemotherapy (CT). PATIENTS AND METHODS: The therapeutic regimen consisted of (1) estrogen suppression by aminoglutethimide (AGL) 1 g/d plus hydrocortisone (HC) 40 mg/d, with surgical castration performed on premenopausal patients; (2) fluorouracil (5-FU) 500 mg/m2, doxorubicin 50 mg/m2, and cyclophosphamide (CPA) 500 mg/m2 (FAC) intravenously (IV) every 3 weeks; (3) following randomization, patients were double-blinded to receive either PL or EE2 50 micrograms exactly 24 hours before receiving FAC. All patients had advanced breast cancer presumably sensitive to endocrine therapy (estrogen receptor-positive [ER+] and/or progesterone receptor-positive [PgR+] status) with measurable lesions; none had received prior systemic antineoplastic therapy for metastatic disease; prior adjuvant hormonal therapy (HT) or CT (without anthracyclines) was allowed if interval since completion was longer than 1 year. RESULTS: Among 154 patients treated according to the protocol, tolerance, response rates, time to progression, and median survival duration were identical in the PL and EE2 groups. Only performance status, dominant metastatic site, and menopausal status seemed to influence response (overall response, 64%), with the highest levels of partial remission (PR) and complete remission (CR) being achieved in premenopausal women (CR plus PR, 26% plus 55%) and in those with dominant soft tissue lesions (CR plus PR, 45% plus 28%). CONCLUSION: We conclude that the validity of the hormonal recruitment concept has not yet been established in clinical practice so that this approach remains experimental. The results achieved by combining (near) complete estrogenic suppression and cyclical FAC chemotherapy are not significantly different from those to be expected with the more conventional use of HT followed by CT in presumably hormone-responsive (ER+) patients.

Assessment of estrogenic recruitment before chemotherapy in advanced breast cancer: preliminary results of a double-blind randomized study of the EORTC Breast Cancer Cooperative Group.

We investigated whether estrogenic recruitment could enhance the antitumor effect of chemotherapy in 165 patients with advanced breast cancer, presumably sensitive to hormonal treatments (ER + and/or PgR + lesions). The therapeutic regimen consisted of: (a) estrogenic suppression by aminoglutethimide 1 g/day + hydrocortisone 40 mg/day; surgical castration in premenopausal patients only; (b) FAC (5FU 500 mg/m2; ADM 50 mg/m2; CPA 500 mg/m2) for 3 weeks; (c) following randomization, exactly 24 h prior to chemotherapy, patients had to take 1 tablet of either placebo (PL) or 50 microgram ethinylestradiol (EE2). Tolerance, responses, time to progression and median survival were identical in both groups. Thus, EE2 before chemotherapy did not contribute to the efficacy of this particular therapeutic regimen, which yielded an overall response rate of 64%. We conclude that the validity of the hormonal recruitment concept has not yet been established in clinical practice, so that this approach remains experimental.

Effect of estradiol on proliferation and on cell loss in the MXT mouse mammary tumor.

The MXT tumor is an experimental mammary tumor, maintained by transplantation in B6D2F1 mice, which contains significant amounts of estrogen and progesterone receptors. The aim of the present work was to study the effect of estradiol (E2) administration on the cell kinetic parameters (thymidine labeling index: TLI, S phase duration: TS, cell cycle duration: Tc, growth fraction: GF, potential doubling time: T, cell loss factor: luminal diameter) of an MXT anaplastic carcinoma strain. These parameters were analyzed using single-, double- or repetitive-tritiated thymidine (3H-TdR) labeling techniques. Uteri were used as controls for the methodology. Our data clearly demonstrate that E2 exerted a marked stimulatory effect on the tumor cell proliferation. This effect is the net result of a shortening of the mean cell cycle duration (Tc) and of a 3-fold increase of the number of cells produced per time unit in 89% of the total cancer cell population. Beside this stimulatory effect, E2 produces a marked increase of the tumoral cell loss. This loss seems to affect a cell fraction which has reached the quiescent state (Q) for a short time.

Phenotypic change of the transplantable MXT mammary adenocarcinoma into mixed bone producing sarcoma-like tumors.

The B6D2F1 mouse mammary adenocarcinoma was adapted to grow in vitro as monolayer. After in vitro passaging of tumor cells, phenotypic changes occurred that were expressed in vivo. Following intraperitoneal inoculation of tumor cells, bone-forming tumors developed. These tumors consisted of undifferentiated adenocarcinoma mixed with large amount of cartilagenous and osseous tissue. The etiology of these phenotypic changes was not yet determined. However, hypothesis of the possible origin of the cartilage and bone forming tissue was formulated. The biologic characterization of the intraperitoneally bone-forming tumor was achieved and the experimental conditions to preserve and induce the reproducible sarcoma-like bone forming tumors were defined. Our data support the usefulness of this new original model for fundamental research as well as for screening of anticancer drugs.

Effects of aminoglutethimide plus hydrocortisone on the genital tract of postmenopausal women with advanced breast cancer. A clinical and cytologic survey.

In this study we compared the effects of either Aminoglutethimide (AGL) or Tamoxifen (TAM) therapy on the genital tract of postmenopausal women with advanced breast cancer. Thus, 15 patients treated with AGL, and 10 patients treated with TAM underwent gynaecological examination, during which vaginal smears were taken. All smears were reviewed in blind by one pathologist for determination of karyopycnotic indices (KI). Under TAM, no significant clinical abnormality was observed, except in one patient who had a small (histologically benign) endocervical polyp, easily removed during the examination. As reported by others, smears made under TAM therapy were generally characterized by high KI, indicating that an hormonal, estrogen-like stimulation remained present in these patients. On the contrary, most women under AGL had some evidence of vulvovaginal atrophy, which was unvariably associated with low KI on smears. Among the latter, four had severe dystrophic lesions consisting of leukoplasia (1), kraurosis (2) or lichen sclerous and atrophicus (1). It is therefore recommended not to neglect the systematic practice of gynaecological examination in patients with advanced breast cancer under endocrine therapy. These observations also indicate that AGL and TAM exert entirely opposite effects on the vaginal mucosa, which is a very sensitive estrogen-target tissue. In good agreement with former endocrine studies, AGL acts as a potent suppressor of estrogens resulting in severe mucosal atrophy. On the contrary, TAM seems nearly always to display some agonistic hormonal stimulation.

Implications of adjuvant hormonal and cytotoxic chemotherapy.

The implications of medical adjuvant treatments of primary breast cancer are best appreciated from an analysis of the available data on rates of local recurrence and survival after mastectomy. Breast cancer may be considered as a systemic disease from the time of clinical presentation in most cases and its cure rate has been estimated at 18% for all cases and 30% for stages I and II combined. Advancing age seems attended by an increasing rate of dying from breast cancer. Local recurrences appear to obey the same mechanisms that govern the occurrence of distant metastases. Their appearance after mastectomy has an average or median delay of one to two years. These data are important factors that condition the indication and planning of reconstructive surgery. They form the background on which adjuvant treatments will exert their influence. Prophylactic castration is of little or no value in altering survival after mastectomy. In contrast, intensive long-term combination chemotherapy of the CMF type seems to increase the relapse-free survival and overall survival, and to decrease the rate of local recurrence specifically in premenopausal patients. It is little or not effective after the menopause. Intensive research is needed to develop better modalities of systemic adjuvant treatment in order to improve the curability of breast cancer.

[Hormonal receptors of malignant breast tumors]. / Les récepteurs hormonaux des tumeurs malignes du sein

Tissue samples from 166 primary and 136 metastatic breast cancers were analysed for the presence of estrogen receptors. It was found by measuring the affinity of the cytoplasmic fraction of these samples for 3H-estradiol-17 beta that receptors were present in 72 p. 100 and 54 p. 100 of primary and metastatic cancers respectively. Receptor concentration varied among sample in an apparently continuous distribution from zero to 2,080 femtomoles per mg tissue protein. This suggests that mammary tumors are different from one another more in a quantitative than in a qualitative way. Detectable amounts of receptors were found in samples from mammary dysplasia, fibroadenomas as well as from one papilloma; none was detected in samples from non-tumorous mammary gland, nipple areola or skin. At mastectomy, no correlation was found between presence or absence of receptors in the primary tumors, and presence or absence of metastatic axillary nodes. On the other hand both the primary and its axillary metastases almost always displayed the same characteristic as far as presence or absence of receptors was concerned. Analysis of clinical studies reported seems to indicate that women with advanced breast cancer respond in a fair proportion of cases to various endocrine treatments when tumor tissue biopsies contain estrogen receptors whereas the probability of a response is very low in their absence.