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L Demers

Publications and source records attributed to L Demers.

At least 19 recordsLinked to original sources
PubMed1992

Synchronization of breast cancer cell proliferation in vivo by combined hormonal and polyamine manipulation.

Optimal synchronization of breast cancer cell proliferation by hormonal means may be limited by cellular heterogeneity in sensitivity to the multistep activation of growth following initial hormone binding to the receptor. We hypothesized that induced synchronous growth may be improved by combined manipulation of the polyamine (PA) pathway since we have previously shown that PAs are distal effectors of hormonal action on proliferation in breast cancer. To test our hypothesis, we induced an initial phase of hormone and PA depletion (castration plus administration of the PA synthesis inhibitor alpha-difluoromethylornithine) in rats bearing N-nitrosomethylurea induced mammary tumors. This was followed by transition phase of hormone repletion in the presence of alpha-difluoromethylornithine (to push the cells into the proliferative cascade up to the distal step controlled by PA) and finally a phase of hormone and PA repletion. Simultaneously, groups of rats were subjected to hormone/PA depletion/repletion individually. The effects of these manipulations on the labeling indices (LIs) of glandular, myoepithelial, and nonepithelial cells were estimated by autoradiography. The combined hormone/PA manipulation yielded the highest degree of synchronization with LIs of the glandular and myoepithelial cells being approximately 2-fold over intact control after only 2 or 3 days of combined repletion. In contrast, hormone treatment alone restored the LIs of glandular cells only to control levels and minimally influenced those of myoepithelial cells. PA manipulation alone failed to affect the LIs of any cell type. Although the rate of tumor regrowth was highest with the combination treatment, the absolute tumor volumes did not differ significantly at the end of the repletion phase between the three regimens. These results indicate that combined hormone/PA manipulation provides the best "therapeutic window" (LI/tumor volume) for implementation of kinetically based cytotoxic chemotherapy.

Animals↗
PubMed1992

Role of polyamines in the growth of hormone-responsive and -resistant human breast cancer cells in nude mice.

Recent in vitro data suggest that at least some hormone-independent breast cancer cells exhibit increased polyamine biosynthesis and resistance to antipolyamine therapy. To address this issue under conditions of in vivo growth, we tested the antiproliferative effect of the polyamine synthetic inhibitor alpha-difluoromethyl-ornithine (DFMO) on hormone-dependent (MCF-7) and -independent (MDA-MB-231, BT-20) breast cancer cell lines growing in nude mice. We observed that DFMO significantly inhibited the growth of established tumors to a similar extent in all cell lines, even though tumor regression was only observed with MCF-7 cells. DFMO, while inhibiting E2-supported MCF-7 breast cancer growth, did not inhibit E2-stimulated progesterone receptor synthesis. Cellular levels of polyamines were highest in MCF-7 cells and lowest in the BT-20 cell line. Tumor content of spermidine was similarly suppressed by DFMO treatment in the 3 cell lines, while the spermine level was unaffected. Cellular putrescine levels were suppressed in MCF-7 and BT-20 cells. Administration of DFMO prior to implantation of fragments of MCF-7 or MDA-MB-231 tumors in nude mice significantly inhibited tumor development to a similar extent. The action of DFMO seemed to be predominantly tumoristatic since new tumors develop in some mice upon discontinuation of the drug. We conclude that the hormone-independent breast cancer cell lines tested do not exhibit increased polyamine biosynthesis or resistance to antipolyamine therapy when grown in vivo in nude mice.

Animals↗
PubMed1992

Elevated soluble c-erbB-2 antigen levels in the serum and effusions of a proportion of breast cancer patients.

PURPOSE: An enzyme-linked immunosorbent assay (ELISA) for the extracellular domain of the c-erbB-2 oncogene product was developed and evaluated to determine if soluble c-erbB-2 could be detected in the serum and effusions of cancer patients. PATIENTS AND METHODS: Sera from 208 previously untreated or progressing cancer patients and 69 healthy controls were assayed in a double-antibody sandwich ELISA that used two monoclonal antibodies to the native extracellular domain of the c-erbB-2 receptor. Fisher's exact test was used to analyze the statistical significance of the frequency of elevated serum c-erbB-2 levels. Immunoprecipitation and Western blotting were used to characterize further the c-erbB-2 immunoreactivity in the serum of four breast cancer patients. RESULTS: Sera from 12 of 53 patients (23%) with metastatic or locally advanced breast cancer, zero of 69 controls, one of 31 patients with ovarian cancer (3%), and two of 124 other cancer patients (2%) had soluble c-erbB-2 values greater than or equal to 5 U/mL. The number of breast cancer patients with elevated serum c-erbB-2 levels was significantly greater than that of the control group (P less than .0001), the ovarian cancer group (P less than .03), and the other cancers group (P less than .0001). Also, two of five effusions (40%) from breast cancer patients had an elevated soluble c-erbB-2 antigen level, compared with zero of 17 effusions from patients with benign diseases. Western blotting of four sera from breast cancer patients with elevated serum c-erbB-2 antigen levels produced bands of approximately 105 kD that seemed to correlate in intensity with increasing ELISA serum levels. CONCLUSION: Serum c-erbB-2 levels are elevated in approximately one fourth of patients with locally advanced or metastatic breast cancer.

Adult↗
PubMed1991

Polyamine involvement in the secretion and action of TGF-alpha in hormone sensitive human breast cancer cells in culture.

These experiments were designed to test polyamine (PA) involvement in the secretion and action of transforming growth factor alpha (TGF-alpha) in hormone responsive MCF-7 breast cancer cells in liquid culture. At the same time, we evaluated the influence of culture conditions (with serum vs. serum depleted) and subclonality of MCF-7 cells on PA involvement in estrogen (E2) and TGF-alpha stimulated cell proliferation. Despite inducing a profound suppression of cellular PA levels and inhibiting basal and E2-stimulated growth, administration of the PA synthesis inhibitor alpha-difluoromethylornithine (DFMO) did not influence either basal or E2-induced TGF-alpha secretion. In the same experiments, on the other hand, addition of DFMO completely blocked the growth stimulatory effect of exogenous TGF-alpha. However, when the culture conditions were changed to serum-free medium, TGF-alpha and E2-induced cell proliferation was affected modestly or not at all by DFMO administration, despite similar suppression of cellular ornithine decarboxylase (ODC) activity and PA levels. In addition, different clones of MCF-7 cells differed in their sensitivity to the antiproliferative effect of DFMO as well as in basal levels of ODC activity and PA. We conclude that PAs are not involved in basal or E2-stimulated TGF-alpha secretion in MCF-7 breast cancer cells. On the other hand, PAs do seem to be important mediators of TGF-alpha and E2-induced breast cancer cell proliferation, though the degree of such involvement appears to be influenced by serum factors and clonal variability of MCF-7 cells.

Breast Neoplasms↗
PubMed1990

Role of insulin-like growth factor-related peptides in the estradiol-, prolactin-, and progesterone-stimulated growth of N-nitrosomethylurea-induced rat mammary tumors in soft agar.

The present experiments were designed to test the role of insulin-like growth factor-related peptides (IGF-RPs) in hormonally stimulated N-nitrosomethylurea (NMU)-induced mammary tumor colony formation in soft agar. To evaluate production of IGF-RP by NMU cells, we tested the abilities of a monoclonal antibody directed against insulin-like growth factor I (alpha sm 1.20B) and of a polyclonal antibody raised against the insulin-like growth factor I (Ab 134) to inhibit the colony-stimulating effects of conditioned media (CM) obtained from estradiol (E2)-, prolactin (PRL)-, and progesterone (Pg)-treated NMU-induced rat mammary tumors. Both Abs abolished the colony-stimulating action of genuine insulin-like growth factor I while having no effect when added alone or with control CM. The addition of either alpha sm 1.20B or Ab 134 (but not that of an irrelevant Ab) consistently blocked the colony-stimulating action of E2-CM, PRL-CM, and Pg-CM, suggesting that IGF-RPs are produced by NMU mammary tumor cells exposed to these hormones. Next, we directly tested the role of IGF-RPs as mediators of hormonally stimulated growth. We indeed observed that the addition of alpha sm 1.20B markedly inhibited the colony-stimulating actions of E2, PRL, and Pg added to NMU mammary tumor cells in soft agar in the absence of serum. We conclude that, in our experimental system, IGF-RPs not only are produced upon exposure to E2, PRL, and Pg, but also are important mediators of hormonally stimulated growth.

Animals↗
PubMed1990

Role of transforming growth factor-alpha-related peptides in the autocrine/paracrine control of experimental breast cancer growth in vitro by estradiol, prolactin, and progesterone.

We have recently suggested that estradiol (E2), prolactin (oPrl), and progesterone (Pg) support the growth of the hormone-responsive N-nitrosomethylurea (NMU) rat mammary tumor in soft agar through autocrine/paracrine mechanisms. To gain insight into the nature of these hormonally regulated growth factors, we tested the ability of two monoclonal antibodies (MAb-425 and 528) directed against the epidermal growth factor receptor (EGF-R) to inhibit the colony-stimulating effects of conditioned media (CM) obtained from E2, oPrl, and Pg-treated NMU rat mammary tumors. Since both MAbs are specific for human EGF-R, MCF-7 breast cancer cells grown in soft agar in the absence of serum were used as our indicator system. Both MAb-425 and 528 totally abolished the colony-stimulating effect of genuine EGF, while having no agonistic/antagonistic action when added alone. Both MAb-425 and 528 markedly inhibited the colony-stimulating effect of rat mammary tumor E2-CM in a dose-dependent fashion. MAb-425 was also found to inhibit the growth-promoting action of Pg-CM, although this effect appeared to be somewhat less consistent and pronounced than that observed with E2-CM. In contrast, the colony-stimulating effect of Prl-CM was only rarely and, usually, modestly affected by the addition of either MAb-425 or 528. Our data suggest that in the NMU mammary tumor grown in soft agar, EGF/TGF alpha-related peptides are produced upon exposure to E2 and possibly Pg but only rarely following Prl administration. The possible role of these growth factors as mediators of hormonal effects in our experimental system remains to be established.

Animals↗
PubMed1990

Interactions between growth factor secretion and polyamines in MCF-7 breast cancer cells.

Polyamines may be involved in hormone-dependent breast cancer cell proliferation. The antiestrogen 4-hydroxy-tamoxifen and the polyamine synthesis inhibitor alpha-difluoromethylornithine (DFMO) inhibited MCF-7 growth, and this effect was additive. Transforming growth factor beta (TGF-beta) levels were increased by both compounds; again the effect was additive. Exogenous putrescine antagonized DFMO but not the antiestrogen. However, exogenous TGF-beta did not inhibit cell growth. Secretion of insulin-like growth factor 1 (IGF-1) was not affected by DFMO-induced polyamine depletion but 4-hydroxytamoxifen increased IGF-1, which suggests an estradiol-like effect. Thus polyamines are involved in basal TGF-beta secretion but do not mediate antiestrogen-induced TGF-beta secretion. IGF-1 secretion by MCF-7 cells is not under polyamine control. The antiproliferative effects of 4-hydroxytamoxifen and DFMO cannot be accounted for by either suppression of IGF-1 secretion (a growth stimulatory factor) or stimulation of TGF-beta production (a growth inhibitory polypeptide).

Breast Neoplasms↗
PubMed1990

Polyamine involvement in basal and estradiol-stimulated insulin-like growth factor I secretion and action in breast cancer cells in culture.

Recent evidence indicates that the polyamine pathway may play a significant role in the autocrine/paracrine control of breast cancer cell proliferation by hormones. To directly test this hypothesis, in the present experiments, we evaluated the polyamine involvement in immunoactive insulin-like growth factor I (IGF-I) secretion and IGF-I action using MCF-7 breast cancer cells cultured in serum-free medium in the presence and absence of estradiol (E2). Administration of the polyamine biosynthetic inhibitor, alpha-difluoromethylornithine (DFMO) induced a marked suppression of cellular ornithine decarboxylase (ODC) activity and polyamine levels which was associated with significant, although partial, inhibition of E2-stimulated growth. Exogenous putrescine administration repleted cellular polyamine pools and completely reversed the growth-inhibitory effect of DFMO. Despite these parallel changes in polyamine levels and proliferative activity, basal as well as E2-stimulated levels of immunoactive IGF-I measured in the conditioned media were unaffected by DFMO with and without exogenous putrescine administration. On the other hand, induction of polyamine depletion and repletion by the same treatments significantly (although partially) affected the proliferative action of exogenously added IGF-I. These findings indicate that polyamines, while not involved in immunoactive IGF-I production, play an important role, at least in part, in IGF-I action in this experimental system. Furthermore, we observed that the administration of a monoclonal antibody directed against IGF-I was able to partially block basal as well as of a monoclonal antibody directed against IGF-I was able to partially block basal as well as E2-stimulated MCF-7 cell proliferation. We conclude that immunoactive IGF-I is an important but not sole mediator of MCF-7 breast cancer growth under our experimental conditions. The polyamine pathway plays an important role in the expression of its proliferative action.

Antibodies, Monoclonal↗
PubMed1990

The pharmacodynamic inhibition of estrogen synthesis by fadrozole, an aromatase inhibitor, and its pharmacokinetic disposition.

In this dose-ranging phase I study, the relationship between in vivo androgen to estrogen conversion kinetics and plasma concentrations of fadrozole was investigated in postmenopausal women receiving therapy with this aromatase inhibitor. Patients received ascending doses, ranging from 0.3-8 mg fadrozole twice daily, each for a period of 2 weeks. Drug kinetics and endocrine effects were evaluated specifically for the 2- and 8-mg twice daily treatment regimens. The in vivo activity of the drug was demonstrated by the suppression of estrone and estradiol biosynthesis from testosterone and androstenedione, respectively. The drug inhibitory constant, KI, for the estrone synthetic pathway, was 3.0 ng/mL (13.4 nmol/L) and was of similar magnitude as that determined under in vitro conditions. The KI for the estradiol synthetic pathway was 5.3 ng/mL (23.7 nmol/L). The disparity between KI values may indicate that the two pathways are not equivalent in terms of their inhibition by fadrozole. Pharmacokinetic data demonstrated tha the drug was rapidly absorbed after oral dosing, with peak plasma concentrations achieved in median times of 1 and 2 h, respectively, for the 2- and 8-mg twice daily treatment regimens. The average half-life and oral clearance values were 10.5 h and 621 mL/min, respectively. Oral clearance was independent of dose.

Aged↗
PubMed1989

Individual and combined effects of alpha-difluoromethylornithine and ovariectomy on the growth and polyamine milieu of experimental breast cancer in rats.

Despite considerable evidence suggesting a critical role of polyamines in the hormonal control of breast cancer growth in vitro, their role in in vivo tumor growth is not established. In these experiments, we evaluated the individual and combined effects of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) and ovariectomy on the growth and cellular levels of ornithine decarboxylase (ODC) and polyamines of N-nitrosomethylurea-induced rat mammary tumors. Despite a similar suppressive effect on ODC activity, the two treatments had a different effect on polyamine levels. As expected, DFMO selectively suppressed putrescine, whereas spermidine and spermine levels were minimally or not affected at all. Since quantitatively putrescine contributes the least to overall polyamine pools, the DFMO effect on this latter parameter was modest. In contrast, ovariectomy, by suppressing the more abundant spermidine and spermine, produced a more profound suppression of total polyamine pools. This finding is in agreement with the notion that hormones not only control ODC activity, but also other enzymes involved in the synthesis of the distal polyamines. Ovariectomy was also more potent than DFMO administration in inhibiting N-nitrosomethylurea-induced mammary tumor growth. No major additive/synergistic effects were observed between DFMO and ovariectomy on tumor growth and cellular levels of ODC activity and polyamines. DFMO administration lowered the tumor level of progesterone receptors and appeared to potentiate the suppressive effect of ovariectomy. In contrast, neither treatment, alone or in combination, altered tumor levels of estrogen receptors. DFMO administration did not affect circulating levels of estradiol and prolactin or uterine and ovarian weights, thus suggesting that its effects were not indirectly mediated through alterations of the endocrine milieu of the host.

Animals↗
PubMed1989

Polyamine involvement in the growth of hormone-responsive and -resistant human breast cancer cells in culture.

Recent evidence indicates that hormone-responsive but not -resistant human breast cancer cells in culture are sensitive to the antiproliferative effect of the polyamine (PA) biosynthetic inhibitor alpha-difluoromethylornithine (DFMO). The present experiments were designed to investigate the potential differential involvement of the PA pathway in the growth of these different biological subtypes of human breast cancer. Thus, we evaluated the effect of DFMO on proliferation, ornithine decarboxylase (ODC) activity, and PA levels of the hormone-dependent MCF-7 and -independent MDA-MB-231 breast cancer cell lines. When tested at comparable cell density, the two cell lines had similar levels of ODC activity and PA. Administration of DFMO (0.01, 0.1, 1, 4 mM) for 6 days caused a similar dose-dependent inhibition of proliferation (up to approximately 15% of control) associated with suppression of ODC activity to undetectable levels at the highest dose. In both cell lines, putrescine and spermidine levels were maximally suppressed by doses of DFMO greater than 0.1 mM. Higher doses of DFMO (1 and 4 mM) also suppressed spermine levels to approximately 60% of control. In detailed time-course studies, DFMO administration (0.1 mM) similarly suppressed by 80% the rise in ODC observed in both cell lines following a medium change. At all time points, putrescine and spermidine levels were likewise suppressed to a similar extent. Addition of putrescine (0.1-2.5 mM) to DFMO-treated cells repleted cellular PA levels and restored growth to approximately 80% of control in both cell lines. We conclude that, under these experimental conditions, PA are similarly involved in the growth of the hormone-responsive MCF-7 and -resistant MDA-MB-231 human breast cancer cell lines.

Biogenic Polyamines↗
PubMed1989

Hormonal control of polyamine pools in experimental breast cancer in vivo: correlation with estrogen and progesterone receptor levels.

The present experiments were designed to test whether, in the hormone responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumor, polyamine levels are under hormonal control and whether they correlate with estrogen (ER) and progesterone (PgR) receptor content. We observed that tumor regression induced by ovariectomy was associated with a decline in putrescine (Pu), spermidine (Sd) and spermine (Sm). Administration of estradiol and perphenazine (to stimulate endogenous prolactin release) to castrated rats restored tumor growth and contents of Pu and Sd to control values in a time dependent fashion while Sm levels were only modestly raised. The hormonal modulation of tumor polyamine levels was particularly obvious when the treatment effects on total pools (i.e. Pu + Sd + Sm) were analyzed. No significant correlation was observed between ER and PgR and polyamines in the tumors of intact rats as well as most of the treated groups. In contrast, a highly significant correlation was observed between ER and PgR levels. We conclude that in this experimental system cellular polyamine levels are hormonally regulated but are not correlated with the ER and PgR content of the tumor.

Animals↗
PubMed1989

Central neurotensin inhibits gastric acid secretion: an adrenergic mechanism in rats.

Although parenteral neurotensin (NT) inhibits stimulated gastric acid secretion, published reports on the effect of centrally administered NT on gastric acid secretion are conflicting. This study provides evidence suggesting that, in chronic gastric fistula rats, intracerebroventricularly administered NT (15-60 micrograms) significantly reduces both basal and pentagastrin-, 2-deoxy-D-glucose-, and carbachol-but not histamine-stimulated gastric acid secretion. Using radioimmunoassay, the concentration of plasma immunoreactive NT increased from 30 to 200 pg/ml at 30 and 60 min, respectively after a single intracerebroventricular (i.c.v.) administration of NT at a dose of 60 micrograms. These serum NT concentrations can be reproduced by a constant NT i.v. infusion at 2 micrograms/kg.h. This parenteral infusion dose does not inhibit acid secretion as does i.c.v. NT. Pretreatment with the i.c.v. dopamine-2 receptor antagonists haloperidol or domperidone totally abolishes the inhibitory effect of i.c.v. NT on pentagastrin-stimulated gastric acid secretion. In contrast, pretreatment with the specific dopamine-1 receptor antagonist SCH 23900 or the specific dopamine-2 receptor antagonist sulpiride does not affect i.c.v. NT-induced inhibition of pentagastrin-stimulated gastric acid secretion. Pretreatment (intracerebroventricularly) with the alpha-adrenergic antagonist phentolamine blocks the antisecretory effect of i.c.v. NT. Administration of 3.0 micrograms NT per side directly into nucleus accumbens (NACB), using a stereotaxic technique, significantly reduces basal gastric acid secretion. This effect of central NT is blocked by pretreatment with intra-NACB haloperidol (0.5 microgram per side). These findings suggest that NT acts centrally to inhibit gastric acid secretion, an effect that may occur within NACB and be mediated by central nervous system alpha-adrenergic receptor activation.

Animals↗
PubMed1988

Involvement of the polyamine pathway in antiestrogen-induced growth inhibition of human breast cancer.

Recent evidence indicates that the antiestrogen tamoxifen (TAM) may inhibit breast cancer cell proliferation, at least in part, through suppression of the polyamine (PA) pathway. To directly test this hypothesis, we evaluated the effect of TAM administration on ornithine decarboxylase (ODC) activity, the rate-limiting enzyme in PA synthesis, as well as cellular PA levels in the hormone-responsive MCF-7 breast cancer cell line in culture. In detailed time course studies, we observed that TAM significantly inhibited the rise in ODC activity observed in control cells following a medium change. Chronic treatment with escalating amounts of TAM caused a dose-related decrease in tumor pools of putrescine and spermidine, while spermine levels were unaffected. The TAM effects on ODC activity and PA pools were reversible with exogenous estradiol administration. However, addition of putrescine to TAM-treated cells did not result in a reversal of the antiproliferative effect of TAM, despite repletion of cellular PA pools. Administration of TAM to the hormone-independent MDA-MB-231 breast cancer cell line did not suppress ODC activity or cellular PA levels despite induction, at high concentrations, of an estradiol-irreversible inhibition of proliferation. We conclude that, in the hormone-responsive MCF-7 breast cancer cell line, TAM causes a significant suppression of the PA pathway, the relation of which, if any, to its antiproliferative action remains obscure. This effect seems to be mediated through the estrogen receptor and does not appear to be a nonspecific consequence of inhibition of cell proliferation.

Biogenic Polyamines↗
PubMed1988

Polyamines and autocrine control of N-nitrosomethylurea-induced rat mammary tumor growth in vitro by progesterone.

These experiments were designed to test whether autocrine/paracrine mechanisms are involved in the growth-promoting action of progesterone (Pg) in the N-nitrosomethylurea-induced rat mammary tumor cultured in vitro in soft agar clonogenic assay. In support of our hypothesis, we observed that conditioned media obtained from Pg-treated tumors (Pg-CM), consistently stimulated colony formation in our system to the same degree as Pg itself (approximately 140% of control). Treatment with heat, trypsin, and concanavalin A abolished the colony-stimulating effect of Pg-CM, thus suggesting the possible glycoprotein nature of the Pg-inducible growth factor(s). The growth-promoting action of Pg-CM was rather specific since CMs obtained from tumors exposed to a variety of other steroid hormones rarely stimulated colony formation and usually only to a modest degree. Administration of the polyamine biosynthetic inhibitor, alpha-difluoromethylornithine, abolished the colony-stimulating effect of Pg-CM. The inhibitory effect of alpha-difluoromethylornithine was reversed in a dose-dependent fashion by exogenous administration of spermidine, which entirely restored the growth-promoting action of Pg-CM. Addition of increasing amounts of spermidine, however, did not potentiate Pg-CM action under our experimental conditions. Our results indicate that autocrine/paracrine mechanisms may mediate, at least in part, the colony-stimulating effect of Pg in our system. The polyamine pathway plays an essential role in the expression of such control of tumor growth by Pg.

Animals↗
PubMed1988

Menstrual status in female athletes: correlation with reproductive hormones and bone density.

To determine whether the changes induced in serum hormone levels by strenuous exercise are accompanied by changes in bone density, we obtained medical, sports, and dietary histories, trabecular bone density, percentage body fat, and serum hormone concentrations in collegiate athletes for comparison with those of college students not involved in organized strenuous exercise. Several important observations emerged from this study of collegiate athletes. First, the eumenorrheic athletes began sports training earlier than the oligomenorrheic athletes. Second, oligomenorrheic athletes displayed significantly lower serum estradiol concentrations during cycle weeks 2 and 3 but had a rise in this hormone concentration during cycle week 4. Significant differences in serum gonadotropin concentrations during week 4 were found in the oligomenorrheic athletes. Finally, no significant difference in spinal mineral density values was found in the three groups.

Adolescent↗
PubMed1986

Autocrine stimulation by estradiol-regulated growth factors of rat hormone-responsive mammary cancer: interaction with the polyamine pathway.

We have recently shown that the integrity of the polyamine pathway is essential but not sufficient for expression of the mitogenic effect of estradiol (E2) in the N-nitrosomethylurea-induced rat mammary tumor grown in vitro in the soft agar clonogenic assay. To elucidate the mechanism of E2 action in this system, we tested whether E2 may stimulate tumor growth through induction of secretory growth factor(s), as already proposed for human breast cancer cell lines in culture. Furthermore, we investigated the potential interaction between such "autocrine" control of tumor growth by E2 and the polyamine pathway. Conditioned medium obtained from E2-treated tumors (E2-CM) but not from control tumors (C-CM) consistently stimulated colony formation when added to N-nitrosomethylurea-mammary tumors plated in soft agar under serum-free media conditions. Such growth-promoting effects of the E2-CM was found to increase with increasing protein concentrations of the medium and was abolished by pretreatment of the medium with concanavalin A, heat, and trypsin. The addition of the polyamine biosynthetic inhibitor alpha-difluoromethylornithine (1 mM) totally abolished the colony-stimulating effect of the E2-CM. Exogenous administration of spermidine (0.1 mM) reversed the inhibitory effect of alpha-difluoromethylornithine on colony formation and restored the action of the E2-CM. Although the addition of polyamines alone did not affect the number of colonies formed, the administration of spermidine was found to significantly enhance in a dose-dependent fashion the colony-stimulating effect of suboptimal concentrations of E2-CM. Attempts to identify the E2-inducible growth factor in the E2-CM and in N-nitrosomethylurea-mammary tumor specimens using monoclonal antibodies raised against the Mr 52,000 E2-inducible protein gave negative results. We conclude that autocrine stimulation of tumor growth by E2 is not limited to human breast cancer cell lines but also occurs in individual experimental mammary tumors grown in soft agar. Our results show that the polyamines must be present for the expression of this "autocrine" control of tumor growth by E2. Finally, the identity of the E2-induced growth factor operating in our system remains to be determined.

Animals↗