The mouse mammary gland in serum-free whole organ culture can be manipulated hormonally to undergo one complete physiological cycle consisting of lobuloalveolar development, functional differentiation and regression, mimicking processes that occur in vivo. A second cycle has not previously been achieved in vitro. The present study has identified a specific requirement for epidermal growth factor (EGF) in the morphological development of mammary lobuloalveoli, allowing two complete cycles of development and regression in culture.

Epidermal growth factor requirement for development of cultured mammary gland.

BACKGROUND Chemoprevention of breast cancer is an active area of investigation. Recent in vivo and in vitro studies have shown that thiazolidinediones (e.g., troglitazone) and retinoids are able to inhibit the growth of breast cancer cells. Troglitazone mediates its action via peroxisome proliferator-activated receptor gamma (PPARgamma). We evaluated the ability of troglitazone, alone or in combination with retinoids, to prevent the induction of preneoplastic lesions by 7,12-dimethylbenz[a]anthracene (DMBA) in a mouse mammary gland organ culture model. METHODS Mammary glands of BALB/c mice were treated with DMBA (2 microg/mL) to induce preneoplastic lesions in organ culture. Effects of troglitazone, all-trans-retinoic acid (retinoic acid; ligand for retinoic acid receptor [RAR] alpha), and LG10068 (ligand for retinoid X receptors [RXRs]), singly or in combination, on the development of lesions were evaluated. Expression of retinoid receptors (RARalpha and RXRalpha) and PPARgamma was determined by western blot analysis. Statistical significance was determined by generalized chi-squared analysis using the GENCAT software program and Bonferroni correction. All P values are two-sided. RESULTS Troglitazone (at 10(-5) M) or retinoic acid (at 10(-6) M) markedly inhibited the development of mammary lesions (both P values <.05); however, together they did not enhance the effectiveness of the other. In contrast, LG10068 (at 10(-7) M or 10(-8) M) alone had very little ability to inhibit development of these lesions, but a combination of LG10068 (at 10(-8) M) and troglitazone (at 10(-5) M or 10(-6) M) almost completely inhibited (by 85% and 100%, respectively; both P values <. 05) the development of mammary lesions. The expression of PPARgamma and RXRalpha remained unchanged with the various treatments, whereas the expression of RARalpha was substantially reduced after treatment with the combination of retinoic acid and troglitazone. CONCLUSIONS To our knowledge, this is the first report showing the possibility of a PPARgamma ligand having chemopreventive activity. Furthermore, an RXR-selective retinoid, LG10068, appears to enhance this activity.

A ligand of peroxisome proliferator-activated receptor gamma, retinoids, and prevention of preneoplastic mammary lesions.

β-carotene reduces sister chromatid exchanges induced by chemical carcinogens in mouse mammary cells in organ culture

Vitamin D, a steroid hormone and exerts its biological effects through its active metabolite 1α, 25 dihydroxyvitamin D3 [1,25(OH)2D3]. Like steroid hormones, 1,25(OH)2D3 is efficacious at very low concentrations and serves as a ligand for vitamin D receptors (VDR), associating with VDR very high affinity. Despite its potent property as a differentiating agent, its use in the clinical practice is hampered by the induction of hypercalcemia at a concentration required to suppress cancer cell proliferation. Therefore nearly 400 structural analogs of vitamin D3 have been synthesized and evaluated for their efficacy and toxicity. Among these analogs, relatively less toxic but highly efficacious analogs, EB1089, RO24-5531, 1α-hydroxyvitamin D5 and a few others have been evaluated in a preclinical toxicity and in Phase I clinical trials for dose tolerance in advanced cancer patients. Clinical trials using vitamin D analogs for prevention or therapy of cancer patients are still in their infancy. Vitamin D mediates its action by two independent pathways. Genomic pathway involves nuclear VDR and induces biological effects by interactions with hormone response elements and modulation of differential gene expressions. Evidence also suggests that vitamin D analogs also interact with steroid hormone(s) inducible genes. The non-genomic pathway is characterized by rapid actions of vitamin D. It involves interactions with membrane-VDR interactions and its interactions with protein kinase C and by altering intracellular calcium channels. Thus, the development of nontoxic analogs of vitamin D analogs and understanding of their molecular mechanism(s) of action are of significant importance in the prevention and treatment of cancer by vitamin D.

Vitamin D and cancer.

Background The form of vitamin D (vitamin D3) in fortified milk and the provitamin D produced by the body undergo metabolic activation to a biologically active form, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. This compound can induce cell differentiation and can prevent proliferation of cancer cells. However, because 1alpha,25(OH)2D3 is hypercalcemic (effective in increasing serum calcium level), it is not suitable for use in cancer prevention or cancer therapy trials. Purpose We synthesized a vitamin D5 series analogue, 1alpha-hydroxy, 24-ethyl-cholecalciferol, or 1alpha-hydroxyvitamin D5 [1alpha(OH)D5], and evaluated its chemopreventive activity in carcinogen-treated mammary glands in organ culture experiments. Methods The analogue 1alpha(OH)D5 was synthesized from sitosterol acetate and was characterized by nuclear magnetic resonance. Its purity was evaluated by high-pressure liquid chromatography. The calcemic activities of vitamin D3 and D5 analogues were determined in vitamin D-deficient Sprague-Dawley rats. Mammary glands of BALB/c mice were placed in organ culture and treated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to induce preneoplastic lesions. Vitamin D analogues were added to the culture medium at four different concentrations, and formation of mammary lesions was evaluated. The effects of 1alpha(OH)D5 and 1alpha,25(OH)2D3 on the expression of vitamin D receptors (VDRs) and transforming growth factor-beta1 (TGF-beta1) were studied by immunohistochemistry. Statistical significance was determined by the chi-squared test. All reported P values were two-sided. Results 1alpha,25(OH)2D3 was fourfold more calcemic than 1alpha(OH)D5 at a dose of 0.042 microg/kg per day in rats. Both 1alpha,25(OH)2D3 and 1alpha(OH)D5 inhibited the development of DMBA-induced preneoplastic lesions in mouse mammary glands compared with untreated glands. The effect of the vitamin D3 analogue was observed at a much lower concentration (0.01 microM). Treatment with 1alpha(OH)D5 resulted in a dose-related (0.01-10.0 microM) inhibition without any toxicity, whereas the vitamin D3 analogue was highly potent but toxic at concentrations of 1.0 microM or higher. Normal mouse mammary glands poorly express VDR and TGF-beta1; incubation with 1alpha(OH)D5 or 1alpha,25(OH)2D3 dramatically induced their expression. Conclusions This is the first report showing the possibility of chemoprevention by a vitamin D5 series compound. We conclude that 1alpha(OH)D5 is less calcemic than 1alpha,25(OH)2D3. It is nontoxic at a wide range of concentrations, but it is potent in inhibiting the development of preneoplastic lesions in mammary glands in organ culture. In addition, we show for the first time the induction of TGF-beta1 in normal mammary tissues by a chemopreventive agent. Implications 1alpha(OH)D5 is a good candidate for in vivo chemoprevention studies. It may mediate its action by inducing expression of VDR and of TGF-beta1, as is seen in other systems.

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Prevention of Preneoplastic Mammary Lesion Development by a Novel Vitamin D Analogue, 1α-Hydroxyvitamin D5

Biological characteristics of nodule-like alveolar lesions (NLAL) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in organ culture of whole mammary gland (BALB/c female mice) were assessed after transplantation into gland-free mammary fat pads of syngeneic virgin mice. (i) Tissue-fragment explants from NLAL areas of the gland produced abnormal lobuloalveolar (LA) outgrowths in 3 of 10 fat pads. (ii) Transplantation of dissociated cells of NLAL-derived LA outgrowths into 36 fat pads showed 100% LA outgrowths and 3 (8%) of these 36 outgrowths produced mammary carcinomas. (iii) The explants of dissociated cells from whole mammary glands treated with DMBA in culture produced full or partial LA structures in 2 of 56 outgrowths. (iv) The explants of dissociated cells prepared from outgrowths derived from outgrowths derived from explants as in iii produced 9 LA outgrowths in 16 instances; mammary tumor incidence in these outgrowths was 3 of 16 (18%). (v) The explants of tissue fragments from LA outgrowths as in iv produced LA outgrowths in 20 of 20 fat pads; mammary carcinomas appeared in 16 of 20 (80%) of these outgrowths. No NLAL was detectable in control glands treated with dimethyl sulfoxide (solvent for DMBA); explants of the control glands consistently produced ductal outgrowths and no tumor. This accomplishment of chemical carcinogen-induced neoplastic transformation of epithelial cells in vitro provides a model for studying carcinogenesis in an entire isolated organ.

/pdf/neoplastic-transformation-of-epithelial-cells-in-whole-4ldze04r3i.pdf

Neoplastic transformation of epithelial cells in whole mammary gland in vitro

The second thoracic mammary glands of Immature female BALB/c mice were cultivated to alveolar development In a chemically defined medium with Insulin plus prolactin plus aldosterone plus hydrocortisone. (IPAH medium). The glands were treated with labeled PH or 14C} or unlabeled 7,12-dlmethylbenz[a]anthracene (DMBA) In dimethyl sulfoxide (DMSO) for 24 hours between days 3 and 4 of culture. For binding studies, isolated mammary gland DNA was treated with RNase and pronase and the radioactivity In DNA was measured. The DNA of glands treated with [14C]DMBA was also analyzed In a CsCI density gradient. The DNA of glands treated with labeled DMBA was found to contain bound radioactivity. UV absorption spectral analysis also showed the presence of the DMBA nucleus at the 310-nm region In the DNA of DMBA-treated glands. Nearly 100% of the Initial (3H]DMBA-DNA radioactivity was retained up to 5 months, and at 8 months 72% of the radioactivity was present In the DNA. For morphologic studies, observation of the glands after treatment with equlmolar concentrations (0.004 M) of DMBA, dibenz[a,h]anthracene, benz[a]anthracene, or anthracene In IPAH medium revealed that DMBA was most potent in Inducing nodulelike alveolar lesions (NLAL) In the mammary glands In culture. Thus the binding of DMBA to DNA corresponds to Its high nodullgenlc action In the mammary glands in the present organ culture model. The results appear to resemble DMBA-DNA Interaction associated with the carcinogenic action of DMBA. The biologic characteristics of the NLAL are being assessed by mammary fat pad transplantation.-J Natl Cancer Inst 61: 465-

Nityanand T. Telang

Papers

Neoplastic transformation of epithelial cells in whole mammary gland in vitro

Binding of 7,12-Dimethylbenz[a]anthracene to BALB/c Mouse Mammary Gland DNA in Organ Culture