David C. Spink

A coculture system was developed to investigate the interactions between MCF‐10A breast epithelial cells and MCF‐7 breast cancer cells stably expressing the green fluorescent protein (MCF‐7‐GFP). Studies with this MCF‐10A/MCF‐7‐GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF‐7‐GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF‐7‐GFP and markedly increased MCF‐10A cell proliferation, greatly inhibited MCF‐7‐GFP cell proliferation in MCF‐10A/MCF‐7‐GFP cocultures. 17β‐Estradiol, which stimulated MCF‐7‐GFP but not MCF‐10A cell proliferation in monoculture, inhibited MCF‐7‐GFP cell proliferation in MCF‐10A/MCF‐7‐GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF‐10A/MCF‐7‐GFP cellular interactions were observed in real time that resulted in the formation of acinus‐like structures. These results indicate a role of normal epithelial cells in inhibiting tumor‐cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.

Cytochrome P450 (CYP)1A1 and CYP1B1, which are under the regulatory control of the aryl hydrocarbon (Ah) receptor (AhR), catalyze the metabolic activation of numerous procarcinogens and the hydroxylation of 17β-estradiol (E ₂ ) at the C-2 and C-4 positions, respectively. There is evidence of cross-talk between estrogen receptor α (ERα)- and AhR-mediated signaling in breast and endometrial cells. To further examine these interactions, we investigated the short- and long-term effects of E ₂ exposure on Ah responsiveness in MCF-7 human breast cancer cells. Short-term exposure to 1 nM E ₂ elevated the ratio of the 4- to 2-hydroxylation pathways of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD)-induced E ₂ metabolism and the ratio of the induced CYP1B1 to CYP1A1 mRNA levels, as determined by real-time PCR. Cells maintained long-term (9–12 months) in low-E ₂ medium progressively lost Ah responsiveness, as indicated by diminished rates of TCDD-induced E ₂ metabolism and ethoxyresorufin O -deethylase activity, and the reduced expression of the CYP1A1 and CYP1B1 mRNAs and proteins levels. These E ₂ -deprived cells showed elevated levels of ERα mRNA, depressed levels of AhR mRNA, and unchanged levels of the AhR nuclear translocator mRNA. Transient transfection studies using a CYP1B1 -promoter-luciferase reporter construct showed that reduced CYP1B1 promoter activity in E ₂ -deprived cells could be restored by co-transfection with an AhR expression construct, indicating that AhR expression was limiting in these cells. The reduced Ah responsiveness of E ₂ -deprived cells was reversed by culture for four passages in medium supplemented with 1 nM E ₂ ; ERα and AhR mRNAs returned to near-normal levels and the inducibility of the CYP1A1 and CYP1B1 mRNAs, proteins, and E ₂ metabolic activities by TCDD was restored. These studies indicate that the continued presence of estrogen is required to maintain high levels of AhR expression and inducibility of the procarcinogen-bioactivating enzymes, CYP1A1 and CYP1B1, in MCF-7 cells.

Polycyclic aromatic hydrocarbons (PAHs) and metals are often environmental cocontaminants, yet there have been relatively few studies of combined effects of PAHs and metals on cytochrome P450 (P450)-catalyzed metabolism. We examined the effects of NaAsO₂ in combination with benzo[a]pyrene (BAP) on CYP1A1 and CYP1B1 in T-47D human breast cancer cells by using estrogen metabolism as a probe of their activities. Exposure to BAP caused elevated rates of the 2- and 4-hydroxylation pathways of estrogen metabolism, indicating induction of both CYP1A1, an estradiol 2-hydroxylase, and CYP1B1, an estradiol 4-hydroxylase. BAP-induced metabolism peaked 9 to 16 h after exposure and returned to near-basal levels by 48 h. Concentration-response studies showed maximal induction of the 2- and 4-hydroxylation pathways at 3 μM BAP; higher levels caused reduced rates of metabolism due to inhibition of CYP1A1 and CYP1B1. NaAsO₂ caused pronounced decreases in the induction of CYP1A1 and CYP1B1 by 3 μM BAP because cotreatment with 10 μM NaAsO₂ inhibited the rates of the 2- and 4-hydroxylation pathways by 86 and 92%, respectively. Western immunoblots showed diminished levels of BAP-induced CYP1A1 by coexposure to NaAsO₂. The levels of the CYP1A1 and CYP1B1 mRNAs induced by BAP were not significantly affected by coexposure to NaAsO₂; however, heme oxygenase 1 mRNA levels were markedly induced by coexposure to BAP and NaAsO₂. These results indicate a post-transcriptional inhibitory effect of arsenite on the expression of CYP1A1 and CYP1B1 in T-47D cells, possibly resulting from reduced heme availability.

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17α- and 17β-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17β-dihydro form, with minimal production of the 17α-dihydro isomer. Both equilenin and 17β-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17β-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4‘,5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17β-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17β-dihydroequilenin, whereas with 17β-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17β-dihydroequilenin in extrahepatic, estrogen-responsive tissues.