[논문]TERT mRNA Expression is Up-Regulated in MCF-7 Cells and a Mouse Mammary Organ Culture (MMOC) System by Endosulfan Treatment

Je Kang Hoon; Kim Ki Nam; Nam Kung Woo; Cho Myung Haing; Mar Woong Chon

doi:10.1007/bf02977804

TERT mRNA Expression is Up-Regulated in MCF-7 Cells and a Mouse Mammary Organ Culture (MMOC) System by Endosulfan Treatment 원문보기

Archives of pharmacal research : a publication of the Pharmaceutical Society of Korea, v.28 no.3, 2005년, pp.351 - 357

Je Kang Hoon (Natural Products Research Institute, Seoul National University) , Kim Ki Nam (Natural Products Research Institute, Seoul National University) , Nam Kung Woo (Natural Products Research Institute, Seoul National University) , Cho Myung Haing (NIAST, RDA and College of Veterinary Medicine, Seoul National University) , Mar Woong Chon (Natural Products Research Institute, Seoul National University, College of Pharmacy, Seoul National University)

Abstract ▼ AI-Helper

Endosulfan is one of the organochlorine pesticides, which are well-known endocrine disruptors (EDs), and it acts as an estrogen agonist. Estrogen is a group of hormones that play an important role in mammary gland function and are implicated in mammary carcinogenesis. In the present study, we studied the effects of endosulfan on nodule like alveolar lesion (NLAL) formation in mouse mammary gland development using a mouse mammary gland organ culture (MMOC) system. Although endosulfan-treated mammary glands did not form NLALs, more alveolar buds were formed in this group than in the negative control (vehicle-treated) group. In addition, telomerase reverse transcriptase (TERT) mRNA expression levels were increased in endosulfan-treated mammary glands in a dose-dependent manner. Telomerase can be activated by estrogen, therefore, we examined the effects of endosulfan on telomerase activity, and found that the telomerase activity in estrogen receptor-positive MCF-7 cells was up-regulated by endosulfan treatment. Moreover, this activation was accompanied by the upregulation of the TERT mRNA expression. Also, transient expression assays using CAT reporter plasm ids containing various fragments of the TERT promoter showed that this imperfect palindromic estrogen-responsive element is almost certainly responsible for the transcriptional activation by endosulfan. These results may help elucidate the endocrine disrupting mechanism of endosulfan.

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가설 설정

This strategy appears to mimic the in vivo formation of female hormone related cancers, such as breast cancer. Therefore, using this method, we examined whether endosulfan has the ability to form precancerous lesions. DM BA-treated mammary glands developed al.

제안 방법

, 1999). To examine the effect of endosulfan on the transcriptional activity of the TERT promoter, we cloned the TERT promoter region from MCF-7 genomic DNA, and constructed a serial-deletion TERT promoter-CAT reporter vector (Fig. 4B). Transient expression assays using CAT reporter plasmids containing various fragments of the TERT promoter showed that the CAT reporter plasmid, containing an imperfect palindromic ERE, is associated with telomerase activation.
Briefly, TERT and p-actin mRNAs were amplified using the following primer pairs: 51- CTCAGGAACACCAAGAAGTTCATC-31 (sense primer) and 5-GGATGAAGCGGAGTCTGG A-3' (anti-sen오야 primer) for TERT, and S-GACCCAGATCATGEGAGA-3 (sense primer) and S'-GCTTGCTGATCCACATCTGC-S1 (antisense primer) for p-actin. Total RNA was extracted using an Uttraspec™ solution (BIOTECX, USA) according to the manufacturer's instructions, and cDNA was synthesized from 2 jig of RNA using a Titan™ one tube RT-PCR Kit (Boehringer Mannheim, Germany) according to the manufacturer's instructions with some modification. Briefly, a mixture of 2 jig of RNA and 1 μL of an antisense primer (20 pmol) was heated at 95℃ for 5 minutes then cooled rapidly on ice.

대상 데이터

Waymouth's medium 752/1, E2, progesterone, hydrocortisone, aldosterone, and insulin were purchased from Sigma (USA). Female 21 to 28 day old Balb/c mic으 were purchased from Daehan Biolink (Korea). The animals were pretreated daily with E2 (1 jig) and progesterone (1 mg) for 9 days.
MCF-7 cells were obtained from the American Type Culture Collection (ATCC, USA) and were grown in DMEM with 5% FBS in the presence of 5% CO₂ at 37℃. In induction assays with 17p-estradiol (E2) and endosuIfan, cells were grown in phenol red-free media containing 5% dextran- coated charcoal-treated FBS for 48 h prior to treatment.
001 compared with the control group. Ten inguinal glands per each sample were used to get statistical results in this experiment. The results are expressed as means±SD of determinations.

이론/모형

(C) Various deletion mutants of theTERT-promoter reporter plasmids were transfected into the MCF-7 cells and then treated with endosulfan (10 μM) or E2 (10 nM) for 48 h. Cells were harvested and the CAT assay was performed by the liquid scintillation counting (LSC) method. The resuIts are expressed as means ± SD of determinations.

후속연구

, 2000). The current report intends to explain these observations by characterizing the estrogen mimicking properties of endosulfan.

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