Ki M H, Paik K J, Lee J H, Chung H Y, Lee K H, Kim K W, Kim N D
Department of Pharmacy, Pusan National University, Korea.
Arch Pharm Res. 1998 Jun;21(3):298-304. doi: 10.1007/BF02975291.
Retinoids are applied to not only cancer prevention but also cancer chemotherapy by stimulating differentiation of cells. We studied differentiation inducing effect of all-trans retinoic acid (ATRA) by studying proportion of high dense fractions of stem-like cells and the size of S phase fraction in cell cycle. From mammary organoids obtained from 7- to 8-week old F344 female rat mammary gland, we cultured rat mammary epithelial cells (RMEC) and treated physiological doses of 10(-6), 10(-7), and 10(-8) M ATRA from the first day and then cultured for 4, 7, and 14 days. After that, immunostaining was performed using peanut agglutinin (PNA) and anti-Thy-1.1 monoclonal antibody (Thy-1.1) that can be used as markers of differentiation. We separated four different cell subpopulations by flow cytometry: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). We observed continuous decreases of high dense fractions of stem-like cells (PNA+ subpopulations) for 14 days and as much decreases as high doses of ATRA, which were thought to be proportional to doses of ATRA. We labeled RMEC with bromodeoxyuridine and investigated cell cycle fractions that went through S phase. We observed a tendency of decrease of S phase fraction with time in culture, which is thought to be related to continuous decreases of PNA+ subpopulations and inhibitory role of ATRA on cell cycle. These results suggest that physiological doses of ATRA could stimulate differentiation of RMEC and convert stem-like RMEC to differentiated cells in SFM for a relatively long period of 14 days.
维甲酸不仅应用于癌症预防，还通过刺激细胞分化应用于癌症化疗。我们通过研究干细胞样细胞高密度组分的比例以及细胞周期中S期组分的大小，来研究全反式维甲酸（ATRA）的分化诱导作用。从7至8周龄的F344雌性大鼠乳腺获得乳腺类器官，我们培养大鼠乳腺上皮细胞（RMEC），并从第一天开始用生理剂量的10^(-6)、10^(-7)和10^(-8) M ATRA处理，然后培养4、7和14天。之后，使用花生凝集素（PNA）和抗Thy-1.1单克隆抗体（Thy-1.1）进行免疫染色，它们可作为分化的标志物。我们通过流式细胞术分离出四个不同的细胞亚群：对两种试剂均呈阴性的细胞（B-）、PNA阳性细胞（PNA+）、Thy-1.1阳性细胞（Thy-1.1+）以及对两种试剂均呈阳性的细胞（B+）。我们观察到干细胞样细胞（PNA+亚群）的高密度组分在14天内持续减少，且减少程度与高剂量ATRA相同，这被认为与ATRA的剂量成正比。我们用溴脱氧尿苷标记RMEC，并研究经历S期的细胞周期组分。我们观察到培养过程中S期组分随时间有减少的趋势，这被认为与PNA+亚群的持续减少以及ATRA对细胞周期的抑制作用有关。这些结果表明，生理剂量的ATRA可刺激RMEC分化，并在无血清培养基中将干细胞样RMEC转化为分化细胞，持续时间长达14天。
