Spillane D M, Reagan J W, Kennedy N J, Schneider D L, Chang T Y
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844.
Biochim Biophys Acta. 1995 Feb 9;1254(3):283-94. doi: 10.1016/0005-2760(94)00190-a.
Using a stable cell line 25-RA derived from wild-type Chinese hamster ovary (CHO) cells as the parental cell, this laboratory previously reported the isolation and characterization of CHO cell mutants (cholesterol-trafficking or CT) defective in transporting LDL-derived cholesterol out of the acidic compartment(s) (lysosomes/endosomes) to the endoplasmic reticulum (ER) for esterification. In this report, we show that the CT mutation can be complemented by fusion with human cells; however, attempts to complement the CT defect through DNA transfection have resulted in a collection of stable cell lines designated as ST cells. Under cholesterol starvation condition, the ST cells exhibit an elevated rate of cholesterol ester biosynthesis (by 3- to 5-fold) compared to both the parental CHO cells and the CT cells. The phenotypes of the ST cells are stable. ST cells are thus new cell lines arisen from the CT cells. When the plasma membranes of the parental, CT, and ST cells are labelled with [3H]cholesterol, ST cells show rates of [3H]cholesterol esterification much higher than that observed in CT cells but lower than that observed in the parental CHO cells. This result shows that translocation of plasma membrane cholesterol to the ER for esterification is defective in the CT cells. This result also suggests that ST cells acquire increased cholesterol trafficking activity between the lysosome and the ER without mixing the plasma membrane cholesterol pool. The characteristics of CT cells and ST cells reported here suggest that translocation of both lysosomal LDL-derived cholesterol and plasma membrane cholesterol to the ER for esterification may require common cellular factors involved in cholesterol egress from the acidic compartment(s) (lysosomes/endosomes).
以源自野生型中国仓鼠卵巢（CHO）细胞的稳定细胞系25 - RA作为亲本细胞，本实验室先前报道了CHO细胞突变体（胆固醇转运或CT）的分离和表征，这些突变体在将低密度脂蛋白（LDL）衍生的胆固醇从酸性区室（溶酶体/内体）转运至内质网（ER）进行酯化方面存在缺陷。在本报告中，我们表明CT突变可通过与人类细胞融合得到互补；然而，试图通过DNA转染来互补CT缺陷却产生了一系列被命名为ST细胞的稳定细胞系。在胆固醇饥饿条件下，与亲本CHO细胞和CT细胞相比，ST细胞的胆固醇酯生物合成速率有所提高（提高了3至5倍）。ST细胞的表型是稳定的。因此，ST细胞是由CT细胞产生的新细胞系。当用[3H]胆固醇标记亲本、CT和ST细胞的质膜时，ST细胞的[3H]胆固醇酯化速率远高于CT细胞，但低于亲本CHO细胞。这一结果表明，CT细胞中质膜胆固醇向内质网的酯化转运存在缺陷。这一结果还表明，ST细胞在不混合质膜胆固醇池的情况下，溶酶体与内质网之间的胆固醇转运活性增加。本文报道的CT细胞和ST细胞的特征表明，溶酶体LDL衍生的胆固醇和质膜胆固醇向内质网的酯化转运可能都需要参与胆固醇从酸性区室（溶酶体/内体）排出的共同细胞因子。
