Obata M, Imamura E, Yoshida Y, Goto J, Kishibe K, Yasuda A, Ogawa K
Department of Pathology, Asahikawa Medical College, Asahikawa, Japan.
Mol Carcinog. 2001 Sep;32(1):9-18. doi: 10.1002/mc.1059.
Primary cultured mouse hepatic cells become senescent within a short period, although rare cells form colonies from which continuously proliferating cell lines can be established. In contrast, hepatic tumor (HT) cells show little senescence and higher colony-forming capacity. To assess this difference, we investigated p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression in primary normal and HT cells, together with cell lines established from both. In primary normal cells, p16(Ink4a)/p19(Arf) were expressed only in association with senescence and disappeared at later stages of colony formation. In contrast, primary HT cells showed sustained p16(Ink4a)/p19(Arf) expression from the beginning. No p16(Ink4a)/p19(Arf) alterations, such as deletion, mutations, or hypermethylation, were detected in the primary HT cells, although most cell lines derived from either normal or HT cell colonies lost p16(Ink4a) or p19(Arf) expression owing to hypermethylation or homozygous deletion of p16(Ink4a)/p19(Arf). On the other hand, primary normal and HT cells and most cell lines showed constitutively elevated expression of p53/p21(Waf1/Cip1), with a further increment after ultraviolet ir-radiation, indicating a functionally normal p53 pathway. These results indicate that primary HT cells are resistant to senescence despite retaining p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression and that loss of p16(Ink4a)/p19(Arf) function is associated only with establishment of the cell lines.
原代培养的小鼠肝细胞在短时间内会衰老，尽管少数细胞能形成集落，从中可建立持续增殖的细胞系。相比之下，肝肿瘤（HT）细胞几乎没有衰老现象，且具有更高的集落形成能力。为评估这种差异，我们研究了原代正常细胞和HT细胞以及由两者建立的细胞系中p16（Ink4a）/p19（Arf）/p53/p21（Waf1/Cip1）的表达情况。在原代正常细胞中，p16（Ink4a）/p19（Arf）仅在衰老相关时表达，并在集落形成的后期消失。相反，原代HT细胞从一开始就持续表达p16（Ink4a）/p19（Arf）。在原代HT细胞中未检测到p16（Ink4a）/p19（Arf）的改变，如缺失、突变或高甲基化，尽管从正常或HT细胞集落衍生的大多数细胞系由于p16（Ink4a）/p19（Arf）的高甲基化或纯合缺失而失去了p16（Ink4a）或p19（Arf）的表达。另一方面，原代正常细胞和HT细胞以及大多数细胞系均显示p53/p21（Waf1/Cip1）的组成性表达升高，紫外线照射后进一步增加，表明p53通路功能正常。这些结果表明，原代HT细胞尽管保留了p16（Ink4a）/p19（Arf）/p53/p21（Waf1/Cip1）的表达，但对衰老具有抗性，并且p16（Ink4a）/p19（Arf）功能的丧失仅与细胞系的建立有关。
