Grobelny J V, Godwin A K, Broccoli D
Fox Chase Cancer Center, Philadelphia PA 1911, USA.
J Cell Sci. 2000 Dec;113 Pt 24:4577-85. doi: 10.1242/jcs.113.24.4577.
Telomere maintenance is essential for the unlimited proliferative potential of human cells, and hence immortalization. However, a number of tumors, tumor-derived cell lines and in vitro immortalized cell lines have been described that do not express detectable telomerase activity. These lines utilize a mechanism, termed Alternative Lengthening of Telomeres (ALT), to provide telomere maintenance. A subset of the cells in each ALT cell line contain a novel form of the promyelocytic leukemia nuclear body (PML NB) in which telomeric DNA and the telomere binding proteins TRF1 and TRF2 co-localize with the PML protein, termed ALT-associated PML bodies (AA-PBs). In contrast, in non-ALT, telomerase-positive cell lines these telomeric proteins and the PML NB occupy distinct and separate subnuclear domains. PML NBs have been implicated in terminal differentiation, growth suppression and apoptosis. The role, if any, of AA-PBs in telomere maintenance or culture viability in telomerase negative cell lines is unclear, but it has been suggested that cells containing these structures are no longer viable and are marked for eventual death. We utilized a series of human ovarian surface epithelium (HOSE) cell lines that use ALT for telomere maintenance to determine if AA-PBs are indeed markers of cells in these cultures that are no longer cycling. We show that AA-PB positive cells incorporate BrdU and thus are able to carry out DNA replication. In addition, AA-PBs are present in mitotic cells and the frequency of cells containing these structures is increased when cultures are enriched for cells in the G(2)/M phase of the cell cycle suggesting that the formation of AA-PBs is coordinately regulated with the cell cycle. Finally, we demonstrate that the majority of the AA-PB positive cells in the culture are not destined for immediate apoptosis. Taken together the data argue against AA-PBs marking cells destined for death and, instead, raise the possibility that these structures may be actively involved in telomere maintenance via the ALT pathway.
端粒维持对于人类细胞无限增殖潜能以及永生至关重要。然而，已描述了许多不表达可检测到的端粒酶活性的肿瘤、肿瘤衍生细胞系和体外永生化细胞系。这些细胞系利用一种称为端粒替代延长（ALT）的机制来维持端粒。每个ALT细胞系中的一部分细胞含有一种新型的早幼粒细胞白血病核体（PML NB），其中端粒DNA以及端粒结合蛋白TRF1和TRF2与PML蛋白共定位，称为ALT相关PML小体（AA-PBs）。相比之下，在非ALT、端粒酶阳性细胞系中，这些端粒蛋白和PML NB占据不同且分开的核内亚结构域。PML NB与终末分化、生长抑制和细胞凋亡有关。AA-PBs在端粒维持或端粒酶阴性细胞系的培养生存力中的作用（如果有）尚不清楚，但有人提出含有这些结构的细胞不再具有活力，并注定最终死亡。我们利用一系列使用ALT维持端粒的人卵巢表面上皮（HOSE）细胞系来确定AA-PBs是否确实是这些培养物中不再循环的细胞的标志物。我们发现AA-PB阳性细胞掺入BrdU，因此能够进行DNA复制。此外，有丝分裂细胞中存在AA-PBs，当培养物富集处于细胞周期G(2)/M期的细胞时，含有这些结构的细胞频率增加，这表明AA-PBs的形成与细胞周期协同调节。最后，我们证明培养物中大多数AA-PB阳性细胞并非注定立即凋亡。综上所述，这些数据反对AA-PBs标记注定死亡的细胞，相反，增加了这些结构可能通过ALT途径积极参与端粒维持的可能性。
