ALT-associated PML bodies are present in viable cells and are enriched in cells in the G(2)/M phase of the cell cycle

Telomere maintenance is essential for the unlimited proliferative potential of human cells, and hence immortalization, However, a number of tumors, tu mor-derived cell lines and in vitro immortalized cell lines have been descr ibed that do not express detectable telomerase activity. These lines utiliz e a mechanism, termed Alternative Lengthening of Telomeres (ALT), to provid e telomere maintenance, A subset of the cells in each ALT cell line contain a novel form of the promyelocytic leukemia nuclear body (PML NE) in which telomeric DNA and the telomere binding proteins TRF1 and TRF2 co-localize w ith the PML protein, termed ALT-associated PML bodies (AA-PBs), In contrast , in non-ALT, telomerase-positive cell lines these telomeric proteins and t he PML NE occupy distinct and separate subnuclear domains. PML NBs have bee n implicated in terminal differentiation, growth suppression and apoptosis, The role, if any, of AA-PBs in telomere maintenance or culture viability i n 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 (H OSE) cell lines that use ALT for telomere maintenance to determine if AA-PB s 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 ca rry out DNA replication, In addition, AA-PBs are present in mitotic cells a nd the frequency of cells containing these structures is increased when cul tures are enriched for cells in the G(2)/M phase of the cell cycle suggesti ng that the formation of AA-PBs is coordinately regulated with the cell cyc le. Finally, we demonstrate that the majority of the AA-PB positive cells i n the culture are not destined for immediate apoptosis, Taken together the data argue against AA-PBs marking cells destined for death and, instead, ra ise the possibility that these structures may be actively involved in telom ere maintenance via the ALT pathway.