Lack of TRF2 in ALT cells causes PML-dependent p53 activation and loss of telomeric DNA.

Alternative lengthening of telomere (ALT) tumors maintain telomeres by a telomerase-independent mechanism and are characterized by a nuclear structure called the ALT-associated PML body (APB). TRF2 is a component of a telomeric DNA/protein complex called shelterin. However, TRF2 function in ALT cells remains elusive. In telomerase-positive tumor cells, TRF2 inactivation results in telomere de-protection, activation of ATM, and consequent induction of p53-dependent apoptosis. We show that in ALT cells this sequence of events is different. First, TRF2 inactivation/silencing does not induce cell death in p53-proficient ALT cells, but rather triggers cellular senescence. Second, ATM is constitutively activated in ALT cells and colocalizes with TRF2 into APBs. However, it is only following TRF2 silencing that the ATM target p53 is activated. In this context, PML is indispensable for p53-dependent p21 induction. Finally, we find a substantial loss of telomeric DNA upon stable TRF2 knockdown in ALT cells. Overall, we provide insight into the functional consequences of shelterin alterations in ALT cells.

Mutation mechanisms that underlie turnover of a human telomere-adjacent segmental duplication containing an unstable minisatellite.

Subterminal regions, juxtaposed to telomeres on human chromosomes, contain a high density of segmental duplications, but relatively little is known about the evolutionary processes that underlie sequence turnover in these regions. We have characterized a segmental duplication adjacent to the Xp/Yp telomere, each copy containing a hypervariable array of the DXYS14 minisatellite. Both DXYS14 repeat arrays mutate at a high rate (0.3 and 0.2% per gamete) but linkage disequilibrium analysis across 27 SNPs and a direct crossover assay show that recombination during meiosis is suppressed. Therefore instability at DXYS14a and b is dominated by intra-allelic processes or possibly conversion limited to the repeat arrays. Furthermore some chromosomes (14%) carry only one copy of the duplicon, including one DXYS14 repeat array that is also highly mutable (1.2% per gamete). To explain these and other observations, we propose there is another low-rate mutation process that causes copy number change in part or all of the duplicon.

Molecular characterization of inter-telomere and intra-telomere mutations in human ALT cells.

Telomeres in most immortal cells are maintained by the enzyme telomerase, allowing cells to divide indefinitely. Some telomerase-negative tumors and immortal cell lines maintain long heterogeneous telomeres by the ALT (alternative lengthening of telomeres) mechanism; such tumors are expected to be resistant to anti-telomerase drug therapies. Occasionally telomerase-negative Saccharomyces cerevisiae mutants survive, and 10% of them (type II survivors) have unstable telomeres. As in human ALT+ cells, short telomeres in yeast type II survivors lengthen abruptly; in yeast, this is dependent on the recombination proteins Rad52p and Rad50p. In human cells, ALT involves copying of sequence from a donor to a recipient telomere. We have characterized for the first time a class of complex telomere mutations seen only in ALT+ cells. The mutant telomeres are defined by the replacement of the progenitor telomere at a discrete point (fusion point) with a different telomere repeat array. Among 19 characterized fusion points, one occurred within the first six repeats of the telomere, indicating that these recombination-like events can occur anywhere within the telomere. One mutant telomere may have been involved in a secondary recombination-like mutation event, suggesting that these mutations are sporadic but ongoing in ALT+ cells. We also identified simple intra-allelic mutations at high frequency, which evidently contribute to telomere instability in ALT+ cells.

Contact with a solid substratum induces cysts in axenic cultures ofPhysarum polycephalum amoebae: mannitol-induced detergent-resistant cells are not true cysts.

Previous workers reported thatPhysarum polycephalum amoebae cultured in liquid axenic medium were induced to form cysts by the addition of mannitol. Their criterion for encystment was the formation of detergent (Triton)-resistant cells (TRC). In this study the frequencies of TRC in suspensions of amoebae from various treatments were compared with counts of cell types identified by transmission electron microscopy. Amoebae treated with mannitol in axenic liquid culture formed 50% TRC after 17 h but no walled cysts were found. It was concluded that TRC induced by mannitol were dense, rounded cells without walls. In contrast, TRC formed after growth to stationary phase on bacterial lawns were walled cells. When resuspended in growth medium, most mannitol-induced TRC reverted to active amoebae within a few minutes, whereas TRC formed on bacteria remained Triton resistant for many hours. It was concluded that delayed reversion of TRC was a more reliable indication of wall formation than Triton resistance alone. Transfer of amoebae from liquid culture to the surface of diluted axenic agar medium resulted in the formation of walled cysts identical in appearance with those formed on bacterial lawns. The results indicated that efficient encystment requires a solid substratum as well as nutrient deprivation.