Frequent recombination in telomeric DNA may extend the proliferative life of telomerase-negative cells

Abstract

For cells on the path to carcinogenesis, the key to unlimited growth potential lies in overcoming the steady loss of telomeric sequence commonly referred to as the 'end-replication problem' that occurs with each cell division. Most human tumors have reactivated telomerase, a specialized reverse transcriptase that directs RNA-templated addition of telomeric repeats on to chromosomal termini. However, approximately 10% of tumors maintain their telomeres through a recombination-based mechanism, termed alternative lengthening of telomeres or ALT. Here we demonstrate that telomeric DNA undergoes a high rate of a particular type of recombination visualized cytogenetically as sister chromatid exchange (SCE), and that this rate is dependent on genotype. A novel model of ALT is presented in which it is argued that telomeric exchanges, if they are unequal and occur at a sufficiently high frequency, will allow cells to proliferate indefinitely without polymerase-mediated extension of telomeric sequence.

Figure 1

Method for detecting SCE within telomeres. (A) Mitotic cells are collected after culture in bromo-substituted nucleotides for a single cell cycle. Fixed cells on microscope slides are stained with the DNA-binding fluorescent dye Hoechst 33258. Exposure to UV light nicks the substituted strand, and exonuclease III digests it. The process effectively removes the newly synthesized DNA strands and leaves behind the two parental strands that are now located on sister chromatids. A single-stranded probe hybridizes to complementary telomeric DNA on one chromatid of each chromosome arm producing a two-signal pattern instead of the four signals seen with ordinary FISH. (B) The expected effect of an SCE within telomeric DNA is to split the hybridization signal. (C) An example of a three-signal hybridization pattern like that expected of a T-SCE. (D) Sequential CO-FISH detection of a T-SCE with the C-rich telomere probe (i), then the G-rich telomere probe (ii), demonstrating the reciprocal pattern with each. This pattern is only produced by true SCE.

Figure 2

Unequal T-SCE delay senescence in telomerase-negative cells. This is an example of a single cell whose shortest telomere is just long enough to permit two cell divisions before senescence. As shown on the left side of the figure, if there is no T-SCE the cell produces an 8-cell colony at the time of clonal senescence. In contrast, the fate of the same cell would be quite different if an unequal T-SCE transferred most of the telomeric DNA to one daughter cell. The right side of the figure shows that one daughter cell senesces immediately, but the other cell proliferates for an additional two divisions and yields a 17-cell colony by the time proliferation ceases. Thus the effect of an unequal T-SCE is to delay clonal senescence.

Figure 3

Unequal T-ICE may also delay senescence. The figure depicts an ICE between two telomeres of different chromosomes. In this example, the exchange lengthens the shortest telomere in the cell at the expense of truncating a longer telomere on a different chromosome. Four possible pairings of chromatids from the two chromosomes can be produced during cell division (AB, Ab, aB and ab). Colonies arising from each of the two possible daughter cell pairs have extended proliferative lives because one daughter cell in each pair inherits a lengthened version of the shortest telomere.

Figure 4

T-ICE can transfer markers to new chromosomes. A marker placed into telomeric DNA can be transferred to a new chromosome if an exchange occurs centromeric to the marker's location. Depending on segregation of chromatids during cell division, a cell may inherit a copy of the marker on both chromosomes (AB), the original chromosome only (Ab), the new chromosome only (aB) or neither chromosome (ab).