Chk2 and p53 Are Haploinsufficient with Dependent and Independent Functions to Eliminate Cells after Telomere Loss

The mechanisms that cells use to monitor telomere integrity, and the array of responses that may be induced, are not fully defined. To date there have been no studies in animals describing the ability of cells to survive and contribute to adult organs following telomere loss. We developed assays to monitor the ability of somatic cells to proliferate and differentiate after telomere loss. Here we show that p53 and Chk2 limit the growth and differentiation of cells that lose a telomere. Furthermore, our results show that two copies of the genes encoding p53 and Chk2 are required for the cell to mount a rapid wildtype response to a missing telomere. Finally, our results show that, while Chk2 functions by activating the p53-dependent apoptotic cascade, Chk2 also functions independently of p53 to limit survival. In spite of these mechanisms to eliminate cells that have lost a telomere, we find that such cells can make a substantial contribution to differentiated adult tissues

Telomere Loss Provokes Multiple Pathways to Apoptosis and Produces Genomic Instability in Drosophila melanogaster

Telomere loss was produced during development of Drosophila melanogaster by breakage of an induced dicentric chromosome. The most prominent outcome of this event is cell death through Chk2 and Chk1 controlled p53-dependent apoptotic pathways. A third p53-independent apoptotic pathway is additionally utilized when telomere loss is accompanied by the generation of significant aneuploidy. In spite of these three lines of defense against the proliferation of cells with damaged genomes a small fraction of cells that have lost a telomere escape apoptosis and divide repeatedly. Evasion of apoptosis is accompanied by the accumulation of karyotypic abnormalites that often typify cancer cells, including end-to-end chromosome fusions, anaphase bridges, aneuploidy, and polyploidy. There was clear evidence of bridge–breakage–fusion cycles, and surprisingly, chromosome segments without centromeres could persist and accumulate to high-copy number. Cells manifesting these signs of genomic instability were much more frequent when the apoptotic mechanisms were crippled. We conclude that loss of a single telomere is sufficient to generate at least two phenotypes of early cancer cells: genomic instability that involves multiple chromosomes and aneuploidy. This aneuploidy may facilitate the continued escape of such cells from the normal checkpoint mechanisms

Healing of Euchromatic Chromosome Breaks by Efficient de novo Telomere Addition in Drosophila melanogaster

Previously, we observed that heterochromatic 4 and Y chromosomes that had experienced breakage in the male germline were frequently transmitted to progeny. Their behavior suggested that they carried functional telomeres. Here we show that efficient healing by de novo telomere addition is not unique to heterochromatic breaks

Primary spermatocyte cysts following dicentric chromosome induction.

<p>Phase contrast views of a normal testis and <i>yw/DcY(H1); hsFLP</i>2B/+ testis five days after heat shock. The apical portion of a normal testis (A) is filled with cysts, with primary spermatocyte cysts occupying most of the volume. Stem cells are located at the left tip. After dicentric induction (B) very few primary spermatocyte cysts are found (none in this particular testis). Instead, elongating spermatid cysts, derived from cells which were beyond the heat shock responsive stage <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004130#pgen.1004130-Golic4" target="_blank">[65]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004130#pgen.1004130-Bonner1" target="_blank">[66]</a>, occupy the entire length of the testis. (C) The primary spermatocyte cyst population after heat shock induction of dicentric chromosomes. Flies that do not make dicentrics (no FLP control, ▴) show no reduction of primary spermatocyte cysts after heat shock. After dicentric induction there is a reduction in primary spermatocyte cysts, followed by recovery in wildtype males (•), but not in <i>p53</i> mutants (▪). The <i>lok</i> mutant males (▾) showed no reduction in primary spermatocyte cysts after dicentric induction. Dotted lines with open symbols represent data only for testes that had at least one primary spermatocyte cyst. Error bars indicate ±1 SEM.</p

Viability of eggs fertilized by <i>y w 70FLP3F/DcY, H1; lok/lok</i> males.

<p><i>w¹¹¹⁸/H1; lok^P6</i> males were crossed to either.</p>a<p><i>y w 70FLP; lok^P6/Cy lok⁺</i>, or</p>b<p><i>y w 70FLP; lok^P6/lok^P6</i> females and their progeny were heat-shocked (or not) at 38° for one hour during the first 24 hrs. of development. The <i>y w 70FLP/H1; lok/lok</i> males that eclosed were then crossed to <i>y w</i> females and egg to adult survival of their progeny was scored.</p><p>FR, fragment ratio; SR, sex ratio.</p

<i>FrY</i> recovery: No heat shock controls.

<p>Males were testcrossed individually to <i>y w</i> females. Genotypes of males tested were:</p>a<p><i>y w 70FLP</i>3F/<i>DcY, H1</i>.</p>b<p><i>y w 70FLP</i>3F/<i>DcY, H1; lok^P6</i> (from <i>lok^P6</i> homozygous mothers).</p>c<p><i>y w 70FLP</i>3F/<i>DcY, H1; p53^5A-1-4</i> (from <i>p53^5A-1-4</i> homozygous mothers).</p

Dicentric bridge frequency in Meiosis II.

<p>MII dicentric bridges were scored in testes dissected from wildtype or <i>lok</i> males, using the same protocol as for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004130#pgen-1004130-g003" target="_blank">Figure 3</a>.</p

Sperm head displacement following dicentric chromosome induction.

<p>HS, heat shock; N, number of elongated post-meiotic cysts scored.</p

Frequency distribution of <i>FrY</i> offspring produced by individual heat-shocked <i>y w 70FLP/DcY, H1</i> males.

<p>Frequency distribution of <i>FrY</i> offspring produced by individual heat-shocked <i>y w 70FLP/DcY, H1</i> males.</p

<i>FrY</i> recovery from wildtype and mutant males (38° 1 hr. heat shock at 0–72 hours of development).

<p>Males were testcrossed individually to <i>y w</i> females. Genotypes of males tested:</p>a<p><i>y w 70FLP</i>3F•<i>YL/DcY, H1</i>.</p>b<p><i>y w 70FLP</i>3F•<i>YL/DcY, H1; lok^P6</i> (from <i>lok^P6/+</i> heterozygous mothers).</p>c<p><i>y w 70FLP</i>3F•<i>YL/DcY, H1; p53^5A-1-4</i> (from <i>p53^5A-1-4</i> homozygous mothers).</p