Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumors

Abstract

Although mechanisms for chromosomal instability in tumors have been described in animal and in vitro models, little is known about these processes in man. To explore cytogenetic evolution in human tumors, chromosomal breakpoint profiles were constructed for 102 pancreatic carcinomas and 140 osteosarcomas, two tumor types characterized by extensive genomic instability. Cases with few chromosomal alterations showed a preferential clustering of breakpoints to the terminal bands, whereas tumors with many changes showed primarily interstitial and centromeric breakpoints. The terminal breakpoint frequency was negatively correlated to telomeric TTAGGG repeat length, and fluorescence in situ hybridization with telomeric TTAGGG probes consistently indicated shortened telomeres and >10% of chromosome ends lacking telomeric signals. Because telomeric dysfunction may lead to formation of unstable ring and dicentric chromosomes, mitotic figures were also evaluated. Anaphase bridges were found in all cases, and fluorescence in situ hybridization demonstrated extensive structural rearrangements of chromosomes, with terminal transferase detection showing fragmented DNA in 5-20% of interphase cells. Less than 2% of cells showed evidence of necrosis or apoptosis, and telomerase was expressed in the majority of cases. Telomeric dysfunction may thus trigger chromosomal fragmentation through persistent bridge-breakage events in pancreatic carcinomas and osteosarcomas, leading to a continuous reorganization of the tumor genome. Telomerase expression is not sufficient for completely stabilizing the chromosome complement but may be crucial for preventing complete genomic deterioration and maintaining cellular survival.

Figure 1

(A) Chromosome breakpoint profiles of 102 PC (blue line) and 140 OS (red line). Cases with <10 aberrations preferentially showed breakpoints in telomeric bands (red type), whereas cases with ≥10 aberrations preferentially had breakpoints in interstitial bands (black type); only the 15 most frequent breakpoints have been indicated. (B) Evolution of breakpoints along chromosomes in OS. For each chromosome arm, the median of the total number of aberrations per case has been indicated for tumors with terminal breakpoints (red bars), interstitial breakpoints (black bars), and chromosome arm losses (circles). In all arms, except 1p, terminal breaks occurred at lower levels of cytogenetic complexity than interstitial breaks. Telomeric associations (tas) occurred in tumors with few aberrations, followed by rings (r) and dicentrics (dic), gains, and finally losses of chromosome arms. (C) The mean number of whole chromosome losses (red) increased in parallel to the total cytogenetic complexity in OS, whereas the number of whole chromosome gains (green) remained constant; vertical bars represent confidence intervals.

Figure 2

Hybridization with TTAGGG probes to (A) normal fibroblast metaphase cells resulted in signals of similar intensity at all chromosome termini. (B) Identical pattern in a liposarcoma cell with a t(12;16); the der(16) is identified by a 16q12 probe and diamidinophenylindol banding (red; arrow). (C) LPC6 exhibited several TTAGGG-negative chromosome ends and dicentric chromosomes (arrow). (D) Similar telomeric hybridization pattern in OS3 with ring (arrow) and dicentric (arrowhead) chromosomes, and with aneusomy for chromosome 17 (centromeric probes in red). (E and F) Anaphase bridges in LPC6 and OS4. (G) Whole chromosome paint shows extensive structural rearrangements of chromosomes 13 (green) and 17 (red) in OS7. (H) Four-color FISH with probes in 13q (Inset) demonstrates amplification of 13q31 sequences (red) in a marker chromosome in OSA. (I) Fragmented DNA (green) in a nuclear bleb and a micronucleus in LPC6, shown by the terminal transferase reaction. (J and K) Broken DNA in chromatin bridges in OSA and LPC10, respectively. (L) Apoptotic cells in A23187-exposed OSA cultures with fragmentation of whole nuclear DNA content (green). (M) Multiple dying cells (arrows) in A23187-exposed OSA cultures shown by Fluoro-Jade staining. (N) Low prevalence of dying cells in cultures not exposed to A23187.

Figure 3

TERT RNA expression measured by reverse transcriptase–PCR with primers outside the alternatively spliced region (TERT3′, Left) and primers for four alternative splice products (TERT, Right). TERT3′ expression as well as two major (239 and 421 bp) and two minor (275 and 457 bp) TERT products are present in thymus (T) and the cell lines OSA, LPC3, LPC6, and LPC10, but not in kidney (K), pancreas (P), or LPC13; β-actin (ACTB) was used as internal control.