Non-Random Telomere Lengthening at Specific Chromosome Ends as a Novel Clonal Event in Chronic Myeloid Leukemia

It is widely accepted that chromosomal telomere dysfunction caused either by telomere shortening or lesions in the capping machinery is an important factor in carcinogenesis. However, in our recent study using quantitative FISH (Q-FISH), telomere restriction fragment (TRF) analysis and fiber FISH techniques on the measurement of telomere length in 32 cases of chronic myeloid leukemia (CML), we found that telomere lengthening at some specific chromosome ends is apparently a non-random event showing a clonal nature. Methods: TRF: genomic DNA was digested by frequently cutting restriction enzymes. After gel electrophoresis and Southern blotting, the blotted DNA was hybridized to a digoxigenin (DIG)-labeled probe specific for telomeric repeats. A DIG-specific antibody covalently coupled to alkaline phosphate followed by the chemiluminescence detection was used to detect telomere signals. The quantitative measurements of mean TRF length can be reached by scanning the signals on the film and analyzing them with the computer software. Q-FISH: chromosomes on cytogenetic slides were hybridized with a peptide nucleic acid (PNA) telomere probe (Panagene, Korea). The leukemia cells can be traced by the particular chromosome rearrangement presented in the metaphase cells, e.g. t(9;22). The signal intensity, which is proportional to telomere length, of each individual telomere was automatically measured with the software of the imaging system (ISIS 2 MetaSystems, Belmont, MA). The relative telomere length was calculated using the ratio of individual signal intensity to the standard deviation. fiber FISH: fiber FISH was performed with cohybridization of a specific subtelomere probe (with a known size and close to the telomere site of interest) and the telomere probe to the DNA/chromatin fibers preparation on the same slide. Results: The telomere lengthening at short arm of a X chromosome (Xp) was the most frequent event (seen in about 50% of CML cases we studied) in leukemia cells that can be identified by the chromosome 9 and 22 translocation [t(9;22)(q34;q11.2)]. The longest telomere length at Xp end for the CML cases reached 200 kb, which is about 20-fold longer than in normal cells. The other telomeres involved in the non-random lengthening include 18p (7/32), Yp (4/32), 4q (4/32), 5p (3/32), 7q (3/32), and 15p (3/32). Conclusion: While the relationship between the sequence organization for telomere shortening and their function is relatively clear, it has not been well stated if telomere lengthening at specific chromosome ends could be involved in cell proliferation, in maintenance of the genome stability and in evolution of the cancer. Our findings have shown the first evidence that the telomere lengthening at some specific chromosome ends is such a salient clonal event. Further investigation on picking up specific individual telomere lengthening in leukemia cells would greatly aid studies of chromosomal stability, telomerase activity, proliferative capacity and the evaluation of clinical status and thus one can use telomere length as an indicator to follow-up the cancer progression, to evaluate the treatment efficacy and to predict the prognosis in cancer.

Reed-Sternberg Cells Have Shortened Telomere Length.

As cells age, the ends of their chromosomes, the telomeres, become shortened. The chromosomal length eventually shortens to a critical point, which is thought to lead to chromosomal instability and/or cell death. Neoplastic cells have increased amounts of telomerase, an enzyme that prevents this shortening; this may be one mechanism by which tumor cells grow. Due to the scarcity of neoplastic Reed-Sternberg cells in tissues of Hodgkin Lymphoma, studies of telomere length and telomerase expression have been difficult and few. These few studies have tended to show an increase in telomerase expression in tissues from Hodgkin Lymphoma. In order to determine telomere length in Reed-Sternberg cells, we performed an immunofluorescence/FISH assay using a PNA pantelomeric probe and a CD30 antibody with which to identify the Reed-Sternberg cells (Am J Pathol 160:1259–68, 2002). Six cases were studied, with patient ages ranging from 18– 51 years. Telomere length was assessed on a semiquantitative scale. Of the six cases, five had evaluable hybridization signal. In these five cases, 53 CD30-positive cells (between 3–20 per case) were studied. In forty cells, the hybridization signal (telomere length) was less than that of surrounding cells, and in 13 it was the same; in no case was telomere length greater in the Reed-Sternberg cell than in surrounding lymphocytes (p< 0.001, Wilcoxon signed ranks test). There was no correlation between the age of the patient or the age of the sample and telomere probe signal intensity/quantity. Similar to epithelial and soft tissue tumors, it appears that the neoplastic cells in Hodgkin Lymphoma have shortened telomere length as compared to the surrounding normal cells.

Mapping a Telomere Using the Translocation eT1(III;V) in Caenorhabditis elegans

In Caenorhabditis elegans, individuals heterozygous for a reciprocal translocation produce reduced numbers of viable progeny. The proposed explanation is that the segregational pattern generates aneuploid progeny. In this article, we have examined the genotype of arrested embryonic classes. Using appropriate primers in PCR amplifications, we identified one class of arrested embryo, which could be readily recognized by its distinctive spot phenotype. The corresponding aneuploid genotype was expected to be lacking the left portion of chromosome V, from the eT1 breakpoint to the left (unc-60) end. The phenotype of the homozygotes lacking this DNA was a stage 2 embryonic arrest with a dark spot coinciding with the location in wild-type embryos of birefringent gut granules. Unlike induced events, this deletion results from meiotic segregation patterns, eliminating complexity associated with unknown material that may have been added to the end of a broken chromosome. We have used the arrested embryos, lacking chromosome V left sequences, to map a telomere probe. Unique sequences adjacent to the telomeric repeats in the clone cTel3 were missing in the arrested spot embryo. The result was confirmed by examining aneuploid segregants from a second translocation, hT1(I;V). Thus, we concluded that the telomere represented by clone cTel3 maps to the left end of chromosome V. In this analysis, we have shown that reciprocal translocations can be used to generate segregational aneuploids. These aneuploids are deleted for terminal sequences at the noncrossover ends of the C. elegans autosomes.

Identification of healed terminal DNA fragments in linear minichromosomes of Schizosaccharomyces pombe

The minichromosome Ch16 of the fission yeast Schizosaccharomyces pombe is derived from the centromeric region of chromosome III. We show that Ch16 and a shorter derivative, Ch12, made by gamma-ray cleavage, are linear molecules of 530 and 280 kilobases, respectively. Each minichromosome has two novel telomeres, as shown by genomic Southern hybridization with an S. pombe telomere probe. Comparison by hybridization of the minichromosomes and their chromosomal counterparts showed no signs of gross rearrangement. Cosmid clones covering the ends of the long arms of Ch16 and Ch12 were isolated, and subcloned fragments that contained the breakage sites were identified. They are apparently unique in the genome. By hybridization and Bal 31 digestion, the ends appear to consist of the broken-end sequences directly associated with short stretches (about 300 base pairs) of new DNA that hybridizes to a cloned S. pombe telomere. They do not contain the telomere-adjacent repeated sequences that are present in the normal chromosomes. The sizes of the short telomeric stretches are roughly the same as those of the normal chromosomes. Our results show that broken chromosomal ends in S. pombe can be healed by the de novo addition of the short telomeric repeats. The formation of Ch16 must have required two breakage-healing events, whereas a single cleavage-healing event in the long arm of Ch16 yielded Ch12.

Identification of healed terminal DNA fragments in linear minichromosomes of Schizosaccharomyces pombe.

The minichromosome Ch16 of the fission yeast Schizosaccharomyces pombe is derived from the centromeric region of chromosome III. We show that Ch16 and a shorter derivative, Ch12, made by gamma-ray cleavage, are linear molecules of 530 and 280 kilobases, respectively. Each minichromosome has two novel telomeres, as shown by genomic Southern hybridization with an S. pombe telomere probe. Comparison by hybridization of the minichromosomes and their chromosomal counterparts showed no signs of gross rearrangement. Cosmid clones covering the ends of the long arms of Ch16 and Ch12 were isolated, and subcloned fragments that contained the breakage sites were identified. They are apparently unique in the genome. By hybridization and Bal 31 digestion, the ends appear to consist of the broken-end sequences directly associated with short stretches (about 300 base pairs) of new DNA that hybridizes to a cloned S. pombe telomere. They do not contain the telomere-adjacent repeated sequences that are present in the normal chromosomes. The sizes of the short telomeric stretches are roughly the same as those of the normal chromosomes. Our results show that broken chromosomal ends in S. pombe can be healed by the de novo addition of the short telomeric repeats. The formation of Ch16 must have required two breakage-healing events, whereas a single cleavage-healing event in the long arm of Ch16 yielded Ch12.