Makoto Kammori

Regulation of Murine Telomere Length by Rtel: An Essential Gene Encoding a Helicase-like Protein

Little is known about the genes that regulate telomere length diversity between mammalian species. A candidate gene locus was previously mapped to a region on distal mouse Chr 2q. Within this region, we identified a gene similar to the dog-1 DNA helicase-like gene in C. elegans. We cloned this Regulator of telomere length (Rtel) gene and inactivated its expression in mice. Rtel(-/-) mice died between days 10 and 11.5 of gestation with defects in the nervous system, heart, vasculature, and extraembryonic tissues. Rtel(-/-) embryonic stem cells showed telomere loss and displayed many chromosome breaks and fusions upon differentiation in vitro. Crosses of Rtel(+/-) mice with Mus spretus showed that Rtel from the Mus musculus parent is required for telomere elongation of M. spretus chromosomes in F1 cells. We conclude that Rtel is an essential gene that regulates telomere length and prevents genetic instability.

Correlation of telomere lengths in normal and cancers tissue in the large bowel.

The hypothesis that telomeres in colorectal cancer cells exhibit age-related shortening, as in normal cells of the colorectal epithelium, was tested with samples of non-cancerous mucosa and cancer tissue from 124 patients (aged 29-97 years). Shortening with aging could be demonstrated for both normal and cancer tissues; regression analysis showed rates for length reduction of 44 and 50 base pair/year, respectively. Straight, essentially parallel, lines were obtained for the two cases, normal tissue values being about 2 kilobase pairs (kbp) higher, with a significant correlation between data at the individual patient level.

Telomerase activity and telomere length in benign and malignant human thyroid tissues

Several studies have demonstrated that telomerase is activated and telomere length is altered in various types of tumors. In this study, we investigated telomerase activities and telomere length in 21 thyroid tumors. Telomerase activity was detected in 11 of 12 thyroid cancers and three of nine follicular adenomas. The mean telomere lengths in the cancers tissue and follicular adenomas were lower than in the respective background tissues, the differences being significant (P=0.0055 and P<0.006), respectively. Our findings suggest that change in telomerase activity and telomere length may be important for development of thyroid tumors.

Telomere shortening in gastric carcinoma with aging despite telomerase activation

Abstract In the present study, we analyzed both telomere length and telomerase activity in surgical and autopsy samples of non-neoplastic mucosa and carcinomas of the stomach. Telomere length, determined by Southern blot analysis, demonstrated progressive shortening with age in non-neoplastic gastric mucosal specimens from 38 human subjects aged between 0 and 99 years, with an average annual loss rate of 46 base pairs (bp). The mean (±SD) telomere length in 21 gastric carcinomas was 7.0u2009±u20091.6u2009×u2009103 base pairs (1.6u2009kbp). In 20 (95%) of the 21 subjects, the values were smaller than those in the non-neoplastic gastric mucosa (mean shortening 1.8u2009kbp), although a strong correlation was observed for the paired data (ru2009=u20090.69, Pu2009=u20090.0004). Similarly, telomere lengths in carcinomas were shorter than those for intestinal metaplasia (a mean difference of 1.1u2009kbp). Telomerase activity, estimated using the telomeric repeat amplification protocol assay, was positive in 18 (86%) of the 21 gastric carcinomas, without significant differences among the three histological types (well, moderately, and poorly differentiated adenocarcinomas) or with sex or age. The results suggest that telomere length and possibly shortening rates vary with the individual, and that examination of both non-neoplastic mucosa and tumors is necessary to improve our understanding of the significance of telomerase in neoplasia.

Basal cells have longest telomeres measured by tissue Q-FISH method in lingual epithelium

We investigated the telomere lengths of individual cell types in lingual mucosa using an improved tissue quantitative fluorescence in situ hybridization (Q-FISH) method. Our tissue Q-FISH method compensates for partially cut nuclei in a tissue section by using the telomere:centromere ratio (TCR). We normalized our TCR measurements (NTCR) using a section from a block of cultured cells placed on the same slide, thus improving the accuracy and reproducibility of the results. Normal lingual mucosa was obtained from 21 autopsied individuals. Immunohistochemistry showed positivity mainly for p27, p63, and CK19 in basal cells, and for Ki-67 in parabasal cells. Q-FISH revealed that NTCR was significantly highest in basal cells and lowest in prickle cells, and also that telomere length regressed at a certain rate in each cell type, firstly. Significant correlations of NTCR among the three epithelial cell types were demonstrated. The present findings appear to support the theory that stem cells exist in the basal layer of the lingual epithelium. The reduction of telomere length with age and in each cell layer is consistent with the telomere biology theory of cell proliferation and differentiation in oral mucosa.

Unusual distribution pattern of telomeric repeats in the shrews Sorex araneus and Sorex granarius

Sorex araneus and Sorex granarius are sibling species within the Sorex araneus group with karyotypes composed of almost identical chromosome arms. S. granarius has a largely acrocentric karyotype, while, in S. araneus, various of these acrocentrics have combined together by Robertsonian (Rb) fusions to form metacentrics, with the numbers and types of metacentrics differing between chromosomal races. Our studies on telomeric sequences in S. araneus and S. granarius revealed differences between chromosomes and between species. In S. araneus (the Novosibirsk race), hybridization signals were present on the telomeres of all the chromosomes after FISH with a PCR-generated telomeric probe. In addition, hybridization signals were observed at high frequencies in the pericentric regions of some but not all metacentrics formed by Rb fusion. There were fewer signals on those metacentrics formed earlier in the evolution of S. araneus. This suggests that S. araneus chromosomes retain at least some telomeric repeats during Rb fusion, but that these repeats are lost or modified over time. These results are critical for the interpretation of the well-studied hybrid zones between chromosomal races of S. araneus, given that Rb fission has been postulated in such hybrid zones and that the likelihood of Rb fission will relate to presence/absence of telomeric sequences at the centromeres of metacentrics. In S. granarius, there were strong signals at the proximal (centromeric) telomeres of the acrocentrics after FISH with a DNA telomeric probe. FISH with a PNA telomeric probe on S. granarius acrocentrics showed that the proximal telomeres were 213xa0kb on average, while the length of the distal telomeres was 3.8xa0kb on average. Two-colour FISH, using a telomeric DNA probe and a microdissected probe generated from the pericentric regions of the S. granarius chromosomes a and b, revealed regions on distinct chromatin fibres where telomeric and microdissected probes were colocalized or localized sequentially. The proximal telomeres of S. granarius are highly unusual both in their large size and their heterogeneous structure relative to the telomeres of other mammals.

Oncocytic Adenocarcinoma of the Stomach: Parietal Cell Carcinoma

We report 10 cases of an unusual type of gastric adenocarcinoma that occurred in elderly patients 58–81 years of age. Histologically, the tumors were well to moderately differentiated tubular adenocarcinomas with very eosinophilic, finely granular cytoplasm. Immunohistochemical stains for antimitochondrial antibody were strongly positive. Ultrastructurally, the tumor cells had numerous mitochondria in their cytoplasm and occasional intracytoplasmic lumina with associated long microvilli. These histologic and ultrastructural features are similar to those of parietal cells in normal gastric fundic mucosa, but immunohistochemical staining of the tumors using four different antiparietal cell antibodies (anti-H+-K+-adenosine triphosphatase antibodies) was negative in all cases. Therefore, we think that these tumors were not parietal cell carcinomas but could be termed oncocytic adenocarcinomas, or adenocarcinomas with oncocytic differentiation. Previously reported cases of parietal cell carcinoma have been said to have a favorable prognosis, but it will be necessary to study a larger number of cases to determine the prognosis of oncocytic adenocarcinoma.

Luminal and cancer cells in the breast show more rapid telomere shortening than myoepithelial cells and fibroblasts

Critically shortened, dysfunctional telomeres may play a role in the genetic instabilities commonly found in cancer. We analyzed 30 surgical specimens of invasive breast carcinoma from women aged 34 to 91 years and estimated telomere lengths as telomere-to-centromere ratio values in the 5 different cell types comprising breast tissue in order to clarify telomere length variations within and between individuals using our tissue quantitative fluorescence in situ hybridization method. We obtained 3 novel findings. (1) In corresponding normal tissues, telomere length decreased in the order myoepithelial cells > normal-appearing fibroblasts > luminal epithelial cells, and telomere lengths were characteristic in these 3 cell types within each individual. (2) As expected, cancer cells had significantly shorter telomeres than myoepithelial cells (P < .0001) and normal-appearing fibroblasts (P = .0161), but there was no significant difference in telomere length between luminal cells and cancer cells (P = .6270). (3) Fibroblasts adjacent to cancer had longer telomeres than normal-appearing fibroblasts distant from cancer (P < .0001). This study, which represents the first reported assessment of telomere length variations in the 5 cell types comprising breast tissue within and between individuals, revealed that normal luminal epithelial cells and cancer cells had the shortest telomeres. Our new findings indicate that telomeres of background luminal cells are as short as those of cancer cells. Tissue quantitative fluorescence in situ hybridization, applicable to analysis of individual cells in tissue sections, is considered to be a powerful technique with considerable promise for studies in oncology.

Telomere shortening with aging in human esophageal mucosa.

Progressive telomere shortening with aging was studied using normal esophageal mucosal specimens from 177 human subjects aged between 0 and 102 years (yrs). We observed age-related shortening of the telomere, at a rate of 60 base pairs (bp) per year (yr). The mean telomere length of 12 neonates was 15.2 kilobase pairs (kbp) and that of 2 centenarians was 9.3 kbp. Mean (±SD) telomere lengths were 14.9±1.3, 14.0±1.8, 10.1±3.7, 10.4±3.3 and 9.5±3.1 kbp for the age groups less than 2 yrs, 2–20 yrs, 21–60 yrs, 61–80 yrs and 81–102 yrs, respectively. The variation in telomere length among individuals in the same age group was greater for the 3 older groups than for the 2 younger groups, as shown by the SDs. Furthermore, older individuals had greater telomere length variation than younger individuals, based on the lengths of DNA digested smears. Although the telomere length decreased significantly with aging at the rate of 60 bp per yr, differences in the mean telomere lengths between the 3 older age groups were not significant. Rapid shortening occurred in the young generations and there was no further substantial decrease in the esophageal mucosa after 60 yrs of age. Compared to the very rapid renewal rate of the esophageal epithelial cells, the annual reduction rate in telomere length was very low. These findings support the hypothesis that germ cells in the esophageal epithelium have a mechanism to lengthen telomeres.

Chromosomal instability and telomere lengths of each chromosomal arm measured by Q-FISH in human fibroblast strains prior to replicative senescence

We monitored the telomere lengths and chromosomal instability characteristics of fibroblasts at different population doubling levels (PDLs) to gain further insight into the role of telomere shortening in chromosomal instability. We used 7 normal diploid human fibroblast strains (TIG-1, 3, 7, 103, 104, 112, and 114) and a quantitative fluorescence in situ hybridization method to measure telomere lengths of the p- and q-arms of individual chromosomes. We also enumerated morphologic signs of chromosomal instability, including fusion or loss of chromosomes, and anaphase bridges. In strains TIG-1, 3, 7, 103, and 114 at the late (phase 3) stage (≧40PDLs), 29 (96.6%) of 30 fusions were associated with one or both of the chromosomal arms that bear significantly shorter telomeres in those populations. In TIG-1 at 62PDL, 6 fusions were associated with Xq (n=3), 21q (n=3), and other (n=6) chromosomes. Xq and 21q had significantly shorter telomeres, and anaphase bridges were often associated with chromosomes X and/or 21 (74.6%). Our results indicate that chromosomes having excessively shortened telomeres at late PDLs begin to show features of instability such as fusions and anaphase bridges.