Telomere length and telomerase activity predict survival in patients with B cell chronic lymphocytic leukemia

The telomere-telomerase hypothesis states that the vast majority of human tumors have a prolonged replicative life span throughout expressing telomerase, which compensates the cell division-associated loss of telomere DNA. The use of telomere length and telomerase expression as new biological markers in cancer patients requires their correlation with disease prognosis. We, therefore, correlated the mean telomere length based on a telomere restriction fragment assay and the activity of telomerase measured with a telomeric repeat amplification protocol with clinical data and overall survival in 58 patients with B cell chronic lymphocytic leukemia (B-CLL). Telomere length showed a highly inverse correlation to telomerase activity. Patients with telomeres below 6.0 kb were associated with high telomerase activity, whereas patients with a telomere length >6.0 kb generally showed low enzyme activity (P <0.001). Patients in Binet A exhibited significantly longer telomeres and had less telomerase activity than did patients in Binet B or Binet C, where significantly shorter telomeres and higher telomerase activity were observed (P=0.031). Short telomere length and high telomerase activity were significantly associated with a shorter median survival (P=0.02 and P <0.001), and telomerase activity was the most significant prognostic factor for overall survival in B-CLL (P <0.001). Our data provide evidence that telomere length, as well telomerase activity, exerts a strong impact on the survival of B-CLL patients and that telomerase activity can be used as a new prognostic marker in this disease.     

Telomere, telomerase and cytogenetic changes in myelodysplastic syndromes

Myelodysplastic syndrome (MDS) is a heterogenous but clonal disorder characterized by cytopenia and dysplastic features. Telomere length in MDS vary but some of them show shortened telomeres. Telomerase activity in MDS also vary but about 60% of them show slightly elevated telomerase activity. According to the disease progression of MDS, MDS patients categorize into 3 groups, i.e., (1) normal telomere length before and after disease progression, (2) short telomere length before and after progression, and (3) shortened telomere with disease progression. Telomerase change with disease progression is not obscure, indicating impairment of telomere dynamics in MDS. These observations may indicate that some MDS show telomerase upregulation possible due to telomere shortening, while the another pathway without telomerase upregulation associated with complex chromosome changes may link to the pathogenesis of MDS.     

Regulation of retinoblastoma gene expression in a mouse mammary tumor model

We initiated studies to investigate the involvement of the murine retinoblastoma (RB) gene in mammary carcinogenesis using cell lines derived from mammary glands of irradiated mice. We found that the RB mRNA levels as well as the amounts of the nuclear phosphoprotein were significantly reduced as the cells progressed in vitro from non-tumorigenic to tumorigenic stages. To further investigate RB gene expression with cellular development and tumorigenicity, we transfected malignant cells with expression vectors containing the murine RB cDNA driven by either the SV40 or the mouse metallothionein promoter sequences. The neomycin resistant gene was included in both vectors and was used as a selective marker for the transfected cells. Cells with reduced levels of endogenous RB mRNA were stably transfected and showed increased expression of RB. In addition, the morphology of these cells were altered and their growth rates in culture were reduced. Injection of the transfected cells into host mice resulted in a delayed onset of tumors compared with nontransfected parental cells. Our studies provide experimental data to confirm that loss of RB gene activity is involved in neoplastic transformation of cells and support the multistep theory of carcinogenesis.     

Spatial and temporal expression of angiogenic molecules during tumor growth and progression

The growth and metastasis of cancer directly correlates with tumor angiogenesis. A better understanding of the expression of regulatory factors controlling angiogenesis is important in exploiting this process therapeutically. Our present study demonstrates that small tumors (3-4 mm in diameter) express more basic fibroblast growth factor (bFGF) and interleukin 8 (IL-8) than large tumors (> 10 mm in diameter), whereas more vascular endothelial growth factor (VEGF) is expressed in large tumors. Immunostaining showed a heterogeneous distribution of angiogenic factors within the tumor; expression of bFGF and IL-8 was highest on the periphery of a large tumor, where cell division is maximum. VEGF expression was higher in the center of the tumor. In vitro studies demonstrated that sparse cultures of tumor cells expressed higher levels of bFGF and IL-8 than confluent cultures. In contrast, the expression of bFGF and IL-8 was not diminished in tumor cells growing on confluent monolayers of normal cells. VEGF expression was upregulated by cell density irrespective of contact with tumor cells or normal cells. These results demonstrate that the expression of different angiogenic factors in tumor cells can be regulated by their proximity to other tumor cells or host cells.     

Analysis of glutaminase activity and RNA expression in preimplantation mouse embryos

The efficiency of the American Optical Company (Hardy, Rand and Rittler) (HRR) plates for screening, grading and classifying red-green colour deficiency was examined for 401 male colour deficient subjects previously identified and diagnosed with Nagel anomaloscope. There were 83 protanopes, 30 protanomalous trichromats, 96 deuteranopes and 192 deuteranomalous trichromats. Screening sensitivity was found to be 100% for dichromats and 96.4% for anomalous trichromats based on one screening error (35 subjects, including 7 dichromats, where identified by a single error). Thirty subjects (13.5%) made errors on screening plates only and were identified as having minimal colour deficiency. The HRR grading system did not distinguish dichromats and anomalous trichromats; 54% of dichromats were graded as having moderate rather than severe colour deficiency. Protan/deutan classification was correct for 95% of subjects who failed grading plates. HRR grades for anomalous trichromats were compared with the anomaloscope matching range and with pass or fail of the D15 test. The results show that only two rather than four grading categories can be distinguished by the HRR plates and that both the D15 and the HRR plates are needed in a vocational test battery to establish the severity of colour deficiency.     

Telomere length regulation during postnatal development and ageing in Mus spretus

Telomere shortening has been causally implicated in replicative senescence in humans. To examine the relationship between telomere length and ageing in mice, we have utilized Mus spretus as a model species because it has telomere lengths of approximately the same length as humans. Telomere length and telomerase were analyzed from liver, kidney, spleen, brain and testis from >180 M.spretus male and female mice of different ages. Although telomere lengths for each tissue were heterogeneous, significant changes in telomere lengths were found in spleen and brain, but not in liver, testis or kidney. Telomerase activity was abundant in liver and testis, but weak to non-detectable in spleen, kidney and brain. Gender differences in mean terminal restriction fragment length were discovered in tissues from M.spretus and from M.spretus xC57BL/6 F1 mice, in which a M. spretus -sized telomeric smear could be measured. The comparison of the rank order of tissue telomere lengths within individual M. spretus showed that certain tissues tended to be longer than the others, and this ranking also extended to tissues of the M.spretus xC57BL/6 F1 mice. These data suggest that telomere lengths within individual tissues are regulated independently and are genetically controlled.     

Decrease of telomere length in thyroid adenomas without telomerase activity

In somatic cells, telomeres shorten with population doubling, thus limiting their capacity to divide. Telomerase, which synthesizes telomeric repeats, can compensate for such shortening. Telomerase activity is known to be absent from most somatic differentiated cells but is present in germline cells, immortal cell lines, or a large majority of malignant tumors. Autonomous thyroid adenomas are benign tumors composed of highly differentiated cells characterized by TSH-independent function and growth. Telomere length and telomerase activity were measured in autonomous and hypofunctioning adenomas and their surrounding tissues. A significant decrease of 3.8+/-1.0 kilobases (kb) was observed in the length of the terminal restriction fragments (TRF) in 12 autonomous adenomas (8.6+/-1.1 kb), compared with the TRF length of their surrounding tissues (12.4+/-1.6 kb). The same kind of decrease, 3.5+/-1.2 kb, was also observed in 16 hypofunctioning adenomas (12.3+/-1.7 kb in surrounding tissue and 8.8+/-1.6 kb in the adenomas). No telomerase activity was detected either in the 12 autonomous adenomas studied or in most of the quiescent tissues (10 of 12). Most of the hypofunctioning adenomas tested (15 of 16) did not display telomerase activity. These results suggest that the cells have undergone a higher number of cell divisions in the adenomas than in the surrounding tissue. Moreover, there is a larger spread of the TRF length distribution in autonomous adenomas than in the collateral tissue. This could reflect the heterogeneity in proliferation status of the cells in the nodule, some of which have reached the end of their life span, whereas others are still proliferating (but with no malignant potential for the autonomous adenomas). In conclusion, benign adenomas exhibit a shorter and more variable telomere length than the normal collateral quiescent tissue, with no telomerase activity to compensate this loss in telomere length.     

The mouse telomerase RNA 5"-end lies just upstream of the telomerase template sequence

Telomerase is a ribonucleoprotein enzyme with an essential RNA component. Embedded within the telomerase RNA is a template sequence for telomere synthesis. We have characterized the structure of the 5' regions of the human and mouse telomerase-RNA genes, and have found a striking difference in the location of the template sequence: Whereas the 5'-end of the human telomerase RNA lies 45 nt from the telomerase-RNA template sequence, the 5'-end of the mouse telomerase RNA lies just 2 nt from the telomerase-RNA template sequence. Analysis of genomic sequences flanking the 5'-end of the human and mouse telomerase RNA-coding sequences reveals similar promoter-element arrangements typical of mRNA-type promoters: a TATA box-like element and an upstream region containing a consensus CCAAT box. This putative promoter structure contrasts with that of the ciliate telomerase-RNA genes whose structure resembles RNA polymerase III U6 small nuclear RNA (snRNA) promoters. These and other comparisons suggest that, during evolution, both the RNA-polymerase specificity of telomerase RNA-gene promoters and, more recently, the position of the template sequence in the telomerase RNA changed.     

Comparison of human telomerase RNA and telomerase activity in urine for diagnosis of bladder cancer

Currently the demand for transplant organs far outstrips the supply in the UK. This problem is even more severe for the Asian population, who have been shown to be disproportionately over-represented on transplant waiting lists in some regions of the UK. Several commentators have suggested that religious and cultural traditions may be the major determinant preventing Asians from donating organs. An exploratory qualitative study was undertaken with the aim of examining the influence of religious beliefs, amongst other things, on the extent and direction of public attitudes towards organ donation in a cross-section of the Asian population in Luton. This study indicates that, in the population studied, culture and religion play a much less prohibitive part in determining the level of organ donation than previously suggested. However, there is a desire to be aware of the religious stances so that people can make a more informed decision. The emphasis should clearly been a reconsideration of the presently inadequate approaches to organ procurement and on devising and supplementing these with more appropriate ones. An example of the failure to inform effectively the relevant populations about important developments is that only two of the 32 Muslims in the survey had heard of the 'fatwa' by the Muslim Legislative Council permitting organ donation.     

Amplification of Ki-ras and elevation of MAP kinase activity during mammary tumor progression in C3(1)/SV40 Tag transgenic mice

We have previously documented that transgenic mice expressing SV40 Tag regulated by the rat prostatic steroid-binding protein C3(1) 5'-flanking region display multistage mammary tumorigenesis. To delineate genetic changes associated with mammary tumor progression, comparative genomic hybridization (CGH) was performed. CGH revealed a consistent gain of the telomeric region of chromosome 6. This region contains the Ki-ras proto-oncogene. Analyses of genomic DNA by Southern blot demonstrated up to 40-fold amplification of the Ki-ras gene. Ki-ras amplification was detected in 12, 46 and 68% of tumors from 4, 5 and 6 month old mice, respectively, whereas no amplifications were found in any preneoplastic mammary tissues. Tumors bearing Ki-ras gene amplification exhibited high levels of Ki-ras RNA and protein. The over-expressed Ki-Ras protein in these tumors appeared functionally active as indicated by the elevated MAP kinase activity. These data demonstrate that while Ki-ras amplification might not be an early event, there is a strong association between Ki-ras amplification and over-expression and mammary tumor progression in this model. This study also shows that CGH is a powerful and useful technique for identifying chromosomal copy number changes during tumor progression, and that this model may provide a predictable in vivo system for studying gene amplification.     

Loss of endogenous mouse mammary tumor virus superantigen increases tumor resistance

From a cross between a tumor-susceptible mouse strain (DBA/2; D) and a tumor-resistant MHC-identical strain (B10.D2; D2) new recombinant inbred mouse strains were established over many generations of inbreeding and tumor resistance selection. Since resistance to the highly metastatic DBA/2 lymphoma variant ESb had an immunologic basis, and the two parental strains differed in endogenous viral superantigens (vSAGs), DNA of three D2 x D recombinant inbred mouse lines was typed for endogenous mouse mammary tumor viruses using mouse mammary tumor virus long terminal repeat- and env gene-specific probes. The resistant D2 x D mice were very similar to the susceptible parental strain D in their Mtv Southern blots, except for the lack of a single band corresponding to Mtv-7, the provirus coding for the strong DBA/2 superantigen Mls-1a. A backcross analysis revealed that Mtv-7-negative F2 mice were significantly more resistant than Mtv-7-positive F2 mice. When Mtv-7 was reintroduced into the resistant lines by crossing them with either CBA/J or BALB/D2.Mls-1a, the mice became again more tumor susceptible. Finally, we demonstrate the ability to transfer immunoresistance and graft-vs-leukemia reactivity from tumor-resistant to tumor-susceptible mice.     

Severe growth defect in mouse cells lacking the telomerase RNA component

A common belief is that one tablet or suppository containing, e.g. 100 mg of a drug can be substituted, without any changes in the therapeutic effect, with two units of the same brand containing 50 mg of the drug. In the present study a single dose of paracetamol was administered to healthy volunteers as (a) two tablets of 500 mg, (b) two suppositories of 500 mg, and (c) one suppository of 1000 mg. There were statistically significant differences in all bioavailability parameters (t(max), C(max) and AUC) between the three treatments. The relative bioavailability of the 500 mg suppositories was 77% and that of the 1000 mg suppositories 66%. The absorption rate from suppositories was markedly lower than from the tablets. Especially low absorption rate was obtained with the suppository of 1000 mg. The two strengths, although having the same trade name, were not therefore bioequivalent.     

B and T cells are required for mouse mammary tumor virus spread within the mammary gland

Mouse mammary tumor virus (MMTV) is an infectious retrovirus transmitted through milk from mother to newborns. MMTV encodes a superantigen (SAg) whose activity is indispensable for the virus life cycle, since a genetically engineered virus with a mutation in the sag gene neither amplified in cells of the immune system of suckling pups nor infected their mammary glands. When wild-type MMTV was injected directly into the mammary glands of uninfected pubescent mice, their lymphoid as well as mammary gland cells became virus infected. To test whether this infection of lymphoid cells was dependent on SAg activity and required for virus spread within the mammary gland, we performed mammary gland injections of wild-type MMTV(C3H) into two strains of transgenic mice that lacked SAg-cognate, V beta 14+ T cells. Neither the MTV-ORF or LEL strains showed infection of their mammary glands. Moreover, no MMTV infection of their peripheral lymphocytes was detected. Similar experiments with mice lacking B cells (mu-chain knockouts) showed no detectable virus spread in the mammary glands or lymphoid tissues. These data suggest that SAg activity and MMTV-infected lymphocytes are required, not only for initial steps of viral infection, but also for virus spread within the mammary gland. Virus spread at late times in infection determines whether MMTV induces mammary tumors.     

Diagnostic significance of telomerase activity and telomere length in endoscopic sample of colorectal cancer

Telomeres are located on both ends of individual chromosomes in eukaryotes. It has been reported that telomerase activity and telomere reduction can be detected in most human cancers. We examined telomerase activity and telomere length in colorectal cancer tissues obtained by colonoscopy. Telomerase activity was examined by the TRAP (telomeric repeat amplification protocol) assay and was detected in 21 of 26 (81%) primary colorectal carcinoma tissues. Two of 9 (22%) colorectal polyp were telomerase positive. Telomere length was analyzed by Southern blotting and there was reduction in telomere lengths in 12 of 15 (80%) primary colorectal carcinoma and 3 of 6 colorectal polyp, compared to the corresponding normal colonic mucosa. Therefore, telomerase activity and telomere length may serve as an useful tool for preoperative cancer diagnosis.     

Effect of oncogene expression on telomerase activation and telomere length in human endothelial, fibroblast and prostate epithelial cells

Although strong evidence is mounting that telomerase reactivation and the thereof resulting stabilization of telomeres is a major mechanism for human cells to overcome replicative senescence, a causal relationship linking telomerase activation conclusively to tumorigenesis remains to be established. Thus, the possibility exists that telomerase activation is passively co-selected as tumors develop. To elucidate the function of telomerase during tumorigenesis, we followed telomerase reactivation during immortalization of human primary cell types with in vitro transforming agents and determined the tumorigenic potential of these cells at various stages of transformation. The effects of SV40, v-Ki-ras, HPV-18 and HPV-16 E6/E7 oncoproteins on telomerase expression was examined in primary and immortalized human prostate epithelial (HPE), human prostate fibroblast (HPF), and umbilical vein endothelial cells (HUVEC). All of five SV40-transformed HPE and HPF lines were telomerase positive and had shorter telomeres than primary cells. The two HPV-18 immortalized HPE cell lines also expressed telomerase activity. In contrast, E6 or E7 alone could not produce immortalized HUVEC and did not reactivate telomerase. Life-span, however, was extended. The E6/E7 immortalized HUVEC had telomerase activity and short but stable telomeres. HPE, HPF or HUVEC cells which had been transformed by one oncoprotein alone were not tumorigenic although they had overcome cellular senescence and re-activated telomerase. However, if these cells were transformed by a second agent, either infection with v-Ki-ras or X-ray treatment, they were able to form tumors in nude mice. This suggests that tumorigenesis is a multistep process and that telomerase activation alone is not sufficient for malignant transformation in human cells.