Inactivation Of Telomerase Research Articles

Inhibition of Telomerase Activity Enhances Hyperthermia-Mediated Radiosensitization

Hyperthermia is a potent sensitizer of cell killing by ionizing radiation (IR); however, hyperthermia also induces heat shock protein 70 (HSP70) synthesis and HSP70 expression is associated with radioresistance. Because HSP70 interacts with the telomerase complex and expression of the telomerase catalytic unit (hTERT) extends the life span of the human cells, we determined if heat shock influences telomerase activity and whether telomerase inhibition enhances heat-mediated IR-induced cell killing. In the present study, we show that moderate hyperthermia (43 degrees C) enhances telomerase activity. Inhibition of telomerase activity with human telomerase RNA-targeted antisense agents, and in particular GRN163L, results in enhanced hyperthermia-mediated IR-induced cell killing, and ectopic expression of catalytic unit of telomerase (TERT) decreased hyperthermia-mediated IR-induced cell killing. The increased cell killing by heat and IR exposure in telomerase-inhibited cells correlates with delayed appearance and disappearance of gamma-H2AX foci as well as decreased chromosome repair. These results suggest that inactivation of telomerase before combined hyperthermia and radiotherapy could improve tumor killing.

Telomerase‐independent cell survival in Arabidopsis thaliana

Telomerase is the reverse transcriptase responsible for the maintenance of telomeric repeat sequences in most species that have been studied. Inactivation of telomerase causes telomere shortening and results in the loss of the telomere's protective function, which in mammals leads to cell-cycle arrest and apoptosis. Experiments performed on Arabidopsis thaliana mutants lacking telomerase activity revealed their unusually high tolerance for genome instability. Here we present molecular and cytogenetic analysis of two cell lines (A and B) derived from seeds of late-generation telomerase-deficient A. thaliana. These cultures have survived for about 3 years and are still viable. However, neither culture has adapted mechanisms to maintain terminal telomeric repeats. One culture (B) suffers from severe growth irregularities and a high degree of mortality. Karyological analysis revealed dramatic genomic rearrangements, a large variation in ploidy, and an extremely high percentage of anaphase bridges. The second cell line (A) survived an apparent crisis and phenotypically appears wild-type with respect to growth and morphology. Despite these indications of genome stabilization, a high percentage of anaphase bridges was observed in the A line. We conclude that the restructured chromosome termini provide the A line with partial protection from end-joining repair activities, thus allowing normal growth.

Activation of a LTR-retrotransposon by telomere erosion.

Retrotransposons can facilitate repair of broken chromosomes, and therefore an important question is whether the host can activate retrotransposons in response to chromosomal lesions. Here we show that Ty1 elements, which are LTR-retrotransposons in Saccharomyces cerevisiae, are mobilized when DNA lesions are created by the loss of telomere function. Inactivation of telomerase in yeast results in progressive shortening of telomeric DNA, eventually triggering a DNA-damage checkpoint that arrests cells in G2/M. A fraction of cells, termed survivors, recover from arrest by forming alternative telomere structures. When telomerase is inactivated, Ty1 retrotransposition increases substantially in parallel with telomere erosion and then partially declines when survivors emerge. Retrotransposition is stimulated at the level of Ty1 cDNA synthesis, causing cDNA levels to increase 20-fold or more before survivors form. This response is elicited through a signaling pathway that includes Rad24, Rad17, and Rad9, three components of the DNA-damage checkpoint. Our findings indicate that Ty1 retrotransposons are activated as part of the cellular response to telomere dysfunction.

Inhibition of c-Myc Oncoprotein Limits the Growth of Human Melanoma Cells by Inducing Cellular Crisis

Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated beta-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with L-buthionine sulfoximine decreases GSH to levels of c-Myc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-L-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with L-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress.

Antisense hTERT inhibits thyroid cancer cell growth.

Activation of telomerase represents an early step in carcinogenesis. Increased telomerase expression in malignant thyroid tumors suggests that inactivation of telomerase may represent a potential chemotherapeutic target. The purpose of this study was to inhibit the protein component of telomerase, hTERT, in a human thyroid cancer cell line in vitro and in vivo using an antisense strategy. A 235-bp fragment of hTERT cDNA was subcloned, and sense and antisense hTERT expression vectors were constructed. These vectors were transfected into a human thyroid carcinoma cell line (FRO). Tumorigenic potential was determined by cellular growth assay, rate of apoptosis, anchorage-independent growth, and tumor growth in a nude mouse model. Significant down-regulation of hTERT expression was seen in the antisense transfected cells, compared with control and those transfected with the sense vector. A decrease in telomerase activity by TRAP assay was observed in the antisense hTERT cells but not in cells transfected with the sense hTERT construct. Inhibition of cell growth was observed after approximately 20 population doublings in the antisense-hTERT clones and was associated with an increase in the rate of apoptosis and a change in cellular morphology. Moreover, anchorage-independent growth was reduced in vitro, and tumor growth rate was diminished in vivo in the antisense hTERT clones. Inhibition of telomerase activity with antisense hTERT in human thyroid cancer cells is achievable and may represent a novel target to inhibit tumor growth.

Inhibition of Telomerase Activity by a Hammerhead Ribozyme Targeting the RNA Component of Telomerase in Human Melanoma Cells

Reactivation of telomerase, an RNA-dependent DNA polymerase that synthesizes new telomeric repeats at the end of chromosomes, is a very common feature in human cancers. Telomerase is thought to be essential in maintaining the proliferative capacity of tumor cells and, as a consequence, it could represent an attractive target for new anti-cancer therapies. In this study, we generated a hammerhead ribozyme composed of a catalytic domain with flanking sequences complementary to the RNA component of human telomerase and designed to cleave specifically a site located at the end of the telomerase template sequence. In vitro the ribozyme induced cleavage of a synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The extent of cleavage was dependent on the ribozyme/substrate ratio as well as the Mg2+ concentration. Moreover, when added to cell extracts from two human melanoma cell lines (JR8 and M14), or three melanoma surgical specimens, the ribozyme inhibited telomerase activity in a concentration- and time-dependent manner. When the ribozyme was delivered to growing JR8 melanoma cells by (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate-mediated transfer, a marked inhibition of telomerase activity was observed. Next, the ribozyme sequence was cloned in an expression vector and JR8 cells were transfected with it. The cell clones obtained showed a reduced telomerase activity and telomerase RNA levels and expressed the ribozyme. Moreover, ribozyme transfectants had significantly longer doubling times than control cells and showed a dendritic appearance in monolayer culture. No telomere shortening, however, was observed in these clones. Overall, our results indicate that the hammerhead ribozyme is a potentially useful tool for the inactivation of telomerase in human tumors.

Increased levels of human telomerase activity have been demonstrated in a wide variety of human tumours

We have used a 19mer, antisense oligonucleotide directed against the human telomerase RNA component (hTR) to inactivate this enzyme. These oligos are linked to a 2',5'-oligoadenylate moiety which activates RNAase L, resulting in degradation of the target RNA. Targeting hTR in variety of human tumour cells from different origins in vitro results in drastically reduced cell viability. In vivo tumours grown subcutaneously in nude mice reduce in size following treatment with the anti-hTR oligos. Normal fibroblast and epithelial cells do not show any reduction in viability following treatment. TUNEL assays demonstrate that the cytotoxic effects are mediated through apoptotic pathways where the maximum effect is seen after only 4 days of treatment. This response is p53 independent since the majority of the tumour cells used are deficient in p53 function. It also seems that telomere shortening is not the cause of cell death since the cells do not go through sufficient rounds of division for this to happen. In order to gain more insight into the pathways which are affected by the 2-5A anti-hTR treatment we investigated the changes in gene expression which are produced in the human glioma cell line U373 following 2-5A anti-hTR treatment using Affymetrix GeneChip microarray technology. We compared gene expression levels at 4 different time points after treatment using the Affymetrix HU6800 chips. The data demonstrates dysregulation of a significant number of genes implicating the possible involvement of a number of different pathways. The role of these genes in the response to inactivation of telomerase is currently under investigation.

Telomerase-independent senescence of human immortal cells induced by microcell-mediated chromosome transfer.

Maintenance of telomeres, commonly through expression of telomerase activity, is necessary but may not be sufficient for human cells to escape from the cellular senescence program and become immortal. We report here that human tumor cells could undergo cellular senescence in the presence of telomerase activity when a specific normal human chromosome was introduced via microcell-mediated chromosome transfer. The cell models studied include SiHa (uterine cervical carcinoma cells expressing E6 and E7 oncoproteins of human papillomavirus type 16) with a transferred chromosome 2, CC1 (choriocarcinoma cells expressing an amino-terminally truncated p53 protein) with a transferred chromosome 7, and JTC-32 (bladder carcinoma cells) with a transferred chromosome 11. The microcell hybrids with the indicated chromosomes ceased to divide after five to 10 population doublings and showed senescence-associated beta-galactosidase activity but still expressed the genes encoding three components of human telomerase, consistent with the retention of telomerase activity. These results are evidence for barriers to human cell immortalization, which involve activation of unidentified senescence-inducing genes that function independently of inactivation of telomerase.

Telomerase in tumors: facts and perspectives

Recent reports have indicated the ribonucleoprotein enzyme telomerase to play an important role in tumorigenesis. Activation of this enzyme is known to prevent progressive shortening of the end of the chromosomes, or telomeres, and hence to be critical in maintaining chromosomal integrity. The telomerase expressing cells require immortality. Supported by recent findings which suggest that telomerase activity is expressed in virtually all cancers but not in normal tissues, except those of the germline, hope grew up toward a potentially important new therapeutic target in the fight against cancer. An emerging hypothesis is that the inactivation of telomerase results in the death of immortal cells. If telomerase activation represents a tumor-specific feature, (gene) therapeutic applications would become most promising in regard to an effective anticancer therapy, possibly with limited side-effects. However, most recent studies report on telomerase activity to be also expressed in normal, non-neoplastic tissues as well as in non-neoplastic hyperproliferative lesions. The questions whether telomerase activity is tumor-specific or associated with (physiologic or pathologic) hyperproliferation is intriguing and remains to be clarified.

Telomerase activity in bladder cancer, bladder washings and in urine

With only a few exceptions, the ribonucleoprotein telomerase has been found in malignant, but not in benign tissues. Telomerase is thus a potentially new diagnostic marker. Carcinoma of the urinary bladder is the most frequent malignant tumor of the urinary tract and, after prostatic carcinoma, the second most common malignancy of the genitourinary system. In order to evaluate the diagnostic capabilities of telomerase in bladder carcinomas, four cell lines derived from human urothelial carcinomas of the bladder, 75 tissue samples from bladder carcinomas, eight tissue samples of normal bladder urothelium, 40 bladder washings and 30 urine samples were examined for telomerase activity. The four cell lines derived from urothelial carcinoma of the bladder (F975, 582, SCaBER, UM-UC-3) all exhibited high telomerase activity and were thus used as positive controls. Telomerase activity was found in nearly all (96%) tissue samples obtained from histologically confirmed urothelial carcinoma of the bladder. None of the normal tissue samples examined showed telomerase activity. Telomerase activity was similarly found in 73% of bladder washings in patients with histologically confirmed bladder carcinoma. There were no false positive results. The determination of telomerase activity in bladder washing samples thus represents a new diagnostic method for detection of tumor cells in rinsing media. Because of the early inactivation or degradation of telomerase there was no detection of the enzyme in native urine in the present study.