Tumor Cell Immortalization Research Articles

Mechanism of Human Telomerase Inhibition by BIBR1532, a Synthetic, Non-nucleosidic Drug Candidate

Telomerase, a ribonucleoprotein acting as a reverse transcriptase, has been identified as a target for cancer drug discovery. The synthetic, non-nucleosidic compound, BIBR1532, is a potent and selective telomerase inhibitor capable of inducing senescence in human cancer cells (). In the present study, the mode of drug action was characterized. BIBR1532 inhibits the native and recombinant human telomerase, comprising the human telomerase reverse transcriptase and human telomerase RNA components, with similar potency primarily by interfering with the processivity of the enzyme. Enzyme-kinetic experiments show that BIBR1532 is a mixed-type non-competitive inhibitor and suggest a drug binding site distinct from the sites for deoxyribonucleotides and the DNA primer, respectively. Thus, BIBR1532 defines a novel class of telomerase inhibitor with mechanistic similarities to non-nucleosidic inhibitors of HIV1 reverse transcriptase.

Telomerase activity in Kaposi's sarcoma, squamous cell carcinoma, and basal cell carcinoma.

Patients with acquired immune deficiency syndrome (AIDS) often develop Kaposi's sarcoma (KS), an unusual skin tumor. The malignant nature of KS has long been disputed. Telomerase activity that maintains telomere length and ensures chromosomal stability, a frequently appearing marker in human malignancies, has been proposed to play a critical role in supporting continued cell growth, hence formation of tumors. We examined telomerase activity in tissue extracts from 22 KS, 10 squamous cell carcinoma (SCC), and 22 basal cell carcinoma (BCC) using the telomeric repeat amplification protocol (TRAP). All of the tumor tissues were previously cryopreserved at -80 degrees C. In this study, all tumor samples tested were positive for telomerase activity. Consistent with the presence of the enzyme activity, the skin tumors had relatively long telomeres. Inhibitors in the tissue extracts of some samples needed to be diluted or extracted by phenol before the enzyme activity was detected in the TRAP assay. All KS as well as two other skin carcinoma samples revealed positive telomerase activity. Our finding supports telomerase's role in tumor cell immortality and suggests the true neoplastic nature of KS.

Expression of telomerase-associated genes: reflection of telomerase activity in gastric cancer?

Telomerase activation is a characteristic of immortalized tumor cells but not of normal cells. Telomerase activity has been detected in approximately 85% of malignant tumors, and assaying for telomerase activity is thought to be useful for diagnosing cancer. Three telomerase-associated molecules [human telomerase RNA component (hTR), telomerase-associated protein (TEP1), and human telomerase reverse transcriptase (hTERT)] have been cloned. We semiquantitatively measured telomerase activity and the expression of these genes in cancerous and noncancerous regions of gastric cancer patients. We also investigated whether the expression of these genes correlated with telomerase activity. Telomerase activity in cancerous regions was significantly higher than in noncancerous regions, but there was no correlation between telomerase activity and the expression of these genes. Furthermore, no clear difference was observed between cancerous and noncancerous regions. These data indicate that the level of three telomerase-associated genes (i.e., hTR, TEP1 mRNA, hTERT mRNA), do not reflect telomerase activity, and the RNA levels of these genes are not useful markers for diagnosing gastric cancer.

On the significance of telomerase activity in human malignant glioma cells

Telomerase is critical for tumor cell immortalization and is a novel target for cancer chemotherapy. Here, we examined whether telomerase is expressed in glioma cell lines, whether telomerase activity is regulated by bcl-2 or p53, and whether telomerase activity predicts response to chemotherapy. Further, we characterized the effects of a candidate telomerase inhibitor, penclomedine, in glioma cells. All 12 human malignant glioma cell lines examined were telomerase positive. Telomerase activity was not modulated during cell cycle progression, did not correlate with p53 status or bcl-2 family protein expression, and did not predict drug sensitivity, except for an association with resistance to carmustine. Ectopic bcl-2 expression did not enhance telomerase activity. Wild-type p53 reduced telomerase activity in cell lines retaining p53 activity but not in p53-mutant cell lines. Penclomedine killed glioma cells via an apoptotic, but death receptor-, bcl-2- and caspase-independent pathway, but did not inhibit telomerase and did not act synergistically with cytotoxic drugs. We conclude that telomerase activity does not account for the differential chemosensitivity of human glioma cells and that penclomedine kills glioma cells via a telomerase-independent pathway.

Replicative senescence and oxidant-induced premature senescence. Beyond the control of cell cycle checkpoints.

Normal human diploid fibroblasts (HDFs) undergo replicative senescence inevitably in tissue culture after a certain number of cell divisions. A number of molecular changes observed in replicative senescent cells occur in somatic cells during the process of aging. Genetic studies on replicative senescence indicate the control of tumor suppression mechanisms. Despite the significance of replicative senescence in aging and cancer, little is known about the central cause of the complex changes observed in replicative senescent cells. The interest in the phenomenon has intensified in recent years, since damaging agents, certain oncogenes and tumor suppressor genes have been found to induce features of senescence in early passage young HDFs or in immortalized tumor cells. The reported features of senescence are summarized here in order to clarify the concept of replicative senescence or premature senescence. The experimental results of extending the replicative life span by reducing ambient oxygen tension or by N-tert-butyl-alpha-phenylnitrone (PBN) argue a role of oxidative damage in replicative senescence. By inducing premature senescence with a pulse treatment of H2O2, we can study the role of the cell cycle checkpoint proteins p53, p21, p16 and Rb in gaining each feature of senescence. Although p53 and Rb control G1 arrest and Rb appears to control cell enlargement, activation of the senescent associate beta-galactosidase, loss of cell replication and multiple molecular changes observed in premature senescent or replicative senescent cells are likely controlled by mechanisms beyond the cell cycle checkpoints.

Telomerase activity as an indicator of malignant potential in iodine-nonreactive lesions of the esophagus

Iodine-nonreactive lesions of esophageal epithelium often are associated with dysplasia and carcinoma. The authors examined the usefulness of telomerase activity as an indicator for esophageal carcinogenesis in such lesions. Telomerase activity was measured using the telomeric repeat amplification protocol assay in 18 samples of iodine-nonreactive lesions apart from the primary tumor in surgically resected specimens obtained from patients with esophageal squamous cell carcinoma (ESCC) and 55 endoscopic punch biopsies of iodine-nonreactive lesions obtained from 25 patients with ESCC and 30 patients who had undergone endoscopic examination for other reasons. Ten of 18 iodine-nonreactive samples (56%) obtained from surgically resected specimens showed telomerase activity. In all ten telomerase positive samples, carcinoma in situ (CIS) was observed in iodine-nonreactive mucosa by light microscopy. In eight telomerase negative samples, no tumor tissue was observed in iodine-nonreactive lesions. In a parallel study, telomerase activity was detected in 28 of 55 endoscopic punch biopsy specimens (51%). CIS was observed in 25 of 28 iodine-nonreactive lesions with positive telomerase activity (89%), and tumor tissue was not observed in the other 3 samples (11%), which included 2 cases of severe dysplasia and 1 case of moderate dysplasia. No tumor tissue was observed in any of the 27 telomerase negative samples. Positive and negative telomerase activity was found to be correlated with the presence and absence, respectively, of immortalized tumor cells in iodine-nonreactive lesions. The measurement of telomerase activity in iodine-nonreactive lesions independently contributes to the selection of an appropriate therapeutic strategy.

Prognostic impact of telomerase activity in non-small cell lung cancers.

To evaluate the clinical significance of telomerase activity, particularly in terms of prognostic impact, in non-small cell lung cancer (NSCLC). Telomerase activity has been found in various tissues. The activation of telomerase is considered necessary for the immortalization of human tumor cells, including NSCLC. The authors studied 103 NSCLC specimens using a polymerase chain reaction based on a telomeric repeat amplification protocol assay. Telomerase activity was detected in 85 (82.5%) of 103 NSCLC specimens but in none of the paired normal lung tissue specimens. More cases of positive telomerase activity were observed in the group with advanced disease and in the group with poorly differentiated tumors. Such factors as the mean age at surgery, sex, smoking, histologic type, and size of tumor extension did not correlate with the telomerase activity. The Kaplan-Meier survival curves in all patients with NSCLC demonstrated that patients with telomerase-positive tumors survived for a significantly shorter period than those with a telomerase-negative tumor (p = 0.0058). According to a multivariate analysis, telomerase activity was identified as an independent prognostic factor (RR = 8.62, p = 0.035). Telomerase activity was one of the most important prognostic factors in patients with NSCLC, and its potential prognostic implication was independent of tumor stage.

Telomerase activity in hepatocellular carcinoma and adjacent liver tissues.

Activation of telomerase and stabilization of telomeres are considered necessary for immortalization of tumor cells. Telomerase activity was analyzed in 69 hepatocellular carcinomas and adjacent chronic liver disease tissues. The telomerase activity level was examined in relation to clinicopathologic features. Telomerase activity was determined by a telomeric repeat amplification protocol. Immature and mature leukocytes were removed from homogenized tissue of adjacent livers using anti-CD45 and anti-CD15 monoclonal antibody-coated magnetic beads. Telomerase activity was detected in hepatocellular carcinomas and leukocytes, but not in liver cells from adjacent chronic liver disease tissues after the separation of leukocytes. All hepatocellular carcinomas displayed telomerase activity, and the activity level correlated with the degree of differentiation (P=0.021) and patient survival (P=0.039). These results indicate that activation of telomerase may be required as a critical step in hepatocarcinogenesis and tumor development, and detection of telomerase activity with removal of contaminating leukocytes may be useful in the characterization or prognostication of hepatocellular carcinoma.

The implication of telomerase activity and telomere stability for replicative aging and cellular immortality (Review).

Telomerase and telomeres have been shown to be involved in the control of cell proliferation, the regulation of cell senescence and the unlimited proliferation capacity of malignant cells. Human telomeres are specialized chromosomal end structures composed of TTAGGG repeats. They function to protect chromosomes from degradation, fusion and recombination. Since the termini of linear molecules are replicated only in the 5'-3' direction by conventional DNA polymerases and require an RNA primer to initiate DNA synthesis, the removal of the RNA primer results in DNA loss with each cell division. To date, telomere shortening has been observed in most dividing somatic cells, eventually leading to cell senescence when critically short telomeres are reached. Telomerase has been identified as a ribonucleoprotein enzyme that can synthesize telomeric repeats onto chromosomes. Borderline telomerase activity has been detected in human primitive hematopoietic cells and in stimulated lymphocytes which increased with cytokine induced ex vivo expansion. However, in most other normal somatic cells, telomerase has not been detected, and consequently telomere shortening can be anticipated after a limited number of population doublings. In contrast, spontaneously immortalized tumor cell lines and the majority of malignant tumors demonstrate high telomerase activity, stable telomere length and unlimited proliferative potential. Mechanisms for telomerase and telomere length regulation are under extensive investigation. These have included the cloning of the RNA component and telomerase associated proteins, antisense experiments that have demonstrated progressive telomere length shortening in the absence of telomerase, and the identification of telomere binding proteins which may regulate telomerase by creating a negative feedback signal. This review aims to summarize important results in the rapidly moving field of telomeres and telomerase.

Synthetic 2'-O-methyl-modified hammerhead ribozymes targeted to the RNA component of telomerase as sequence-specific inhibitors of telomerase activity.

Telomerase is a ribonucleoprotein that synthesizes tandem arrays of the hexameric DNA sequence TTAGGG at chromosome termini using its RNA component as a template. As most normal cells lack telomerase activity, a progressive shortening of chromosome length occurs with each cell division because of incomplete DNA replication. Cell senescence ensues when a critical telomere length is reached, but importantly, senescence bypass and life span extension occur in normal cells transfected with functional telomerase activity. Almost 90% of all tumors express telomerase activity, implying that telomerase is an important determinant in tumor progression and cell immortalization. However, the exact role and regulation of the individual components of the telomerase complex are not fully understood and would benefit from the availability of specific inhibitors. In this study, we investigated the potential use of chemically stabilized, catalytic RNA molecules (hammerhead ribozymes) to inhibit telomerase activity by cleaving the RNA component in a sequence-specific manner. Catalytically competent (active) hammerhead ribozymes containing 2'-O-methyl ribonucleotides for enhanced biologic stability and designed to be complementary to the RNA component of human telomerase exhibited dose-dependent inhibition of telomerase activity in human glioma U87-MG cell lysates with an IC50 of around 0.4 microM. Catalytically incompetent (inactive) ribozymes or mismatched ribozymes with reduced hybridization capability to telomerase RNA did not inhibit telomerase activity, as detected by a PCR-based telomeric repeat amplification protocol (TRAP) assay. In vitro cleavage reactions using short substrates and RT-PCR analyses of the full-length RNA substrate in U87-MG cell lysates confirmed a sequence-specific catalytic cleavage of the targeted RNA component of telomerase. Exogenously administrable, synthetic ribozymes may have important uses in further understanding the role and regulation of this ribonucleoprotein in normal and diseased tissues as well as in the potential therapy of telomerase-positive tumors.

High reactivity of monoclonal antibody (TO73) with human malignant tumor cell line.

The biological characteristics of a new monoclonal antibody (TO73) reacting with a vincristine-resistant human leukemic cell line (KY-VCR) were evaluated. Immunological and electron-immunological studies showed that TO73 reacted with the surface glycoprotein of KY-VCR. TO73 was found to have no effect on cell growth and intracellular uptake of vincristine. In human neoplastic cell lines, TO73 was found to react with 11 of 27 (41%) cell lines. With regard to de novo primary tumor with one exception, TO73 did not react with any of the examined primary tumor cells. The patient with TO73-positive leukemia died of induction failure due to drug resistance. Complete remission was achieved in the other leukemic patients. These results indicate that TO73 antigen may be associated with immortalization of tumor cells and poor prognosis in some cases.

Progressive telomere shortening and telomerase reactivation during hepatocellular carcinogenesis

Telomeres shorten progressively with age in normal somatic cells in culture and in vivo. The maintenance of telomere length is assumed to be an obligatory step in the progression and immortalization of most human tumor cells. To understand the role of telomere dynamics in the development of hepatocellular carcinoma (HCC), we examined the length of terminal restriction fragment (TRF), as an indicator for telomere length, in HCC and surrounding tissues with chronic active hepatitis (CAH) or liver cirrhosis (LC). The study was performed in 12 hepatitis C virus (HCV) antibody-positive, 12 hepatitis B virus (HBV) antigen-positive tissues, and 4 tissue samples from virus-negative patients with HCC. The peak TRFs in all 3 types of HCC were significantly shorter than those of the surrounding tissues (i.e., LC or CAH). TRFs examined in one patient with atypical adenomatous hyperplasia (AAH) also was shortened. Thus, progressive TRF shortening occurs from normal to CAH to LC to HCC(AAH). Telomerase, an enzyme that adds repeated telomere sequences onto the chromosome ends and stabilizes telomere length in immortal cells, also was examined in tissues and detected in high levels almost exclusively in HCCs. Interestingly, the intensity of telomerase activity in the AAH case was similar to that of HCC. In addition, the telomerase activity of biopsy samples with a fine 21-gauge needle also was examined in 10 HCCs, 2 adenomatous hyperplasias (AHs), 2 LCs, and 2 CAHs. We found strong telomerase activity in all the HCCs and surprisingly in the 2 cases that were pathologically diagnosed as AH. Thus, the findings strongly suggest that persistent cell proliferation or rapid cell turnover through damage of hepatic cells result in a process of multistep hepatocellular carcinogenesis. Thus, progressive shortening of telomeres and the activation of telomerase may be a useful marker for the early detection of malignant progression in liver disease.

Dynamic Changes in Nuclear Architecture during Mitosis: On the Role of Protein Phosphorylation in Spindle Assembly and Chromosome Segregation

During mitosis, the vertebrate cell nucleus undergoes profound changes in architecture. At the onset of mitosis, the nuclear envelope breaks down, the nuclear lamina is depolymerized, and interphase chromatin is condensed to chromosomes. Concomitantly, cytoplasmic microtubules are reorganized into a mitotic spindle apparatus, a highly dynamic structure required for the segregation of sister chromatids. Many of the above events are controlled by reversible phosphorylation. Hence, our laboratory is interested in characterizing the kinases involved in promoting progression through mitosis and in identifying their relevant substrates. Prominent among the kinases responsible for regulating entry into mitosis is the Cdc2 kinase, the first member of the cyclin dependent kinase (Cdk) family. Recently, we found that Cdc2 phosphorylates HsEg5, a human kinesin-related motor protein associated with centrosomes and the spindle apparatus. Our results indicate that phosphorylation regulates the association of HsEg5 with the mitotic spindle and that the function of this plus-end directed motor is essential for centrosome separation and bipolar spindle formation. Another kinase implicated in regulating progression through mitosis is Plk1 (polo-like kinase 1), the human homologue of theDrosophilagene product “polo.” By antibody microinjection we have found that Plk1 is required for the functional maturation of centrosomes and hence for entry into mitosis. Furthermore, we found that microinjected anti-Plk1 antibodies caused a more severe block to cell cycle progression in diploid fibroblasts than in immortalized tumor cells. This observation hints at the existence of a checkpoint linking Cdc2 activation to the presence of functional centrosomes.

WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis.

The Wilms tumor suppressor gene WT1 encodes a developmentally regulated transcription factor that is mutated in a subset of embryonal tumors. To test its functional properties, we developed osteosarcoma cell lines expressing WT1 under an inducible tetracycline-regulated promoter. Induction of WT1 resulted in programmed cell death. This effect, which was differentially mediated by the alternative splicing variants of WT1, was independent of p53. WT1-mediated apoptosis was associated with reduced synthesis of the epidermal growth factor receptor (EGFR), but not of other postulated WT1-target genes, and it was abrogated by constitutive expression of EGFR. WT1 repressed transcription from the EGFR promoter, binding to two TC-rich repeat sequences. In the developing kidney, EGFR expression in renal precursor cells declined with the onset of WT1 expression. Repression of EGFR and induction of apoptosis by mechanism that may contribute to its critical role in normal kidney development and to the immortalization of tumor cells with inactivated WT1 alleles.

The RNA component of human telomerase.

Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

Viral oncolysates as human tumor vaccines.

Postoncolytic immunity entails immune reactions acquired through an oncolytic virus infection or through repeated immunizations with viral oncolysates (or virally modified tumor cell membranes) that are valid and operational also against virally not modified tumor cells of the same type. NK cells react to budding virions, induce target cell lysis primarily but not exclusively by the production of granzymes and pore-forming proteins and operate without direction from memory cells. In contrast, immune T cells (including some TIL) are MHC-restricted, act under the direction of memory cells and lyse target cells primarily but not exclusively by the release of lymphotoxin (TNF beta) causing programmed cell death (apoptosis) through endonuclease activation and target cell DNA fragmentation. This author proposes that it is not NK, but the immune T cells that mediate postoncolytic immunity. Oncogene amplification may protect immortalized tumor cells even when expressing peptide antigens through MHC molecules against lymphotoxin-mediated apoptosis; but virally-infected tumor cells releasing budding virions remain susceptible to NK cells. Highly immunogenic viral oncolysates should present both budding virions for NK cells and processed viral and tumoral peptide antigens co-jointly for immune T cells.

Proliferation, senescence, and neoplastic progression of β cells in hyperplasic pancreatic islets

Three different cases of pancreatic β cell hyperplasia in mice are accompanied by an increase in a subclass of cells expressing tyrosine hydroxylase (TH), a neuronal enzyme. In the nontumorigenic cases of islet growth during normal pregnancy and in the obese mutant mouse, the TH-insulin cells do not divide, in contrast to the “insulin-only” cells. In later stages the number of proliferating insulin-only cells decreases concomitant with an increase in the number of non-dividing TH-insulin cells, suggesting that the TH-insulin cells are on a pathway to senescence. In the presence of an oncoprotein the TH-insulin cells are able to proliferate. The proliferation of this cell type may represent an escape from the senescence pathway and progression to immortal tumor cells.