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Cited by 422Telomeres, Telomerase, and SenescenceDNA and Nucleic Acid ChemistryOrganoselenium and organotellurium chemistry

A highly selective telomerase inhibitor limiting human cancer cell proliferation

Open access
Damm, Klaus, Hemmann, Ulrike, Garin‐Chesa, Pilar, Hauel, Norbert, Kauffmann, Iris, Priepke, Henning, Niestroj, Claudia, Daiber, Christine +13 more·2001·The EMBO Journal
DOI 10.1093/emboj/20.24.6958PMC PMC125790
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Abstract

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports non-nucleosidic small molecules, BIBR1532 and BIBR1591, that selectively inhibit human telomerase and cause progressive telomere shortening in cancer cells, followed by senescence-like growth arrest without acute cytotoxicity. In a mouse xenograft model using telomere-shortened HT1080 cells, oral BIBR1532 reduced tumorigenic potential and tumor incidence.

Key findings

  • BIBR1532 and BIBR1591 inhibited telomerase in vitro with IC50 values of 93 nM and 470 nM, respectively; BIBR1532 did not inhibit a panel of DNA/RNA polymerases, HIV reverse transcriptase, helicases, or translation at concentrations far above its telomerase IC50.
  • Long-term treatment of NCI-H460 lung carcinoma cells shortened average telomere restriction fragments from 4 kb to 1.5 kb by population doubling 140, a loss of about 30 bp per doubling; similar shortening occurred in HT1080, MDA-MB231, and DU145 cells. Growth arrest began after PD135 and was complete after 4–8 additional doublings, including in p53-deficient lines.
  • Treated cells developed senescence features such as enlarged and multinucleated morphology, SA-β-galactosidase activity, altered cell-cycle profiles, telomere dysfunction, and chromosomal end fusions; no increase in apoptosis was detected. Microarray analysis of 6,817 genes found 302 (4.4%) with at least twofold expression changes, including p21 upregulation (12.9-fold) and hTERT downregulation (5-fold).
  • In mice injected with telomere-shortened HT1080 cells, detectable tumors appeared after 16.5 ± 1.8 days versus 4.53 ± 0.6 days for controls (p < 0.0001). Oral BIBR1532 at 100 mg/kg/day prolonged the lag to 26.2 ± 4.3 days (p = 0.0255) and reduced the proportion of animals with tumors >1000 mm³ at day 43 to 32% versus 70% without inhibitor (p = 0.01034).

Methods

  • Telomerase activity was measured using HeLa nuclear extracts and a PCR-based assay, with a direct assay using recombinant hTERT and in vitro transcribed hTR; selectivity was tested against various polymerases, helicases, translation, and HIV reverse transcriptase.
  • Cancer cell lines NCI-H460, HT1080, MDA-MB231, and DU145 were grown long-term with BIBR1532 (10 µM) or BIBR1591 (50 µM); telomere length was assessed by Southern blot and Q-FISH, with FACS, TUNEL, SA-β-galactosidase staining, and Affymetrix HuGeneFL microarray analysis of 6,817 genes.
  • Tumorigenicity was tested by subcutaneous injection of control or inhibitor-treated HT1080 cells into immunodeficient nu/nu NMRI mice; BIBR1532 was given orally by gavage at 100 mg/kg/day, and tumor size was measured by caliper.

Limitations

  • The paper states that telomerase inhibition requires a long lag period before effects appear, so treatment may need to be continuous for weeks to months; standard xenograft models with pre-established tumors are not suitable for showing growth delay, prompting the use of telomere-shortened cells.
  • The paper notes that only eight metaphases from inhibitor-treated cells could be analyzed by Q-FISH because of the low mitotic index, compared with 20 control metaphases.
  • Evident from the text: efficacy and tolerability were tested in immunodeficient mice over a limited period, and BIBR1532 is far more selective for human than mouse telomerase, so host telomerase inhibition was not assessed; clinical safety and long-term benefit remain untested.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1093/emboj/20.24.6958

Cites

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Statistics

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Claims the paper makes

0 of 54 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § Telomerase inhibitors induce telomere shortening in cancer cells

    Untreated cells and solvent-only cells kept a stable TRF size.

    “In contrast, untreated cells or cells exposed to solvent alone maintained a stable TRF size (Figure 2A).”
    Quote in the paperSubstantial claim
  • Findingown data
    § Telomerase inhibitors induce telomere shortening in cancer cells

    In NCI-H460 cells grown with telomerase inhibitor, mean TRF length fell from 4 kb to 1.5 kb by population doubling 140, a loss of 30 bp per PD.

    “The average telomere restriction fragment (TRF) size shortened progressively from 4 to 1.5 kb at population doubling (PD) 140 (Figure 2A), corresponding to a telomere loss of 30 bp/PD.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Microarray analysis of mRNA expression levels in senescent NCI-H460 cells

    In senescent inhibitor-treated NCI-H460 cells, 302 genes (4.4%) differed at least 2-fold in at least three of four independent comparisons.

    “However, in the inhibitor-treated, senescent NCI-H460 cells we identi®ed 302 (4.4%) genes showing at least a 2-fold difference in expression levels in a minimum of three out of four independent comparisons (for complete gene list see Supplementary data available at The EMBO Journal Online).”
    Quote in the paperNot a substantial claim
  • Findingown data
    § Microarray analysis of mRNA expression levels in senescent NCI-H460 cells

    BRCA1, BRCA2 and BARD1 expression was repressed in inhibitor-treated senescent NCI-H460 cells.

    “In contrast, expression of the breast cancer susceptibility genes BRCA1 and BRCA2 as well as BARD1 (BRCA1-associated Ring domain protein), which are thought to play a key role in the cellular DNA damage response, was found to be repressed.”
    Quote in the paperNot a substantial claim
  • Findingown data
    § Abstract

    Treating cancer cells with these inhibitors causes telomeres to shorten progressively.

    “Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression.”
    Quote in the paperSubstantial claim
  • May combine two claims. 39
  • The quote only partly states this claim. 2
  • Classified as probably not a substantial claim. 14

  • May combine two claims.

    Findingown data
    § Abstract

    New chemical compounds inhibit telomerase selectively both in vitro and in vivo.

    “We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Telomerase inhibitors limit cancer cell proliferation

    After PD135, inhibitor-treated NCI-H460 cells grew more slowly and almost stopped proliferating after another 4-8 population doublings.

    “However, after PD135 the inhibitor-treated cells slowed their growth and showed an almost complete inhibition of proliferation after additional 4±8 population doublings (Figure 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Microarray analysis of mRNA expression levels in senescent NCI-H460 cells

    hTERT mRNA fell 5-fold, and TRF1 was among the induced genes.

    “We also observed a 5-fold reduction of hTERT mRNA, encoding the catalytic subunit of telomerase, while the telomere binding protein TRF1 was found among the induced genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Microarray analysis of mRNA expression levels in senescent NCI-H460 cells

    Few differentially expressed genes had prior links to DNA damage or senescence; the most prominent was p21 Waf1, up 12.9-fold.

    “Only a few of these genes have previously been associated with DNA damage or senescence and most prominent among these is the cyclin-dependent kinase inhibitor p21 Waf1 , which we found to be up-regulated 12.9-fold.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Effect of telomerase inhibition on tumorigenic potential

    After subcutaneous injection into immunodeficient mice, control cells with ~4 kb telomeres reached >50 mm2 tumours in 4.53 ± 0.6 days (n=24), while telomere-shortened cells took 16.5 ± 1.8 days (n=27, p<0.0001).

    “After subcutaneous injection into immunode®cient mice, untreated control cells with telomeres of ~4 kb (Figure 6A) reached a detectable tumour size (>50 mm 2 ) in 4.53 6 0.6 days (n = 24) whereas the telomere-shortened cells formed detectable tumours only after a lag period of 16.5 6 1.8 days (n = 27, p <0.0001).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Small molecules selectively inhibit telomerase activity in vitro

    BIBR1532 and BIBR1591 reduce telomerase processivity in vitro in a dose-dependent way, with IC50 values of 93 and 470 nM.

    “Two examples from this class of compounds, designated BIBR1532 {2-[(E)-3-naphtalen-2-yl-but-2enoylamino]-benzoic acid} and BIBR1591 {5-morpholin-4-yl-2-[(E)-3-naphtalen-2-yl-but-2-enoylamino]-benzoic acid}, inhibit the in vitro processivity of telomerase in a dose-dependent manner, with half-maximal inhibitory concentrations (IC 50 ) of 93 and 470 nM, respectively (Figure 1B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Effect of telomerase inhibition on tumorigenic potential

    Average tumour size and incidence up to 36 days after injection were significantly lower in mice given pretreated, telomere-shortened cells.

    “The average tumour size (Figure 6B) and incidence (Figure 6C) up to 36 days after injection was signi®cantly lower in animals bearing pretreated, telomere-shortened cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Telomerase inhibitors induce telomere shortening in cancer cells

    NCI-H460 cells have a heterogeneous telomere size distribution, with a mean length of 4 kb and a main range of 2-6 kb.

    “NCI-H460 cells exhibit a heterogeneous size distribution, with an average telomere length of 4 kb and a predominant range of 2 to 6 kb (Figure 2A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Telomerase inhibitors induce a senescent phenotype

    Growth-arrested NCI-H460 cells were enlarged, often multinucleated, had vacuolated cytoplasm, and showed senescence-associated beta-galactosidase activity.

    “the growth-arrested NCI-H460 cells (Figure 4B) became enlarged, often contained multiple nuclei, had a vacuolated cytoplasm and showed induction of senescence associated b-galactosidase activity (Figure 4C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Abstract

    The model also showed in vivo efficacy, no adverse side effects, and easy oral dosing.

    “This model was also used to demonstrate in vivo ef®cacy with no adverse side effects and uncomplicated oral administration of the inhibitor.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Inhibitor-treated cells exhibit telomere dysfunction

    Treated cells had more chromosome end fusions (0.88 vs 0.55 per metaphase) and more chromosomes lacking telomere signal at both sister chromatids (mean 3.75 vs 1.1 per metaphase) than controls.

    “Furthermore, we observed an increase in chromosome end fusions (0.88/metaphase in treated versus 0.55/ metaphase in control cells) as well as an increased number of chromosomes with no telomere signal at both sister chromatids in treated cells (mean: 3.75/ metaphase) compared with control cells (mean: 1.1/ metaphase).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Inhibitor-treated cells exhibit telomere dysfunction

    The histogram showed accumulation of short telomeres and greater skewness of telomere fluorescence, similar to pre-senescent fibroblasts.

    “The histogram also shows an accumulation of short telomeres and an increased skewness in the distribution of telomere ¯uorescence (Figure 5C) which is very similar to the observations in pre-senescent ®broblasts (Martens et al., 2000).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Martens et al., 2000 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data
    § Effect of telomerase inhibition on tumorigenic potential

    Inhibitor-treated late-passage HT1080 fibrosarcoma cells had a mean TRF of 1.6 kb but grew and formed colonies in vitro like controls.

    “we utilized inhibitor-treated, latepassage HT1080 ®brosarcoma cells with an average TRF size of only 1.6 kb (Figure 6A) but with an in vitro growth rate and colony forming ability similar to control cultures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Telomerase inhibitors limit cancer cell proliferation

    In telomerase-negative normal human lung fibroblasts and ALT-phenotype SAOS-2 osteosarcoma cells, inhibitor treatment did not affect telomere length, growth kinetics or morphology over 8 weeks.

    “Our results showed that inhibitor treatment had no effect on telomere length, growth kinetics and morphology for the entire time of treatment (8 weeks) in either cell line.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Bryan et al., 1995 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data
    § Reversibility of inhibition

    No spontaneous telomere lengthening or telomerase-independent growth from an ALT phenotype was observed.

    “We never observed spontaneous telomere lengthening or telomerase-independent growth attributable to the induction of an ALT phenotype.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Effect of telomerase inhibition on tumorigenic potential

    Continued drug exposure inhibited initial tumour growth and lowered the incidence of tumours >1000 mm3 to six animals (32%) at day 43 versus 19 animals (70%) without inhibitor (p=0.01034).

    “Continued drug exposure inhibited initial tumour growth (Figure 6B) and resulted in a reduced incidence of tumours >1000 mm 3 with only six animals (32%) testing positive at day 43 of treatment compared to 19 animals (70%) not receiving the telomerase inhibitor ( p = 0.01034, Figure 6C).”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data
    § Tumorigenicity assays

    Pre-senescent, telomere-shortened HT1080 cells showed significant differences in tumour size compared to untreated control cells, and BIBR1532 treatment further affected tumour size.

    “Statistical analysis was performed using Fisher's exact test, with signi®cant differences (p <0.05) between untreated control and pre-treated cells (*; p <0.0002) as well as between pre-treated cells in the presence or absence of BIBR1532 (**; p = 0.01034) at the indicated time points.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The compounds BIBR1532 and BIBR1591 were stable under the culture conditions.

  • May combine two claims.

    Findingown data
    § Tumorigenicity assays

    Telomerase inhibitors induce telomere shortening and limit cell proliferation.

    “Telomerase inhibitors induce telomere shortening and limit cell proliferation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The compounds BIBR1532 and BIBR1591 were stable under the culture conditions.

  • May combine two claims.

    Findingown data
    § Effect of telomerase inhibition on tumorigenic potential

    Inhibitor treatment was well tolerated, with no acute or chronic toxicity over the full period, which lasted 60 days for mice with no or small tumours.

    “The inhibitor treatment was very well tolerated with no signs of acute and chronic toxicity over the entire treatment period, which lasted 60 days for mice developing no or only small tumours.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingnone given
    § Effect of telomerase inhibition on tumorigenic potential

    BIBR1532 is highly specific for human telomerase and inhibits mouse telomerase only with IC50 >50 μM.

    “BIBR1532 is very speci®c and selective for human telomerase and inhibits mouse telomerase only with an IC 50 >50 mM.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Tumorigenicity assays

    The mean telomere fluorescence intensity in inhibitor-treated NCI-H460 cells (217 ± 101) was significantly lower than in control cells (321 ± 160), with p < 0.0001.

    “The differences in mean ¯uorescence intensity (arbitrary ¯uorescence units 6 SD) between the control cells (321 6 160) and the treated cells (217 6 101) were highly signi®cant (p <0.0001).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The compounds BIBR1532 and BIBR1591 were stable under the culture conditions.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data
    § Tumorigenicity assays

    The NCI-H460 cell line is hypotriploid with seven marker chromosomes (modal chromosome number 57) and exhibits a characteristic chromosome with interchromosomal telomere signals.

    “The NCI-H460 cell line is hypotriploid with seven marker chromosomes (modal chromosome number 57) and exhibits a characteristic chromosome with interchromosomal telomere signals.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § Abstract

    Most human cancers and germline cells have telomerase activity, whereas normal human somatic tissues generally do not.

    “Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Counter et al., 1994 source

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    • cites Kim et al., 1994 source

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    • cites Shay and Bacchetti, 1997 source

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  • May combine two claims.

    Premisecitation
    § Abstract

    Blocking telomerase can shorten telomeres and stop uncontrolled growth.

    “Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Harley et al., 1990 source

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    • cites Counter et al., 1992 source

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    Premisecitation
    § Introduction

    Most normal human cells have low or undetectable telomerase, and their telomeric DNA is lost at 30-120 bp per replication cycle.

    “In most normal human cells, telomerase activity is low or not detectable, and telomeric DNA is progressively lost at a rate of 30±120 bp with each replication cycle (Harley et al., 1990;Hastie et al., 1990;Counter et al., 1992).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Harley et al., 1990 source

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    • cites Hastie et al., 1990 source

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    • cites Counter et al., 1992 source

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    Premisecitation
    § Introduction

    In cancer cells, telomerase reactivation is thought to stabilize telomere length, offset erosion, and give malignant cells unlimited proliferative capacity.

    “In cancer cells, the reactivation of telomerase is thought to stabilize telomere length, thereby compensating for the cell division-related telomere erosion and providing unlimited proliferative capacity to malignant cells (Counter et al., 1992;Kim et al., 1994).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Counter et al., 1992 source

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    • cites Kim et al., 1994 source

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  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Introduction

    Uncapped chromosomes are prone to degradation and fusion and can trigger DNA damage checkpoints, possibly contributing to replicative senescence and growth arrest in aged primary cultured cells.

    “Uncapped chromosomes are sensitive to degradation and fusion and can activate DNA damage checkpoints, thus potentially contributing to the replicative senescence and growth arrest observed in aged primary cultured cells (Hay¯ick and Moorhead, 1961).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Hayflick and Moorhead, 1961 source

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  • May combine two claims.

    Premisecitation
    § Introduction

    Telomerase activity occurs in immortalized cell lines and in 80-90% of human cancer specimens across many cancer types.

    “Telomerase activity has been demonstrated in immortalized cell lines and in 80±90% of human cancer specimens representing a range of cancer types (Counter et al., 1994;Kim et al., 1994;Shay and Bacchetti, 1997)”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Counter et al., 1994 source

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    • cites Kim et al., 1994 source

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    • cites Shay and Bacchetti, 1997 source

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  • May combine two claims.

    Interpretationown data
    § Telomerase inhibitors limit cancer cell proliferation

    The inhibitor-induced growth arrest appears independent of functional p53, because p53-deficient HT1080, MDA-MB231 and DU145 lines showed similar growth curves with a significant lag before arrest.

    “This telomerase inhibitor-induced growth arrest is apparently independent of functional p53 since similar growth curves, with an onset of cellular growth arrest after a signi®cant lag-phase, were obtained for the p53-de®cient HT1080, MDA-MB231 and DU145 cell lines (Figure 2B).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    Human telomerase is a high-molecular-weight complex containing a template RNA subunit and proteins including hTERT.

    “In human cells, the enzyme comprises a high molecular weight complex with a template-containing RNA subunit (Feng et al., 1995) and protein components including the catalytic subunit human telomerase reverse transcriptase, hTERT (Harrington et al., 1997;Meyerson et al., 1997;Nakamura et al., 1997).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Feng et al., 1995 source

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    • cites Harrington et al., 1997 source

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    • cites Meyerson et al., 1997 source

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    Premisecitation
    § Introduction

    Experiments with a dominant-negative human telomerase showed that telomerase inhibition can cause telomere shortening, then proliferation arrest and apoptotic cell death.

    “Genetic experiments using a dominant-negative form of human telomerase demonstrated that telomerase inhibition can result in telomere shortening followed by proliferation arrest and cell death by apoptosis (Hahn et al., 1999b;Zhang et al., 1999).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Hahn et al., 1999b

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    • cites Zhang et al., 1999 source

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    Premisecitation
    § Introduction

    Telomere length determines how many divisions a cell can undergo before senescence.

    “Indeed, it has been proposed that telomere length speci®es the number of cell divisions a cell can undergo prior to senescence (Cooke and Smith, 1986;Harley, 1991).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Cooke and Smith, 1986 source

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    • cites Harley, 1991 source

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    Premisecitation
    § Introduction

    Telomerase is an RNA-dependent DNA polymerase that maintains TTAGGG repeats at eukaryotic chromosome ends.

    “Telomerase is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric TTAGGG repeats at eukaryotic chromosome ends (Morin, 1989;Blackburn and Greider, 1995).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Morin, 1989 source

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    • cites Blackburn and Greider, 1995

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  • May combine two claims.

    Interpretationown data
    § Reversibility of inhibition

    TRF analysis showed rapid telomere elongation in these cells, indicating that telomerase inhibition is fully reversible.

    “Examination of the TRF sizes also revealed a rapid elongation of the telomeres in these cells (Figure 3B), demonstrating that the telomerase inhibition is fully reversible.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisereasoning
    § Introduction

    Growth arrest dependent on telomere shortening needs many cell divisions to appear, so treatment may need to continue for weeks to months.

    “Cellular growth arrest that depends on telomere shortening will require a series of cell division cycles to become apparent, and treatment may have to be given continuously for weeks to months, potentially in conjunction with other treatment modalities.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning
    § Reversibility of inhibition

    Treated NCI-H460 cells appear to enter senescence sequentially rather than simultaneously, possibly because their telomere lengths are heterogeneous.

    “Apparently, the treated cells enter senescence not in a parallel but rather in a sequential fashion, which may be due to the heterogeneity of their telomere lengths.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data
    § Inhibitor-treated cells exhibit telomere dysfunction

    Because treated cells had reduced proliferation and a low mitotic index, only eight metaphases were obtained and compared qualitatively with 20 control metaphases.

    “Due to the reduced proliferative capacity and the correspondingly low mitotic index only eight metaphases were obtained from the inhibitor-treated cells, and these were compared qualitatively with 20 metaphases from control cultures.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § Discussion

    In mTERC-/- mouse cells, telomere dysfunction is a prominent trait that impairs DNA repair and enhances sensitivity to ionizing radiation.

    “Recent data suggest that in mTERC ±/± mouse cells telomere dysfunction is a prominent trait that impairs DNA repair and enhances sensitivity to ionizing radiation (Wong et al., 2000).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Wong et al., 2000 source

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  • Classified as probably not a substantial claim.

    Study design and conductown data
    § Tumorigenicity assays

    Statistical analysis was performed using Fisher's exact test.

    “Statistical analysis was performed using Fisher's exact test”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Discussion

    Reported telomerase inhibition strategies include peptide nucleic acids and 2'-O-MeRNA oligonucleotides against the telomerase RNA template, telomeric DNA binders such as cationic porphyrins or anthraquinones, and nucleosidic reverse transcriptase inhibitors.

    “Several strategies to inhibit telomerase activity have been reported.These include peptide nucleic acids and 2¢-O-MeRNA oligonucleotides directed towards the telomerase RNA template (Herbert et al., 1999), compounds that target telomeric DNA such as cationic porphyrins or anthraquinones (Sun et al., 1997) and nucleosidic reverse transcriptase inhibitors (Strahl and Blackburn, 1996).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Herbert et al., 1999 source

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    • cites Sun et al., 1997 source

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    • cites Strahl and Blackburn, 1996 source

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  • May combine two claims.

    Interpretationreasoning
    § Discussion

    A significant portion of these TRFs likely consists of subtelomeric sequences rather than TTAGGG repeats, suggesting that the true length of telomeric TTAGGG repeats has been reduced to only several hundred basepairs.

    “A signi®cant portion of these TRFs likely consists of subtelomeric sequences rather than TTAGGG repeats; suggesting that the true length of telomeric TTAGGG repeats has been reduced to only several hundred basepairs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Discussion

    Senescence signs appeared in p53-positive NCI-H460 and p53-deficient HT1080, DU145 and MDA-MB231 lines, suggesting broad therapeutic utility.

    “The phenotypic signs of senescence were observed not only in the p53-positive NCI-H460 cells but also in the p53-de®cient HT1080, DU145 and MDA-MB231 cell lines, consistent with broad therapeutic utility.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The effects of telomerase inhibition observed in human cancer cell lines in vitro and in mouse xenografts are predictive of therapeutic benefit in human cancer patients.

  • May combine two claims.

    Interpretationown data
    § Discussion

    The pharmacological results show that growing human tumour cell lines with selective, non-nucleosidic small-molecule telomerase inhibitors causes progressive telomere shortening, then a senescence phenotype and strong anti-proliferative effects in vitro and in vivo.

    “The pharmacological results presented here demonstrate that extended propagation of human tumour cell lines in the presence of compounds from a novel class of selective, non-nucleosidic small molecule telomerase inhibitors results in progressive telomere shortening followed by the induction of a senescence phenotype and profound anti-proliferative effects in vitro and in vivo.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Analysis of gene expression using oligonucleotide arrays

    Selected genes were required to show at least a 2-fold regulation in at least three out of four pairwise comparisons.

    “Selected genes were required to show at least a 2-fold regulation in at least three out of four pairwise comparisons.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    Dominant-negative hTERT alleles caused cell death in telomerase-positive cancer cell lines.

    “Another approach described recently made use of dominant-negative alleles of hTERT, expression of which resulted in cell death of telomerase-positive cancer cell lines (Hahn et al., 1999b;Zhang et al., 1999).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Hahn et al., 1999b

      Not checked in the free run.

    • cites Zhang et al., 1999 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationnone given
    § Discussion

    The role of telomerase in the immortalization of rodent cells may not be identical to human cells in all respects.

    “The role of telomerase in the immortalization of rodent cells may not be identical to human cells in all respects”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationcitation
    § Discussion

    Telomere-positive cells may not possess a telomere maintenance mechanism other than telomerase.

    “A recent analysis of telomeric recombination mechanisms, which are apparently the mechanistic basis for ALT, suggests that telomere-positive cells may not possess a telomere maintenance mechanism other than telomerase (Dunham et al., 2000).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Dunham et al., 2000 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    An ALT mechanism has been described for virus-transformed human cells and a small proportion of established human cancer cell lines that lack telomerase activity.

    “A similar ALT mechanism has been described for virustransformed human cells and a small proportion of established human cancer cell lines that lack telomerase activity (Bryan et al., 1995).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Bryan et al., 1995 source

      Not checked in the free run.

48 of 48

  • The paper relies on this without support.

    The effects of telomerase inhibition observed in human cancer cell lines in vitro and in mouse xenografts are predictive of therapeutic benefit in human cancer patients.

    “These ®ndings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.”

  • The paper relies on this without support.

    The human cancer cell lines used in this study are representative of human cancers in general.

    “Telomerase inhibition by BIBR1532 or BIBR1591 results in a continuous erosion of the telomeres in human cancer cell lines derived from ®brosarcoma, lung, breast and prostate carcinoma.”

  • The paper relies on this without support.

    The mouse xenograft model with immunodeficient mice is appropriate for assessing the tumorigenic potential and in vivo efficacy of telomerase inhibitors in humans.

    “This model was also used to demonstrate in vivo ef®cacy with no adverse side effects and uncomplicated oral administration of the inhibitor.”

  • The paper relies on this without support.

    The absence of adverse side effects in mice over the treatment period predicts tolerability in humans.

    “The inhibitor treatment was very well tolerated with no signs of acute and chronic toxicity over the entire treatment period, which lasted 60 days for mice developing no or only small tumours.”

  • The paper relies on this without support.

    Defective telomeres are unable to cap chromosomes effectively.

    “Defective telomeres are unable to cap chromosomes effectively”

  • The paper relies on this without support.

    The compounds BIBR1532 and BIBR1591 were stable under the culture conditions.

    “The compounds were stable under these conditions.”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Treatment of HT1080 fibrosarcoma cell lines with BIBR1532 results in progressive telomere shortening, induction of a senescence phenotype and inhibition of tumor growth when transplanted into recipient mice.

    “treatment of HT1080 fibrosarcoma cell lines with the telomerase inhibitor BIBR1532 results in progressive telomere shortening, induction of a senescence phenotype and inhibition of tumor growth when transplanted into recipient mice65.”

    From Telomere dysfunction and tumour suppression: the senescence connection (2008) · cites this paper as [65]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

  • Up- and down-regulated gene counts exceed the stated total

    Minor

    The reported 166 down-regulated and 142 up-regulated genes sum to 308, not the stated total of 302 genes with altered expression.

    Reported 302 · recomputed 308

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Blackburn E and Greider C (eds) (1995) Telomeres. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, NY.

Cited by

Sorted by citations

Related

Authors
Damm, K., Hemmann, U., Garin‐Chesa, P., Hauel, N., Kauffmann, I., Priepke, H., Niestroj, C., Daiber, C., Enenkel, B., Guilliard, B., Lauritsch, I., Müller, E., Pascolo, E., Sauter, G., Pantic, M., Martens, U. M., Wenz, C., Lingner, J., Kraut, N., Rettig, W. J., & Schnapp, A.
Affiliations
  • Boehringer Ingelheim (Germany) (DE)
  • University of Freiburg (DE)
  • University Medical Center Freiburg (DE)
  • ISREC Foundation (CH)
  • Boehringer Ingelheim (Austria) (AT)
Corresponding author
Damm, Klaus
ORCID
Lingner, Joachim
Venue
The EMBO Journal
Publisher
Springer Science and Business Media LLC
Published
2001-12-17
Volume
20
Issue
24
Pages
6958-6968
ISSN
  • 0261-4189 (ISSN-L)
  • 1460-2075
DOI
10.1093/emboj/20.24.6958
PMC
PMC125790
OpenAlex
W2145344158
Cited by
422
References
45
Citations by year
2026: 10 · 2025: 12 · 2024: 16 · 2023: 11 · 2022: 8 · 2021: 14 · 2020: 16 · 2019: 11 · 2018: 13 · 2017: 14 · 2016: 14 · 2015: 13 · 2014: 11 · 2013: 16 · 2012: 22
FWCI
19.53
Type
journal article
Language
en
Open access
Licence
Locations
  • The EMBO Journal (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • FreiDok plus (Universitätsbibliothek Freiburg) (submittedVersion)
  • Infoscience (Ecole Polytechnique Fédérale de Lausanne) (submittedVersion)
  • Infoscience (Ecole Polytechnique Fédérale de Lausanne) (submittedVersion, cc-by-nc-nd)
  • IRIS (submittedVersion)
  • SERVAL (Université de Lausanne) (submittedVersion)
Funders
  • National Science Foundation
  • European Commission
  • Deutsche Forschungsgemeinschaft
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
Topics
  • Health Sciences › Medicine › Physiology › Telomeres, Telomerase, and Senescence
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Organoselenium and organotellurium chemistry
Keywords
telomerase inhibitors, telomere shortening, tumor cell proliferation, cellular senescence, tumorigenicity, oral administration, xenograft models, chromosomal abnormalities, mitotic abnormalities, gene expression changes
MeSH terms
Cell Division, Enzyme Inhibitors, Humans, Neoplasms, RNA, Messenger, Tumor Cells, Cultured, Telomere, Telomerase, Reverse Transcriptase Polymerase Chain Reaction, Oligonucleotide Array Sequence Analysis, Gene Expression Profiling

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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