Journal of Hepatology
Volume 41, Issue 3 , Pages 491-497, September 2004

Telomeres and telomerase: new targets for the treatment of liver cirrhosis and hepatocellular carcinoma

Department of Gastroenterology, Hepatology, and Endocrinology, Medical School Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

published online 19 August 2004.

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1. Introduction 

Telomeres form the ends of eukoryotic chromosomes. The main function of telomeres is to stabilise chromosome ends thus to prevent chromosomal instability (CIS) and activation of DNA damage response. Telomere shortening due to the end replication problem of DNA polymerase limits the proliferative capacity of human cells to 50–70 cell doublings. The holoenzyme telomerase prevents ongoing telomere shortening by de novo synthesis of telomere repeats. However, in humans, postnatal telomerase expression is suppressed in most somatic tissues—including the liver. Telomere shortening limits the regenerative capacity of hepatocytes during chronic liver disease and hepatocellular senescence is associated with cirrhosis formation. In animal models, it has been shown that telomerase reactivation can improve liver regeneration and prevent cirrhosis formation induced by telomere shortening. The future use of telomerase activation for treatment of chronic liver disease depends on its effect on hepatocarcinogenesis. Telomerase reactivation appears to be required for hepatocellular carcinoma progression whereas telomere shortening is linked to CIS and tumor initiation. This review will focus on the role of telomere shortening and telomerase activation in cirrhosis and hepatocarcinogenesis and the potential use of telomerase activators or inhibitors for these disease stages.

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2. Telomeres and telomerase 

Telomeres are specialized protein-DNA structures at the end of linear eukaryotic chromosomes [1], [2], [3]. The main function of telomeres is to cap chromosomal ends in order to distinguish the chromosomal end from inappropriate DNA double strand breaks induced by DNA-damage. This telomere capping function is necessary to prevent chromosomal fusions and induction of DNA-damage responses (for review see Ref. [4]). Human telomeric DNA consists of conserved tandem repeats (TTAGGGn), which extends up to 10–15 kb [5], [6]. Our current understanding is that telomeres need to have a minimum length to form a tertiary structure, which is necessary for telomere capping function. It has been shown that telomeres form DNA quadruplex structures in vitro [7] or lasso-like structures [8]. The telomeres are associated with several binding proteins known as telomere binding proteins which include TRF1, TRF2, POT1, TIN2, RAP1, Tankyrase etc. (for review see Refs. [9], [10], [11], [12]). These proteins together with their interacting partners stabilize telomeres, regulate telomere length homeostasis, and putatively contribute to signalling processes emanating from the telomere.

The enzyme telomerase synthesises telomere sequence de novo and adds it to the telomere end. The holoenzyme consists of two essential components, the telomerase template RNA component (TERC) [13], which contains the complementary sequence of TTAGGG for the synthesis of telomere sequences and the proteinacious catalytic subunit–the telomerase reverse transcriptase (TERT) [14]. In humans, the expression of telomerase is strictly regulated. Telomerase is un-detectable in most somatic cells but is active in a subset of cells such as germ cells [15], stem cells (for review see Ref. [16]), progenitor-cells [17] and activated lymphocytes [18], [19], [20], [21]. The limiting factor for telomerase activity in humans is the catalytic component TERT, which is not expressed in most somatic cells [22].

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3. Telomere shortening induces replicative senescence and chromosomal instability 

The enzyme DNA-polymerase is unable to fully replicate the terminal portion of linear chromosomes during lagging strand DNA synthesis in the S-phase of the cell cycle [23]. Due to this end-replication problem telomeres shorten during each cell division by 50–100 base pairs [23]. When telomeres reach a critically short length telomeres loose their capping function at the chromosomal end [24]. Dysfunctional telomeres are sensed as DNA-damage and generate a DNA-damage signal [25], [26], [27]. The exact nature of this DNA-damage signal has yet to be identified, however, it is known that activation of ATM, ATR, and p53 and its downstream targets (e.g. p21) play a key role in response to telomere dysfunction [26], [27], [28], [29]. Activation of these DNA-damage responses induces a permanent growth arrest of cells with critically short telomeres—a stage that has been termed replicative senescence [30] (Fig. 1). The activation of this replicative senescence program is mitogen dependent since dysfunctional telomeres do not generate a DNA-damage signal in mitogen-deprived cells [31]. Primary human cells reach the senescence stage after 50–70 cell doublings [32], which has fuelled a debate whether telomere shortening limits the regenerative capacity of tissues and organs in humans. A variety of studies have demonstrated telomere shortening in various human tissues during ageing and chronic disease [33]. Experimental support for the telomere hypothesis of limited organ regeneration has come from studies in the telomerase deficient mouse (mTERC−/−). Late generation of these mice show critical telomere shortening resulting in premature ageing and impaired organ-homeostasis of highly-proliferative organs [24], [34], [35] (Fig. 1). In addition, a mutation of the telomerase RNA component (TERC) has recently been linked to the autosomal dominant form of human Dyskeratosis congenita [36], [37] – a disease characterised by premature ageing phenotypes [38] reminiscent of phenotypes in ageing mTERC−/− mice [35]: skin defects, anaemia, liver cirrhosis and cancer formation at early age.

  • View full-size image.
  • Fig. 1. 

    There are two possibilities for telomerase therapy in liver disease: critical telomere leads to induction of replicative senescence and chromosomal instability. Replicative senescence impairs regeneration during ageing and chronic disease. Chromosomal instability elevates the rate of cancer initiation. In contrast, telomere dysfunction and chromosomal instability inhibit tumor progression and telomere stabilisation (e.g. by telomerase activation) is required for cancer progression. [This figure appears in colour on the web.]

In addition to the activation of replicative senescence, critical telomere shortening and telomere dysfunction induce chromosomal fusions in human [39] and mouse cells [24]. The fusion of chromosomes at dysfunctional telomeres is mediated by DNA-repair proteins that are involved in the repair of DNA double strand breaks by non-homologous end joining [40]. When cells with fused chromosomes enter the cell cycle, the fused chromosomes are pulled to opposite poles of the spindle during mitosis. This leads to disruption of chromosome arms resulting in gains and losses of chromosome fragments in the daughter cells. This mechanism of induction of CIS by telomere dysfunction has been named fusion-bridge-breakage cycle (Fig. 1). In addition, it has been shown in mTERC−/− mice that the rate of non-reciprocal translocations are significantly increased in mice with shortened telomeres [41]. The initiation of CIS by telomere dysfunction in mTERC−/− mice provoked an increased incidence of sporadic cancer during ageing [35] (Fig. 1), a shift in the tumor spectrum in p53 mutant mice [41], and an increased initiation of intestinal tumors in APC mutant mice [42]. In contrast to these effects on tumor initiation, telomere dysfunction in mTERC−/− mice suppressed the progression of tumors [42], [43], [44]. Suppression of tumor progression by telomere dysfunction was linked to an activation of the p53-pathway resulting in increased rates of tumor cell apoptosis, and decreased rates of tumor cell proliferation [42]. Therefore, telomere dysfunction has dual roles in carcinogenesis by increasing the initiation but inhibiting the progression of tumors (for review see Ref. [45]) (Fig. 1).

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4. Telomere shortening in cirrhosis and hepatocellular carcinoma 

A variety of studies have demonstrated telomere shortening in chronic liver disease [46], [47], [48], [49], [50]. Most significant telomere shortening has been detected at the cirrhosis stage [46], [47], [48], [49], [50] and some of these studies have described a correlation between telomere shortening and cirrhosis progression [46], [47], [48], [50]. In addition, it has been shown that senescent cells accumulate in chronic liver disease [51] and at the cirrhosis stage [50]. Currently, hepatocytes were identified as the cell type affected by telomere shortening and senescence at the cirrhosis stage, whereas cells in the fibrosis area of the cirrhotic liver have relatively longer telomeres and do not enter senescence [50]. Experimental evidence for the impact of telomere shortening on liver regeneration and cirrhosis formation has come from studies in telomerase deficient mice (mTERC−/−), which show defects in liver regeneration and an accelerated formation of cirrhosis in response to chronic liver damage [52]. These studies have revealed that telomere shortening limits the number of liver cells participating in organ regeneration by blocking cells with critically short telomeres from entering the cell cycle [53]. According to these studies telomere shortening is heterogeneous between different cells of an organ and the regenerative capacity of an organ depends on the size of the population of cells with sufficient telomere reserves [53]. Together, the current data support a telomere hypothesis of cirrhosis formation. According to this hypothesis chronic liver diseases induce hepatocyte damage resulting in regeneration of remaining hepatocytes and an elevated cell turnover of hepatocytes. This leads to accelerated telomere shortening in hepatocytes and finally to induction of hepatocellular senescence. If at this stage, the organ damage continues other cell types that usually do not participate in organ regeneration, e.g. stellate cells, become activated and form cirrhotic scar tissue.

During hepatocarcinogenesis in humans, telomere shortening appears to have a tumor initiating effect. The risk of HCC formation increases sharply at the cirrhosis stage, which is characterised by hepatocyte telomere shortening (see above). In addition, a variety of studies have demonstrated shortened telomeres in HCC as compared to non-cancerous surrounding liver tissue (for review see [45]). It was recently demonstrated that hepatocytes are the cell type affected by telomere shortening in HCC [54] and that hepatocyte telomere shortening was more pronounced in aneuploid HCC compared to diploid HCC [54]. These data indicate that hepatocyte telomere shortening promotes induction of CIS thereby increasing the risk for initiation of HCC in humans. In mTERC−/−, mice telomere shortening had a diverse impact on HCC formation. Initiation of early pre-malignant liver tumors was increased in response to carcinogen treatment (diethylnitrosamine or carbon tetrachloride) or uPA-transgene expression [55]. Paradoxically, the progression of the initiated tumors towards macroscopic HCC was strongly impaired by telomere shortening in mTERC−/− mice [55]. Together these data indicate that telomere shortening increases HCC initiation by induction of CIS, whereas tumor progression requires telomere stabilisation (for review see Ref. [45]).

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5. Telomerase activity in chronic liver disease, cirrhosis, and hepatocellular carcinoma 

In normal human liver, there is no significant telomerase activity [56], [57], [58]. In contrast, some studies have reported a weak activation of telomerase during chronic viral hepatitis or cirrhosis [59], [60], [61]. However, it is not yet clear whether weak level of telomerase activity in hepatitis samples represent an activation of telomerase in hepatocytes. An alternative explanation could be that telomerase is activated in infiltrating lymphocytes in diseased liver. Activated human lymphocytes are known to be telomerase-positive and are found at a significant level in liver affected by viral hepatitis. In line with this hypothesis hTERT expression in chronic liver disease was only found in infiltrating mononuclear cells but not in hepatocytes [62] or at very low frequency (<1%) in hepatocytes [63]. The data on telomere shortening during chronic liver disease and cirrhosis indicate that even if a low level of telomerase activity is present in chronic liver disease, this level of activity is not sufficient to prevent telomere erosion during chronic liver disease (see above).

In contrast to normal human liver, over 80% of human HCC show high level of telomerase activity (for review see Ref. [45]). Most other human cancer types also display high-level telomerase activity in similar percentages to HCC (for review see Ref. [45]). During hepatocarcinogenesis the activation of telomerase has been linked to the reexpression of hTERT [22], expression of which is normally suppressed in most human somatic tissues after birth. The timing of telomerase activation during hepatocarcinogenesis is still a matter of debate. Some studies have described telomerase activity [59], [64] and expression of hTERT [60] at early pre-malignant stages of hepatocarcinogenesis in regenerative nodules and liver cirrhosis. Other studies indicated that significant activation of telomerase [58], [65], [66], [67], [68] and hTERT-expression [22], [62], [63], [69] occur at later stages of hepatocarcinogenesis in regenerative nodules with high-grade dysplasia or at the carcinoma stage. The level of telomerase activity in HCC [68], [70], [71], [72], [73] or surrounding non-cancerous liver tissue [74] have been identified as a prognostic marker for HCC-recurrence after liver surgery and patient survival. It remains an important question how telomerase reactivation is controlled during hepatocarcinogenesis. One of the known activators of hTERT transcription, the c-myc oncogene, is over-expressed in most human HCC [75]. Some studies have shown that the activation of the myc-pathway is linked to activation of telomerase in human HCC [76]. However, there are indicators that pathways than other than c-myc also contribute to the activation of telomerase in human HCC [77]. In this regard, it is an interesting observation that Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets the human telomerase gene potentially playing a role in the activation of TERT transcription [78], [79].

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6. The use of telomerase activators or inhibitors as therapeutic targets for liver cirrhosis and hepatocellular carcinoma 

The above data suggest that in principle two therapeutic approaches for telomerase inhibition or activation could be used in liver disease (Fig. 2):

1.Telomerase activation for prevention of hepatocellular senescence and improvement of hepatocyte regeneration at the cirrhosis stage.

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  • Fig. 2. 

    The effects of telomere shortening on regeneration and carcinogenesis. 1. Telomerase inhibitors will destabilise telomeres in telomerase-positive HCC thereby inducting tumour cell apoptosis and cell cycle arrest. Telomerase-negative, non-cancerous liver tissue will not be affected by telomerase inhibitors. 2. Telomerase activators will stabilise the telomeres in telomerase-negative cirrhotic liver. Telomere stabilisation will rescue hepatocyte senescence and will improve the regenerative capacity of the cirrhotic liver. This therapy will not affect the growth of telomerase-positive HCC. Senescent hepatocytes are shown as blue circles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Proof of principle for this approach has come from studies in mTERC−/− mice. In mTERC−/− mice telomerase activation by adenoviral mediated transfer of mTERC rescued telomerase activity in mouse liver. In this mouse model telomerase re-activation improved hepatocyte regeneration and prevented premature cirrhosis formation induced by chronic liver damage [52]. In human cells the activation of telomerase is facilitated by transfer of the catalytic subunit of telomerase (hTERT, see above). Expression of hTERT stabilises telomere length in primary human cells and is sufficient for the immortalisation of many human primary celltypes including human fetal hepatocytes [80]. In principle, such cell lines could be useful for artificial liver devices or cell transplantation approaches for the treatment of liver failure. It has yet to be determined whether hTERT-immortalised primary human cells are genetically stable. Initial reports did not detect any adverse effect of hTERT on chromosomal stability and oncogenic transformation [81], [82]. However, recent reports described a pre-malignant phenotype of hTERT-immortalised cells after extensive passage [83]. A different therapeutic approach could be to directly deliver hTERT to the organ affected by regenerative exhaustion in vivo. The problem with this approach in regard to liver cirrhosis is that any hepatotoxicity of a gene delivery system could be deleterious in patients with cirrhosis, therefore, adenoviral mediated gene transfer is not an option for these patients. It has yet to be determined whether other gene or protein transfer systems can be used to safely and efficiently re-activate telomerase in the liver. To avoid an oncogenic risk of telomerase-activation for therapeutic approaches in vivo it appears possible that a transient activation of telomerase could increase the regenerative capacity of an organ without facilitating unlimited growth of pre-malignant cells. Pharmaceutical telomerase activators have not yet been reported.

2. Telomerase inhibition for the treatment of HCC.

The high incidence of telomerase re-activation in human cancer including HCC indicates that tumor growth depends on telomere stabilization (see above). A variety of studies have shown that telomerase inhibition limits the growth of telomerase-positive human cancer cell lines by induction of telomere shortening, apoptosis, and cell cycle arrest (for review see Ref. [84], [85]). Similarly, it has been shown that telomerase inhibition in hepatoma cells induces cell cycle arrest and induction of apoptosis [86], [87], [88]. In addition, telomere shortening increases chemosensitivity of cancer cells [89] indicating that a combination of telomerase inhibition with classical chemotherapy might be an efficient approach for cancer treatment.

One potential escape mechanism of HCC in response to telomerase-inhibitors could be the activation of alternative mechanisms of telomere elongation (ALT). ALT leads to telomere elongation by homologous recombination and copy switching [90], [91], [92], [93]. In yeast two different types of ALT mechanisms are described (for review see Ref. [94]), whereas only the analogous to the type II mechanism has been described for human ALT cells [95]. It is known that a subset of 10–20% of human tumors – including HCC – is telomerase-negative. Although, for HCC the existence of ALT has not been shown directly, it seems likely that some of the telomerase-negative tumors have indeed activated ALT [96]. In addition to this telomerase independent ALT mechanism, it has also reported that a co-existence of ALT and telomerase can exist (for review see Ref. [97]). The clinical relevance of this possible escape mechanism for the treatment of HCC using telomerase inhibitors remains to be investigated.

Telomerase inhibition may have minimal side effects since most of human tissues and organs are telomerase negative (see above). However, the regenerative potential of telomerase-positive stem and progenitor cells might be affected by long-term telomerase inhibition. In addition, it has recently been shown that normal human fibroblasts express telomerase during S-phase of the cell cycle and that this telomerase-expression is important for maintenance of functional telomeres [98]. If this is the case in other human cells telomerase inhibition could induce side effects in a variety of tissues and organs. Another potential problem of telomerase inhibitors for cancer treatment could be that the anti-cancer effect occurs only after a lag period when telomeres reach a critically short length [99]. Therefore, it may be necessary to determine the telomere length of tumors to predict the response towards telomerase inhibition. In this context, it is interesting that HCC are characterised by extremely short telomeres [54] making this cancer a good target for telomerase inhibition. Given the lack of significant telomerase activity in non-cancerous liver tissue, it seems possible that telomerase inhibition would have little effect on cirrhosis progression. Eventually, a sequential treatment with transient telomerase activation followed by telomerase inhibition could be useful for cirrhosis patients with HCC.

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PII: S0168-8278(04)00262-4

doi:10.1016/j.jhep.2004.06.010

Journal of Hepatology
Volume 41, Issue 3 , Pages 491-497, September 2004

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