HTERT

FIGURE 15.1 One-dimensional capillary electrophoresis separation of a protein homogenate prepared from the hTERT cell line. Both separations were preformed in 30 pm ID, 145 pm OD, 20 cm long capillaries at 20,000 V. (a) Micellar electrokinetic chromatography performed with a 100 mM CHES, 100 mM Tris, and 15 mM SDS buffer at pH 8.7. Sample is electro-kinetically injected with 0.25 kV for 1 s (b) Capillary sieving electrophoresis performed in 5% Dextran (513 kDa), 100 mM CHES, 100 mM Tris, 3.5 mM SDS, pH 8.7. 

A relatively new cell line that has not to date been characterised for its use in biopharmaceutics is based on primary airway epithelial cells infected with retroviruses expressing hTERT and HPV-16 E6/E7 (NuLi-1) [54], NuLi-1 cells were cultured on plastic up to passage 30. When grown on collagen-coated, semi-permeable membranes (Millicell-PCF), NuLi-1 TEER decreased only slightly over the 30 passages from 685 31 to 389 21 ohm.cm2. The TEER of NuLi-1 is similar to that observed with the primary bronchial cultures of 532 147 ohm.cm2. Thus, NuLi-1 cells can form an electrically tight airway epithelial barrier that mimics active and passive ion transport properties of primary human bronchial epithelial cells [54],... 

Figure 26.1 Immortalization of human cells Cells enter replicative senescence at mortality stage 1 (Ml Hayflick limit) after about 60 population doublings (PD). The protein p 16 accumulates in senescent cells. The simian virus 40 (SV40) large T antigen as well as the human papilloma virus (HPV) type 16-E6 and E7 proteins sequester the retinoblastoma protein (Rb) and/or p53 constitutively releases the transcription factor E2F. E2F induces expression proteins required for progression through Gl/S transition, thus the cells escape cell cycle arrest. At mortality stage 2 (M2), transformed cells must overcome senescence and crisis before they are immortalized. This is likely to involve the activation of telomerase either by the introduction of hTERT cDNA or by a genetic change that activates telomerase.

Beyond the Hayflick limit, cells that have escaped senescence and/or crisis must progress beyond mortality stage 2 (Figure 26.1). Recent studies have shown that human telomerase reverse transcriptase cDNA (hTERT) in combination with viral oncogenes also has the potential to immortalize human airway epithelial cells [15, 78-82], However, even though hTERT alone will cause enhanced growth potential of a cell line, with the exception of one study [81],... 

Transfect/infect cells with a transforming agent (e.g., SV40, HPV, Adeno-SV40, hTERT/oncogene plasmid)... 

MacKenzie, K. L., S. Franco, C. May, M. Sadelain, and M. A. Moore. 2000. Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation. Exp Cell Res 259(2) 336-50. 

At last, nucleolin might play a specific role in telomeric replication and maintenance, as suggested by two types of data. First, it binds telomeric repeat (TTAGGG)n in vitro (Ishikawa et al, 1993 Pollice et al, 2000), with a marked preference for the single-stranded form. Secondly, it interacts in vitro and in vivo with hTERT (Khurts et al, 2004), the protein catalytic component of human telom-erase. This interaction takes place both in the cytoplasm and in the nucleolus, where it could promote the assembly of hTERT with the RNA subunit hTERC. As a conclusion, many data regarding the involvement of nucleolin in DNA replication are indirect and an experimental demonstration through knockdown or knockout studies is still awaited. 

Hartmann U, Brummendorf TH, Balabanov S et al. Telomere length and hTERT expression in patients with acute myeloid leukemia correlates with chromosomal abnormalities. Haematologica 2005 90 307-316. 

Campbell LJ, Fidler C, Eagleton H et al. hTERT, the catal34ic component of telomerase, is downregulated in the haematopoietic stem cells of patients with chronic myeloid leukaemia. Leukemia 2006 20 671-679. 

Oligonucleotides Peptide nucleic acids (PNA, hTERT) AlPcS, TPPS2a... 

All finally differentiated eukaryotic cells lack telomerase activity. Thus, when cells divide, the ends of the chromosomes can not be duplicated and the telomeres get shorter and shorter with every cell division, until the cell finally dies. Thus, the lack of telomerase activity may determine the number of divisions a cell can go through. And indeed, disruption of the telomerase gene in yeast leads to senescence and death (see Fig. 12.12). The ageing process in the yeast cells was reversed when human telomerase was expressed. Expression of the catalytic subunit of the human telomerase holoenzyme (hTERT) also enabled human cells to avoid senesce and to multiply indefinitely. This raised hopes to immortalize human cells, without interfering with their differentiation. (For more information, see ref. 21 and Chapter 17). 

If the telomerase activity alone were to determine the life span of a cell, one would expect that cells such as muscle cells and neurons, that do not divide, never age. But, this is obviously not the case. This paradox shows that the relationship between control of senescence and cell death is much more complicated. Recent work of T. Kiyono et reviewed by Robert Weinberg,23 with two human epithelial cell types (keratinocytes and mammary epithelial cells), showed that making cells immortal requires not only expression of the hTERT gene and of the telomerase, but also inactivation of the retinoblastoma (RB) tumour-suppressor pathway (the RB pathway halts cells in the Gi phase of the cell cycle in response to a wide range of inhibitory signals, see Fig. 12.12). 

Fig. 12.12 With each cell division, the telomeies at the end of the chromosomes become shortened. The model shows that in order to stop senescence and death, both telomerase activity and blockage of the RB pathway are needed. Telomerase activity and blockage of the RB pathway are intimately linked, because the factors that block the RB pathway (the wyc oncogene and the E6 oncogene of the human papillomavirus, see Part 4) also upr late expression of hTERT.24 In contrast to normal differenbated cells, cancer cells have telomerase adivity. In this way, cancer cells overturn the life-sparvcontrolling clock and become immortal. Since, about 90% of all human cancers are derived from epithelial cells, it follows that in epithelial cancer cells the RB pathway has also been inactivated. To the contrary, when the tumour-suppressor p 53 accumulates, the cells age prematurely.25...

Fig. 17.3 The expression of two oncogenes, SV40 TAg and H-ras, together with the catalytic subunit of human telomerase, hTERT, transforms normal human cells to immortal cells and eventually to tumour cells. (Information from Hahn et a/. t r see also ref. 15.)...

FCS fetal calf serum hTERT human telomerase catalytic subunit... 

The telomerase holoenzyme core consists of a catalytic subunit, the reverse transcriptase protein hTERT (1-3), and an RNA template subunit, hTR (4), which are essential for telomerase activity (5). Otherproteins (6,7) and kinases (8-11) are... 

The hTERT messenger RNA has also been targeted with antisense oligonucleotides that are designed to hybridize with complementary sequences of hTERT mRNA. The recent clinical success of the first antisense drag provides the impetus for further development of these strategies for selective disruption of telomerase expression (42). 

In addition to antisense targeting, hammerhead ribozymes have also been used to inhibit the expression of hTERT messenger RNA. These ribozymes are small catalytic RNA molecules that consist of a catalytic core flanked by antisense sequences that function in the recognition of the target sequence. These RNAs possess endoribonuclease activity that allows for the degradation of target transcripts. A hammerhead ribozyme has recently been used to cleave the hTERT mRNA in breast epithelial cells that inhibited telomerase activity and resulted in shortened telomeres, decreased net growth, and apoptosis (43). 

The intracellular introduction of synthetic molecules that bind to a specific component (or components) of the holoenzyme telomerase can serve to literally switch off the activity of telomerase once it is already in full-swing (as is the case in most cancer cells). To date, the agents employed to successfully down-regulate or inhibit telomerase activity by directly binding to one of its components have mainly targeted hTERT and hTR, the core subunits of telomerase. These inhibition agents are shown in Figures 3A and 3B. 

Modified short DNA and RNA molecules with novel bond linkages between the bases have been designed with the aim to use the antisense approach by binding to the RNA template in the hTR subunit(s) to prevent or halt transcription and thereby act as competitive inhibitors of telomerase activity. Hence, the hTR RNA template is unavailable to hTERT for reverse transcription (52). The various types of sugar phosphodiester backbone modifications in these molecules are intended to confer certain desirable characteristics or properties, such as intracellular penetration, superior binding affinity, and therefore specificity, to the hTR RNA template and in order to enable intact delivery to their target. 

Telomerase activity can also be inhibited by the direct binding of small non-nucleosidic synthetic compounds to the hTERT reverse transcriptase component of telomerase. Schnapp and coworkers have recently reported the first mixed-type noncompetitive (70) catalytic telomerase inhibitor, (2-((E)-3-naphtalen-2-yl-but-2-enoylamino)-benzoic acid) (BIBR1532), which causes telomere shortening and senescence characteristics in various types of cancer cells in vitro and in vivo in mouse xenograft models at nanomolar concentrations (71). 

Last, the hTERT reverse transcriptase inhibitors do not effect their activity by specifically and persistently binding to hTERT rather, they act as competitors for the substrate deoxyribonucleotides used by reverse transcriptases, such as hTERT, to construct DNA chains (or more specifically for hTERT, to construct telomeric DNA extensions). Small nucleoside analogues can act as reverse transcriptase inhibitors, although only some of these compounds, such as 6-thio-2 -deoxyguanosine 5 -triphosphatc (TDG-TP), are selective against the hTERT reverse transcriptase (72). TDG-TP is effective at low micromolar concentrations (72) and stops telomeric DNA extension after incorporation into the DNA (73). 

Kharbanda S, Kumar V, Dhar S, Pandey P, Chen C, et al. 2000. Regulation of the hTERT telomerase catalytic subunit by the c-Abl tyrosine kinase. Curr. Biol. 10 568-75... 

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