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Keywords:

  • bladder cancer;
  • human telomerase reverse transcriptase gene (hTERT);
  • gene therapy

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

OBJECTIVE

To construct a mutant enhanced green fluorescence protein (pEGFP) human telomerase reverse transcriptase (hTERT) gene expression vector (pEGFP-hTERT), to observe its expression in transfected human bladder cancer cell line T24 and its role in the molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer therapy.

MATERIALS AND METHODS

Polymerase chain reaction (PCR) amplification was performed using primers based on the gene sequence of hTERT. The PCR product was cloned into plasmid pGEMT-T Easy and the sequence of mutant hTERT gene analysed. A recombinant mutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. After transfecting the fusion gene into T24 cells by the method of calcium phosphate-DNA co-precipitation, we detected steady expression of the GFP-hTERT fusion protein by fluorescent-light microscopy. Changes in the proliferation of T24 cells were detected by light microscopy, and β-galactosidase staining correlated with senescence.

RESULTS

Identification of pEGFP-hTERT by enzyme digestion showed that the mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. Steady expression of GFP-hTERT fusion protein was located in the nucleus of transfected cells. Positive expression senescence-associated β-galactosidase staining in transfected cells increased gradually with extended cultured time, and their growth was suppressed.

CONCLUSION

The recombinant mutant vector (pEGFP-hTERT) was successfully constructed and expressed steadily in T24 cells. The mutant-type hTERT gene suppresses the proliferation of T24 cells by a competitive effect on telomerase activity. This suggests that the hTERT gene might be a suitable gene target for bladder cancer therapy.


Abbreviations
hTERT

human telomerase reverse transcriptase

pE(GFP)

enhanced (green fluorescence protein)

X-Gal

5-bromo-4-chloro-3-indol-β- d-galactopyranoside

hTR

human telomerase RNA template

TP1/TLP1

telomerase-associated protein

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumour progression. The human telomerase reverse transcriptase gene (hTERT) was identified as a gene essential for telomerase activity. hTERT encoding the telomerase catalytic subunit is expressed at high levels in the great majority of human tumours. Moreover, the message is up-regulated concomitantly with telomerase activation during cellular immortalization and tumour progression. Thus far, over-expression of hTERT has been regarded as the rate-limiting step in telomerase activation [1].

Bladder cancer is thought to be the most common urothelial carcinoma in China. To determine whether disruption of hTERT gene function would limit the growth of bladder cancer cell line T24, to explore the specific regulatory mechanisms by which hTERT participates in telomerase activity function, and to provide a potentially effective target gene for gene therapy of human bladder cancer, we constructed a mutant enhanced green fluorescence protein (pEGFP)-hTERT expression vector (pEGFP-hTERT) and observed its expression in transfected T24 cells. We present evidence to indicate that telomerase activity is inhibited in transfected cells, and indicate the feasibility of hTERT gene therapy for bladder cancer.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The human bladder cancer cell line T24 was obtained from the Department of Urology of Peking University and propagated as a monolayer culture in Dulbecco's modified Eagle's medium (Gibco-BRL, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air, 5% CO2. The medium was changed every 2–3 days.

The vector pC1-Neo-hTERT, generously provided by Dr Elinor Eaton (Massachusetts Institute of Technology, USA), was created by inserting the hTERT (3.45 kb fragment) at the EcoR I and Sal I sites of the vector pC1-Neo. The C terminus of hTERT (730 bp) from clone plasmid pC1-Neo-hTERT was PCR-amplified with oligonudeotide primers synthesized by Sangon Co., Shanghai, China (forward-upstream: 5′-GGA ATT CGA GGT GTC CCT GAG TAT GGC-3′ and reverse-downstream: 5′-GC GTC GAC GTC CAG GAT GGT CTT GAA G-3′). PCR was carried out under the conditions of 35 cycles of 94 °C for 45 s, 65 °C for 45 s and 72 °C for 90 s. The resulting mutant 730 bp fragment was subcloned into the vector pGEMT-T Easy by T4 DNA ligated enzyme (Promega, Madison, WI, USA). After being purified and sequenced correctly, the deletion mutant fragment was inserted into the flanking EcoR I and Sal I restriction sites of the plasmid pEGFP-C1 (Clontech, Palo Alto, CA, USA) carrying the green fluorescent protein (GFP) gene, the reconstructed vector was designated as pEGFP-hTERT (Fig. 1), transformed into JM109, selected by Amp® and extracted using the Wizard Genomic DNA Purification Kit (Promega). This structure was shown by electrophoresis after combination of EcoR I and Sal I enzyme digestion.

image

Figure 1. Construction of mutant recombinant vector pEGFP-hTERT.

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T24 cells were transfected with 5 µg of pEGFP-hTERT plasmid DNA in Opti-MEM® reduced-serum medium using the method of calcium phosphate-DNA co-precipitation according to the manufacturer's instructions. After incubation in the transfection solution for 16 h at 37 °C in a humidified atmosphere of 95% air, 5% CO2, the transfected T24 cells were allowed to recover for 48 h in Dulbecco's modified Eagle's medium and grown under G418 selection (400 ng/L) for the stably transfected T24 cells. Empty-vector and untreated controls were designed for each group.

To observe GFP by fluorescence microscopy, transfected cells were plated on 60-mm tissue-culture plates containing a glass coverslip. The cells were fixed with 4% formaldehyde in PBS, washed twice, air-dried and mounted on glass slides according to the manufacturer's directions. Transfection with pEGFP-C1 was used as a control to discriminate signals from autofluorescence.

To construct growth curves, T24 cells were seeded at 2 × 104 cells/mL in 24-well plates. Group 1 comprised routinely cultured T24 cells; group 2, T24 cells transfected with pEGFP-C1; and group 3, T24 cells transfected with pEGFP-hTERT. Each group had three wells, which were counted under inversion microscopy every other day. For each well, cells were counted three times to construct the cell growth curve.

To detect the induction of senescence, for each time point, each group of cells was propagated as a monolayer culture in tissue culture plates containing glass coverslips. The coverslips were washed twice with PBS and fixed with 3% formaldehyde in PBS for 5 min at room temperature, washed again, and the cells were stained at pH 6.0 with fresh X-Gal (5-bromo-4-chloro-3-indol-β- d-galactopyranoside) solution incubated for 4 h at 37 °C. Morphological and senescent changes were observed under inversion microscopy.

For statistical analysis, groups were compared using Student's t-test. All reported P values were two-sided, and P < 0.05 was considered to indicate statistical significance.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The mutant pEGFP-hTERT was digested with the combination of EcoR I and Sal I enzyme, and two bands were shown by electrophoresis; 730 bp and 4700 bp specific amplification fragments corresponding to hTERT and EGFP were evident in plasmid pEGFP-hTERT, suggesting that the mutant hTERT sequence was subcloned into the EcoR I and Sal I sites of the pEGFP-C1 vector (Fig. 2).

image

Figure 2. Identification of mutant pEGFP-hTERT by enzyme digestion. Lane 1: pEGFP-C1; Lane 2: pEGFP-C1 digested by EcoR I and Sal I; Lane 3: pEGFP-hTERT; Lane 4: pEGFP-hTERT digested by EcoR I and Sal I (730 bp and 4700 bp); Lane 5: DL15000 DNA molecular weight marker (15 000 bp, 10 000 bp, 5000 bp, 2500 bp, 1000 bp and 250 bp).

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Monolayer cultures of T24 cells were transfected separately with pEGFP-C1 and pEGFP-hTERT as described in the Methods. Normal cultured T24 cells were used as a negative control. After a 12-day selection by 400 ng/L G418, one colony was chosen from each transfected culture; the untreated cells died.

GFP was steadily expressed in T24 cells transfected with pEGFP-hTERT. GFP generated a bright green fluorescent signal that was mainly located in the nucleus of cells transfected with pEGFP-hTERT. By contrast, the fluorescent marker was observed in the cytoplasm of cells transfected with pEGFP-C1, and little background fluorescence was observed in untransfected cells (Fig. 3).

image

Figure 3. Fluorescent microscopy analysis of GFP expression in T24 cells. (oil-immersion objective × 40). A, GFP expression was localised in the cytoplasm of cells transfected with pEGFP-C1 (arrow); B, GFP expression was localised in the nucleus of cells transfected with pEGFP-hTERT (arrow).

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After 2 weeks, the population doubling time of cells transfected with pEGFP-hTERT was longer than for untransfected cells or cells transfected with pEGFP-C1; these cells showed slowed growth and eventually stopped proliferating. Parallel cultures of cells expressing pEGFP-C1 or control cells showed no change in growth, and the difference between groups was significant (P < 0.05; Fig. 4).

image

Figure 4. Cell growth curves of the three types of T24 cells (T24, red circles; T24/pEGFP-C1, green squares; T24/pEGFP-hTERT, light red triangles). The symbols indicate mean cell counts (n = 9) and the error bars show the sem.

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Untransfected and transfected T24 cells were assayed for senescence-associated X-Gal activity (Fig. 5). X-Gal staining appeared blue and granular in the cytoplasm; control cells had little staining. T24 cells transfected with pEGFP-hTERT showed denser staining than controls, and the staining intensity gradually increased with time.

image

Figure 5. X-Gal staining of T24 cells (haematoxylin and eosin, × 200). A, No expression in untransfected T24 cells; B, Positive staining in T24 cells transfected with pEGFP-hTERT.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Telomerase is a specific ribonucleoprotein reverse transcriptase that is responsible for maintaining the terminal repeats of telomeres and the stability of human chromosome ends. Activation of telomerase is thought to be essential for immortalized cells, and to contribute to progression towards malignancy [2]. High levels of telomerase activity have been detected in 85–90% of malignant human tumours [3]. Analysis of bladder tumour tissue biopsies found that almost all (90%) expressed telomerase activity [4]. Telomerase is composed of three major subunits: human telomerase RNA template (hTR), TP1/TLP1 (telomerase-associated protein) and hTERT. The expression levels of hTR and TP1/TLP1 are not proportional to telomerase activity, but expression levels of hTERT (a catalytic subunit of telomerase) parallel telomerase activity in the great majority of human tumour samples [5]. The association between hTERT expression and telomerase activity, both of which are present in immortal transformed cells but which are absent in normal, mortal cells, suggests that induction of hTERT expression might underlie the activation of telomerase. Thus up-regulation of hTERT probably plays a role in the progression of human cancers, and induction of hTERT gene expression appears to be the rate-limiting step in the telomerase activation that leads to cellular immortalization and malignant tumour progression [6]. This suggests that the hTERT gene is an attractive target for gene therapy designed to interfere with malignant cell proliferation. An anticancer therapy based on inhibiting hTERT gene expression has the potential to be highly selective for tumour cells, with few toxic side-effects [7]. However, the mechanism by which hTERT participates in telomerase function and tumorigenesis is not defined. Therefore, a systematic study to investigate the association of telomerase activation with hTERT regulatory molecules during tumour promotion or tumour progression is important. Analyses of hTERT gene function are likely to become more common as the regulation of telomerase is understood better. Point mutation analysis has confirmed that deletion mutations in N-terminal regions of hTERT significantly impaired telomerase function [8], but the C-terminal regions of hTERT polypeptide have not been subject to detailed molecular analysis.

The present investigations focused on C-terminal function of the hTERT gene with a view to manipulating cellular telomerase. To determine if the uncharacterized C-terminal regions of the hTERT gene are functionally important in regulating telomerase, we began a mutagenic analysis of the hTERT gene. We constructed a mutant plasmid pEGFP-hTERT. We deleted the N-terminal of the recently identified hTERT sequence, but conserved ≈ 730 bp C-terminal, and introduced a substitution mutation hTERT into the reporter gene plasmid pEGFP-C1. We investigated the effects of this mutation of hTERT on telomerase activity, cellular senescence and growth of the human bladder cancer cell line T24.

GFP from the jellyfish Aequorea victoria is an important reporter molecule for monitoring fusion gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (λmax = 509 nm) when the cells are excited with ultraviolet or blue light (λmax = 395 nm), and this can be detected by fluorescence microscopy. Using GFP fusion proteins not only facilitates simultaneous detection of transcription reporters, but also permits analysis of protein trafficking [9]. The use of GFP allowed real-time analysis of mutant C-terminal function of the hTERT gene. Using fluorescence microscopy, we monitored GFP production from individual transfected cells and observed green fluorescence steadily expressed in T24 cells transfected with pEGFP-hTERT. In transfected cells, the fluorescent marker was located mainly in the nucleus of cells transfected with pEGFP-hTERT, but mainly in the cytoplasm of cells transfected with pEGFP-C1. These observations suggest that the C-terminal region of the hTERT gene encodes a partly catalytic subunit of telomerase which was transcribed and translated in the cytoplasm, then transported into the nucleus, and which was largely responsible for the specific telomerase template RNA binding function, suggesting that telomerase function is controlled at least partly at a post-transcriptional level. Recent studies have indicated that repression of telomerase in normal somatic tissues involves transcriptional regulation of the hTERT gene [10], and transcriptional up-regulation of hTERT might be an important mechanism by which telomerase becomes activated during cellular immortalization and malignant tumour progression [11]. Thus inhibition of hTERT expression at the transcriptional level could be an important antineoplastic strategy. The present results show that complete inhibition of telomerase could be achieved by targeting the active site of the hTERT gene. Further studies will be necessary to define the precise binding target of the hTERT gene.

The proliferative changes of T24 cells were detected by light microscopy and X-Gal stain correlated with senescence. Transfection of the mutant hTERT gene prolonged population doubling times, decreased telomerase activity, caused growth inhibition and induced senescence in T24 cells, and increased rates of apoptosis. By contrast, control cells either showed no growth defects or grew only slightly slower, and showed no senescence. The mechanisms of cellular senescence are not fully known, but at least two mechanisms are involved: the telomere ‘ageing clock’ theory, and the cell-cycle regulatory pathway. In culture, normal human cells have a finite lifespan and undergo cell senescence; this stage of the cell-life cycle is known as the Hayflick limit, or mortality stage 1. Oncogenic transformation can block the progression to senescence, leading to an extended lifespan until cells reach the next crisis (mortality stage 2). Recent studies indicate that high expression of hTERT can activate telomerase, allowing cells to become immortal by maintaining the stability of the ends of chromosomes, and overcome mortality stage 1 arrest or bypass mortality stage 2 [12]. The desired effect of telomerase inhibition would be to shorten telomeres, causing replicative senescence and preferably cell death due to irreparable chromosome damage. Thus, an anticancer therapy might involve inhibition of hTERT expression and telomerase to trigger cell senescence [13].

Although the inhibitory effects of the mutant hTERT gene on the growth of T24 cells are striking, the underlying molecular mechanisms are unclear. The C-terminal region of hTERT has been hypothesized to bind the telomerase RNA template, it is also possible that the deletion mutation might disrupt interactions of hTERT with other components of telomerase; telomerase activity is affected by a balance of competing mutant and wild-type enzyme events, and inhibition of wild-type enzyme activity might contribute to a reduction in telomerase activity. Eventually, the proliferation of T24 cells stops at a point termed ‘senescence’. Recent experiments have confirmed that repression of telomerase activity in tumour-derived cell lines results in progressive telomere shortening and reprogramming of cellular senescence [14]. Activation of endogenous senescence signalling pathways in T24 cells might represent an alternative treatment for bladder cancers.

Taken together, our studies on hTERT are important for understanding the cellular factors involved in controlling telomerase activity. A future goal is to assess the effects of targeting hTERT as a new gene therapy against human bladder cancer, with many potential advantages over current approaches [15].

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

This work was supported by grants from China postdoctoral science foundation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES