Possible Regulation of Telomerase Activity by Transcription and Alternative Splicing of Telomerase Reverse Transcriptase in Human Melanoma
2001; Elsevier BV; Volume: 116; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2001.01343.x
ISSN1523-1747
AutoresRaffaella Villa, Chiara Della Porta, Marco Folini, Maria Grazia Daidone, Nadia Zaffaroni,
Tópico(s)Immunotherapy and Immune Responses
ResumoTo investigate the regulatory mechanisms of telomerase activity in human melanoma cells, we assessed the enzyme's catalytic activity and the expression of the telomerase subunits, the human telomerase RNA, the human telomerase-associated protein, and the human telomerase reverse transcriptase, in 52 melanoma lesions. Eight normal skin specimens were also studied. Telomerase activity was detected in 84.6% of melanomas, whereas all skin specimens were telomerase negative. Human telomerase-associated protein mRNA and human telomerase RNA were constitutively expressed in all melanoma and skin specimens. Although at a variable level of expression, human telomerase reverse transcriptase mRNA was detected in all but one melanomas, whereas it was never present in skin samples. Reverse transcriptase–polymerase chain reaction experiments were performed using primers within the reverse transcriptase domain of human telomerase reverse transcriptase and revealed the presence of multiple alternatively spliced transcripts in melanoma specimens. Among the 44 telomerase-positive melanomas, one showed the full-length transcript alone whereas in all other specimens a full-length message was present with different combinations of alternatively spliced variants. In these tumors the expression of the full-length transcript was generally equal to or higher than that of the alternatively spliced variants. The ratio full-length transcript to alternatively spliced species ranged from 0.6 to 5.26, with a median value of 1.18. Among the seven telomerase-negative melanomas, one displayed the β deletion transcript alone, whereas in the remaining six tumors weak expression of the full-length transcript and a more abundant level of alternatively spliced transcripts were found. In these cases human telomerase reverse transcriptase ratio ranged from 0.09 to 1.1, with a median value of 0.40. The results suggest that transcription and alternative splicing of human telomerase reverse transcriptase are regulatory mechanisms controlling telomerase activity in melanoma. To investigate the regulatory mechanisms of telomerase activity in human melanoma cells, we assessed the enzyme's catalytic activity and the expression of the telomerase subunits, the human telomerase RNA, the human telomerase-associated protein, and the human telomerase reverse transcriptase, in 52 melanoma lesions. Eight normal skin specimens were also studied. Telomerase activity was detected in 84.6% of melanomas, whereas all skin specimens were telomerase negative. Human telomerase-associated protein mRNA and human telomerase RNA were constitutively expressed in all melanoma and skin specimens. Although at a variable level of expression, human telomerase reverse transcriptase mRNA was detected in all but one melanomas, whereas it was never present in skin samples. Reverse transcriptase–polymerase chain reaction experiments were performed using primers within the reverse transcriptase domain of human telomerase reverse transcriptase and revealed the presence of multiple alternatively spliced transcripts in melanoma specimens. Among the 44 telomerase-positive melanomas, one showed the full-length transcript alone whereas in all other specimens a full-length message was present with different combinations of alternatively spliced variants. In these tumors the expression of the full-length transcript was generally equal to or higher than that of the alternatively spliced variants. The ratio full-length transcript to alternatively spliced species ranged from 0.6 to 5.26, with a median value of 1.18. Among the seven telomerase-negative melanomas, one displayed the β deletion transcript alone, whereas in the remaining six tumors weak expression of the full-length transcript and a more abundant level of alternatively spliced transcripts were found. In these cases human telomerase reverse transcriptase ratio ranged from 0.09 to 1.1, with a median value of 0.40. The results suggest that transcription and alternative splicing of human telomerase reverse transcriptase are regulatory mechanisms controlling telomerase activity in melanoma. telomeric repeat amplification protocol human telomerase RNA human telomerase reverse transcriptase human telomerase-associated protein Human telomeres are specialized structures consisting of tandem repeats of the hexanucleotide sequence 5′-TTAGGG-3′ and associated proteins, which cap both ends of chromosomes and protect them from degradation and end-to-end fusion (Blackburn, 1991Blackburn E.H. Structure and function of telomeres.Nature. 1991; 350: 569-573Crossref PubMed Scopus (2906) Google Scholar). Telomeres shorten with each round of DNA replication as a consequence of the inability of conventional DNA polymerases to copy the extreme ends of linear chromosomes (Watson, 1972Watson J. Origin of concatemeric T7 DNA.Nat Biol. 1972; 239: 197-201Crossref Scopus (1298) Google Scholar). In the absence of a compensatory mechanism, telomere shortening results in chromosomal instability, which leads to replicative cellular senescence (Harley, 1991Harley C.B. Telomere loss: mitotic clock or genetic time bomb?.Mutat Res. 1991; 256: 271-282Crossref PubMed Scopus (1062) Google Scholar;Allsopp et al., 1992Allsopp R.C. Vaziri H. Patterson C. et al.Telomere length predicts replicative capacity of human fibroblasts.Proc Natl Acad Sci USA. 1992; 89: 10114-10118Crossref PubMed Scopus (1884) Google Scholar). Telomerase provides the enzymatic activity that compensates for telomere shortening in germ-line cells and cancer cells (Counter et al., 1992Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1880) Google Scholar;Kim et al., 1994Kim N.W. Piatyszek M.A. Prowse K.R. et al.Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6305) Google Scholar;Greider, 1998Greider C.W. Telomerase activity, cell proliferation, and cancer.Proc Natl Acad Sci USA. 1998; 95: 90-92Crossref PubMed Scopus (352) Google Scholar). The almost ubiquitous expression of telomerase in human tumors and not in somatic cells supports the hypothesis that the enzyme is involved in cellular immortality and carcinogenesis (Rhyu, 1995Rhyu M.S. Telomeres, telomerase, and immortality.J Natl Cancer Inst. 1995; 87: 884-894Crossref PubMed Scopus (412) Google Scholar). Moreover,Hahn et al., 1999Hahn W.C. Counter C.M. Lundberg A.S. Beijesbergen R.L. Brooks M.W. Weinberg R.A. Creation of human tumour cells with defined genetic elements.Nature. 1999; 400: 464-468https://doi.org/10.1038/22780Crossref PubMed Scopus (1911) Google Scholar have recently demonstrated that ectopic expression of the catalytic telomerase subunit in combination with two oncogenes (SV 40 large-T and H-ras) resulted in direct tumorigenic conversion of normal human epithelial and fibroblast cells. Human telomerase is a large ribonucleoprotein complex composed of at least three components: the RNA subunit hTR, which contains the template sequence coding for telomere repeats (Feng et al., 1995Feng J. Funk W.D. Wang S.S. et al.The RNA component of human telomerase.Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2017) Google Scholar); the catalytic subunit hTERT, which possesses conserved reverse transcriptase motifs (Harrington et al., 1997aHarrington L. McPhail T. Mar V. et al.A mammalian telomerase-associated protein.Science. 1997; 275: 973-977https://doi.org/10.1126/science.275.5302.973Crossref PubMed Scopus (618) Google Scholar;Meyerson et al., 1997Meyerson M. Counter C.M. Eaton E.N. et al.hEst2 the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization.Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1623) Google Scholar;Nakamura et al., 1997Nakamura T.M. Morin G.B. Chapman K.B. et al.Telomerase catalytic subunit homologs from fission yeast and human.Science. 1997; 277: 955-959https://doi.org/10.1126/science.277.5328.955Crossref PubMed Scopus (2001) Google Scholar); and the telomerase-associated protein hTEP1, which may play a part in co-ordinating the telomerase holoenzyme structure and/or recruiting telomerase regulatory factors (Harrington et al., 1997bHarrington L. Zhou W. McPhail T. et al.Human telomerase contains evolutionarily conserved catalytic and structural subunits.Genes Dev. 1997; 11: 3109-3115Crossref PubMed Scopus (400) Google Scholar). Whereas hTR and hTEP1 are widely expressed in normal cells (Feng et al., 1995Feng J. Funk W.D. Wang S.S. et al.The RNA component of human telomerase.Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2017) Google Scholar;Harrington et al., 1997aHarrington L. McPhail T. Mar V. et al.A mammalian telomerase-associated protein.Science. 1997; 275: 973-977https://doi.org/10.1126/science.275.5302.973Crossref PubMed Scopus (618) Google Scholar), the expression of hTERT is repressed in most human somatic tissues (Nakamura et al., 1997Nakamura T.M. Morin G.B. Chapman K.B. et al.Telomerase catalytic subunit homologs from fission yeast and human.Science. 1997; 277: 955-959https://doi.org/10.1126/science.277.5328.955Crossref PubMed Scopus (2001) Google Scholar) and becomes reactivated in the great majority of human tumors and immortal cell lines (Meyerson et al., 1997Meyerson M. Counter C.M. Eaton E.N. et al.hEst2 the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization.Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1623) Google Scholar;Kyo et al., 1999Kyo S. Kanaya T. Takakura M. Inoue M. Human telomerase reverse transcriptase as a critical determinant of telomerase activity in normal and malignant endometrial tissues.Int J Cancer. 1999; 80: 60-63Crossref PubMed Google Scholar). Moreover, a strong correlation has been observed between telomerase activity and hTERT mRNA expression in many immortalized and tumor cell models (Harrington et al., 1997aHarrington L. McPhail T. Mar V. et al.A mammalian telomerase-associated protein.Science. 1997; 275: 973-977https://doi.org/10.1126/science.275.5302.973Crossref PubMed Scopus (618) Google Scholar;Meyerson et al., 1997Meyerson M. Counter C.M. Eaton E.N. et al.hEst2 the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization.Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1623) Google Scholar;Nakamura et al., 1997Nakamura T.M. Morin G.B. Chapman K.B. et al.Telomerase catalytic subunit homologs from fission yeast and human.Science. 1997; 277: 955-959https://doi.org/10.1126/science.277.5328.955Crossref PubMed Scopus (2001) Google Scholar), which would suggest that expression of hTERT is the rate-limiting step in telomerase activation. The potential mechanisms of regulation of telomerase activity have been addressed in many recent studies. Specifically, it has been demonstrated that the hTERT promoter contains binding sites for several transcription factors (Cong et al., 1999Cong Y.S. Wen J. Bacchetti S. The human telomerase catalytic subunit h TERT: organization of the gene and characterization of the promoter.Hum Mol Genet. 1999; 8: 137-142https://doi.org/10.1093/hmg/8.1.137Crossref PubMed Scopus (405) Google Scholar;Wick et al., 1999Wick M. Zubov D. Hagen G. Genomic organization and promoter characterisation of the gene encoding the human telomerase reverse transcriptase (hTERT).Gene. 1999; 232: 97-106https://doi.org/10.1016/s0378-1119(99)00108-0Crossref PubMed Scopus (0) Google Scholar;Wu et al., 1999Wu A. Ichihashi M. Ueda M. Correlation of the expression of human telomerase subunits with telomerase activity in normal skin and skin tumors.Cancer. 1999; 86: 2038-2044Crossref PubMed Scopus (89) Google Scholar), suggesting that hTERT expression may be subject to multiple levels of control and regulated by different factors in different cellular contexts. Moreover, it has been proposed that, besides gene transcription, alternative splicing of hTERT transcripts can also regulate telomerase activity (Ulaner et al., 1998Ulaner G.A. Hu J.F. Vu T.H. Giudice L.C. Hoffman Ar Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.Cancer Res. 1998; 58: 4168-4172PubMed Google Scholar). The function of the alternatively spliced variants is currently unknown; however, as several of the splice sites remove reverse transcriptase motifs (Figure 1), these transcripts probably do not code for functional reverse transcriptase. Suppression of telomerase activity during fetal kidney development is associated with this mechanism (Ulaner et al., 1998Ulaner G.A. Hu J.F. Vu T.H. Giudice L.C. Hoffman Ar Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.Cancer Res. 1998; 58: 4168-4172PubMed Google Scholar). It has recently been demonstrated that such a mechanism for regulating telomerase activity is also operative in normal and neoplastic ovarian and uterine tissues (Ulaner et al., 2000Ulaner G.A. Hu J.F. Vu T.H. Oruganti H. Giudice L.C. Hoffman A.R. Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium.Int J Cancer. 2000; 85: 330-335Crossref PubMed Scopus (173) Google Scholar). Protein phosphorylation also seems to be an important post-translational mechanism controlling the structure and function of telomerase (Li et al., 1997Li H. Zhao L.L. Funder J.W. Liu J.P. Protein phosphatase 2A inhibits nuclear telomerase activity in human breast cancer cells.J Biol Chem. 1997; 272: 16729-16732Crossref PubMed Scopus (188) Google Scholar;Li et al., 1998Li H. Zao L. Yang Z. Funder J.W. Liu J.P. Telomerase is controlled by protein kinase C alpha in human breast cancer cells.J Biol Chem. 1998; 273: 33436-33442Crossref PubMed Scopus (188) Google Scholar;Kang et al., 1999Kang S.S. Kwon T. Kwon D.Y. Do S.I. Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit.J Biol Chem. 1999; 274: 13085-13090Crossref PubMed Scopus (421) Google Scholar). Moreover, proteins associated with telomeres, such as TRF1 (van Steensel and de Lange, 1997van Steensel B. de Lange T. Control of telomere length by the human telomeric protein TRF1.Nature. 1997; 385: 740-743Crossref PubMed Scopus (1024) Google Scholar) and the poly(adenosine diphosphate-ribose) polymerase tankyrase (Smith et al., 1998Smith S. Giriat I. Schmitt A. de Lange T. Tankyrase, a poly(ADP-ribose) polymerase at human telomeres.Science. 1998; 282: 1484-1487Crossref PubMed Scopus (863) Google Scholar), are thought to play a crucial part in the control of telomerase activity. This study aimed to investigate the possible mechanisms of regulation of telomerase activity in human melanoma tissues by correlating the catalytic activity of the enzyme with the expression of the three telomerase subunits, hTERT, hTR, and hTEP1, as well as with the presence of alternatively spliced hTERT variants. Tumor lesions (36 lymph node metastases and 16 cutaneous/subcutaneous metastases) were obtained from 52 patients with cutaneous melanoma at stages III (43 cases) and IV (nine cases). For four patients, two synchronous metastatic lesions were tested. Forty patients underwent surgery alone, four patients had also been previously treated with immunotherapy (interferon-α or interleukin-12) and eight patients with chemotherapy (dacarbazine). Eight normal skin specimens were also obtained from the abdomen or breast of patients who underwent surgery for aesthetic purposes. Tissue were obtained at the time of surgery and then flash-frozen in liquid nitrogen. All tissues underwent histologic examination and were stored at -80°C until use. In particular, to confirm the representativeness of the tumor tissue sampled for molecular analyses, quality control was performed on frozen sections, stained with hematoxylin–eosin, before performing telomerase analysis. For each case, telomerase activity and telomerase component RNA expression were determined in two contiguous pieces of the same tumor lesion. The human melanoma cell line JR8 (Zupi et al., 1985Zupi G. Mauro F. Balduzzi M.A. Pardini C. Cavaliere R. Greco C. Established melanoma cell lines from different metastatic nodules of a single patient. A useful model for cancer therapy.Proc Am Assoc Cancer Res. 1985; 26: 22Google Scholar) and the immortalized fibroblast cell line GM847 (Perrem et al., 1999Perrem K. Bryan T.M. Englezou A. Hackl T. Moy E.L. Reddel R.R. Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids.Oncogene. 1999; 18: 3383-3390Crossref PubMed Scopus (95) Google Scholar) were used as positive and negative controls for telomerase activity and hTERT gene expression, respectively. Cell extracts were obtained as previously described (Villa et al., 1998Villa R. Zaffaroni N. Folini M. Martelli G. De Palo G. Daidone M.G. Silvestrini R. Telomerase activity in benign and malignant breast lesions: a pilot prospective study on fine-needle aspirates.J Natl Cancer Inst. 1998; 90: 537-539https://doi.org/10.1093/jnci/90.7.537Crossref PubMed Scopus (27) Google Scholar). Telomerase activity was measured by the polymerase chain reaction (PCR) -based telomeric-repeat amplification protocol (TRAP) with some modifications (Kim et al., 1994Kim N.W. Piatyszek M.A. Prowse K.R. et al.Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6305) Google Scholar). Samples containing 0.06, 0.6, and 6 µg of protein were analyzed in the TRAP reaction for each tissue specimen by the TRAPeze kit (Intergen, Oxford, U.K.) according to the manufacturer's protocol. After extension of the substrate TS (5′-AATCCGTCGAGCAGAGTT-3′) oligonucleotide by telomerase, the telomerase products were amplified by PCR in the presence of 5′-[32P]-end-labeled TS primer for 30 cycles and resolved in 10% polyacrylamide gels. Each reaction product was amplified in the presence of a 36 bp internal TRAP assay standard, and each sample extract was tested for RNase sensitivity. A TSR8 quantitation standard (which serves as a standard to estimate the amount of product extended by telomerase in a given extract) was included for each set of TRAP assays. Quantitative analysis was performed with the Image-QuanT software (Molecular Dynamics, Sunnyvale, CA), which allowed densitometric evaluation of the digitized image. Telomerase activity was quantified by measuring the signal of telomerase ladder bands and calculated as the ratio to the internal standard using the following formula:relativetelomeraseactivity:((X−X0)/C)×((R−R0)/Cr)−1(1) where X is the untreated sample, X0 is the RNase-treated sample, C is the internal control of untreated samples, Cr is the internal control of TSR8, R is the TSR8 quantitation control, and R0 is the negative control. Telomerase activity obtained with 1.0 µg of protein extract from the JR8 melanoma cell line was arbitrarily defined as 1.00 unit. The relative telomerase activity of each tissue sample represents the ratio between the arbitrary unit of the tested sample and that of JR8 cells. Total cellular RNA was extracted from frozen samples with the TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Total RNA (0.5 µg) from each sample was used for c-DNA production using the reverse transcriptase–PCR Core kit (Perkin Elmer, Branchburg, NJ) with random hexamers. The cDNA samples were then amplified with the same kit. Specifically, hTR cDNA was amplified using TR-46S (5′-CTAACCCTAACTGAGAAGGGCGTAG-3′) and TR-148AS (5′-GAAGGCGGCAGGCCGAGGCTTTTCC-3′) oligonucleotides with an initial heating at 95°C for 3 min, followed by 28 cycles of 95°C for 30 s, 65°C for 45 s, 72°C for 30 s, and 72° for 5 min. Primers 5899S and 5900AS (Ulaner et al., 1998Ulaner G.A. Hu J.F. Vu T.H. Giudice L.C. Hoffman Ar Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.Cancer Res. 1998; 58: 4168-4172PubMed Google Scholar) were added at 72°C of cycle 7 for the β-actin internal control. TEP1 cDNA was amplified using TEP1-S (5′-TCAAGCCAAACCTGAATC TGAG-3′) and TEP1-AS (5′-CCCCGAGTGAATCTTTCTACGC-3′) oligonucleotides (Ramakrishnan et al., 1998Ramakrishnan S. Eppenberger U. Mueller H. Shinkai Y. Narayanan R. Expression profile of the putative catalytic subunit of the telomerase gene.Cancer Res. 1998; 58: 622-625PubMed Google Scholar) with an initial heating at 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 62°C for 45 s, 72°C for 30 s, and 72°C for 5 min. Primers 774 A and 775AS (Ulaner et al., 1998Ulaner G.A. Hu J.F. Vu T.H. Giudice L.C. Hoffman Ar Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.Cancer Res. 1998; 58: 4168-4172PubMed Google Scholar) were added at 72°C of cycle 10 for the β-actin internal control. The first hTERT cDNA amplification used TERT-1784S (5′-CGGAAGAGTGTCTGGAGCAA-3′) and TERT-1929AS (5′-GGA TGAAGCGGAGTCTGGA-3′) oligonucleotides with an initial heating at 95°C for 3 min, followed by 33 cycles of 95°C for 30 s, 65°C for 45 s, 72°C for 30 s, and 72°C for 5 min. Primers 774S and 775AS were added at 72°C of cycle 13 for internal control. The PCR products were electrophoresed on a 3% agarose gel and visualized with ethidium bromide. Amplification of alternatively spliced hTERT cDNA (Figure 1) was obtained using TERT-2164S (5′-GCCTGAGCTGTACTTTGT CAA-3′) and TERT-2620AS (5′-CGCAAACAGCTTGTTCTCCAT GTC-3′) oligonucleotides with initial heating at 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 62°C for 50 s, 72°C for 50 s, and 72°C for 5 min. The amplification was performed in a mixture containing 0.3 µCi of [α-32P]deoxycytidine triphosphate (300 Ci per mmol; Amersham Pharmacia Biotech, Cologno Monzese, Milan, Italy). Primers 774S and 775AS were added at 72°C of cycle 25 for internal control. Reverse transcriptase–PCR experiments were performed in duplicate. Amplified products were electrophoresed on 5% non denaturing polyacrylamide gel in 1 × Tris–borate ethylenediamine tetraacetic acid buffer (90 mM Tris–borate, pH 8.3, 2 mM ethylene diamine tetraacetic acid). The gel was dried and autoradiographed. All amplified products were analyzed by a ScanJET IIcx/T scanner (Hewlett Packard, Milan, Italy) and quantified by ImageQuant software. To evaluate the relative levels of expression of the target genes, the value of the internal standard (β-actin) in each test-tube was used as the baseline gene expression in that samples and the relative value was calculated for each of the target genes (densitometric value of the target gene/densitometric value of β-actin). The values were then used to compare expression across tested samples. The hTERT ratio (calculated by dividing the expression value of the full-length transcript signal by that of the alternatively spliced species) was also determined for each tissue sample. Small fragments of fresh tumor material were incubated with 3H-thymidine, fixed for 6–10 h in buffered formalin, and processed by autoradiography as previously described (Costa et al., 1990Costa A. Silvestrini R. Mezzanotte G. Vaglini M. Grignolio E. Clemente C. Cascinelli N. Cell kinetics: an independent prognostic variable in stage II melanoma of the skin.Br J Cancer. 1990; 62: 826-829Crossref PubMed Scopus (8) Google Scholar). TLI was assessed by scoring a total of 1000–3000 tumor cells and defined as the percentage ratio between labeled tumor cells and total number of tumor cells. Spearman's regression analysis was used to evaluate the relationship between telomerase activity and hTERT mRNA expression in individual melanoma lesions. Wilcoxon's rank sum test was used to compare the hTERT ratios in the two subsets of telomerase-positive and telomerase-negative tumors. Telomerase activity and expression of the hTR, hTEP1, and hTERT telomerase subunits were determined in a series of 52 human metastatic malignant melanomas (36 lymph node metastases and 16 cutaneous/subcutaneous metastases) and eight normal skin samples. Telomerase activity was measured by the TRAP assay, and the reliability of the assay was investigated by testing three different protein concentrations (0.06, 0.6, and 6 µg) for each tissue sample (Figure 2). We failed to evidence any telomerase activity at any protein concentration in eight of 52 (15.4%) melanomas, whereas the other 44 cases (84.6%) displayed a positive TRAP signal of at least one protein concentration. Specifically, 10 tumors (19.2%) showed positivity only at the highest protein concentration, 14 tumors (26.9%) gave a positive TRAP signal when 0.6 and 6 µg of protein were used, and the remaining 20 tumors (39.4%) were positive at all protein concentrations. In no case was a tumor that showed telomerase activity at the protein concentration of 0.6 µg negative at the highest protein concentration. The finding would exclude the possibility of false-negative results. Moreover, results obtained in individual specimens showed that the extent of the TRAP signal was highly dependent on protein concentration. As a consequence, TRAP results obtained with 6 µg of protein were considered as the best indicator of enzyme activity, and the concentration used to compare telomerase activity across samples. For this purpose, data were expressed as relative values with respect to the activity (arbitrarily defined as 1.00 unit) observed in the JR8 human melanoma cell line, which was used as an external control throughout the study. Results revealed the presence of a variable degree of telomerase catalytic activity, ranging from 0.18 to 1.42 units (with standard deviations, calculated on three replicates of each samples, ranging from 1.0 to 34% of the mean values), in the 44 telomerase-positive melanomas. Conversely, no telomerase activity was found in any of the normal skin samples at any protein concentration tested. No significant differences in the frequency of telomerase-positive lesions or in the level of telomerase activity was observed between the two types of metastasis. Specifically, 30 of 36 (83%) lymph node metastases and 14 of 16 (87.5%) cutaneous metastases were positive for telomerase activity and displayed a median enzyme catalytic activity of 0.48 and 0.45 unit, respectively. Moreover, no difference in the rate of melanoma cell proliferation, determined as TLI, was found as a function of telomerase activity. In fact, the median TLI values were 6.86% and 7.34% in telomerase- positive and telomerase-negative melanomas, respectively. No association was observed when TLI values and the levels of telomerase catalytic activity were matched in individual tumors. The optimal conditions for the detection of hTR and hTEP1 expression by reverse transcriptase–PCR, in terms of PCR cycles and starting RNA amount, were first optimized by using the JR8 human melanoma cell line (data not shown). Reverse transcriptase–PCR experiments carried out on clinical specimens indicated that all melanoma and normal skin samples constitutively expressed hTR and hTEP1 mRNA (Figure 3b). As regards hTERT, the mRNA was initially measured with primers for a region of the transcript upstream from the reverse transcriptase domain (hTERT-1784S and TERT-1929AS) (Figure 1). Also for this gene, the optimal conditions for reverse transcriptase–PCR experiments were defined on JR8 cells; however, as hTERT is generally expressed at very low levels in human tumors, the adequacy of the amount of starting RNA was verified in a subset of 10 melanomas. A progressive increase in the quantity of PCR products by increasing the amount of starting RNA was observed in the range of 0.05–0.8 µg when the target (hTERT) and the reference (β-actin) genes were coamplified in the same tube (Figure 4). Only one of 52 melanomas did not express hTERT mRNA (Figure 3b, sample 5), and there was no telomerase activity in this sample (Figure 3a, sample 5). All the other melanoma samples expressed hTERT mRNA albeit to a variable extent, ranging from 0.10 to 1.20 arbitrary densitometric units relative to the β-actin gene, when evaluated using a starting RNA amount of 0.5 µg. Moreover, a trend towards an association between the level of hTERT mRNA expression and that of telomerase catalytic activity (rs = 0.34; p = 0.08) was evidenced in the 44 telomerase-positive hTERT-expressing melanomas. The association reached statistical significance (rs = 0.49; p = 0.010) in the subset of lymph node metastases. Seven of eight melanomas that were negative for telomerase activity expressed hTERT mRNA (Figure 3b, representative samples 1, 3, and 9), whereas no hTERT mRNA signal was observed in normal skin samples (Figure 3b, representative samples 13–16). No association was observed between proliferative activity, in terms of TLI, and hTERT mRNA levels in individual melanomas.Figure 4Quantification of hTERT gene expression by reverse transcriptase–PCR in human melanomas. (A) Densitometric quantification of PCR products for target (hTERT) and reference (β-actin) genes as a function of the amount of starting RNA in a representative series of melanomas. Data represent mean ± SD obtained from three replicates. (B) Examples of coamplification of hTERT and β-actin genes in melanoma samples. The β-actin primers were added at the 13th cycle.View Large
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