TELOMERASE EXPRESSION IN BENIGN AND MALIGNANT SKIN NEOPLASMS: COMPARISON OF THREE MAJOR SUBUNITS

Transcription

1 TELOMERASE EXPRESSION IN BENIGN AND MALIGNANT SKIN NEOPLASMS: COMPARISON OF THREE MAJOR SUBUNITS Sindy Hu, Heng-Leong Chan, Min-Chi Chen, 1 and Jong-Hwei S. Pang 2 Abstract: Telomerase is a specialized ribonucleoprotein polymerase that plays an important role in maintaining cellular immortality. The multifactorial activation of telomerase, a critical step in human carcinogenesis, is not fully understood. Many studies have shown increased telomerase activity in various skin cancers. To study the regulation of telomerase in cutaneous malignancy, we used the reverse transcription polymerase chain reaction (RT-PCR) with specific primers to analyze the mrna expression of three major telomerase subunits: human telomerase RNA (htr), telomerase-associated protein (TP1) and human telomerase reverse transcriptase (htert). Clinical skin specimens were taken from 62 Taiwanese patients with a variety of skin diseases, including five from normal skin, 27 from benign tumors, 17 from malignant tumors, six from premalignant tumors, and seven from inflammatory skin diseases. TP1 was detected in 61 of 62 specimens (98.4%) and htr was found in 58 specimens (93.6%). htert was more often found in specimens from malignant and premalignant tumors (73.9%, 17/23) compared to those from benign tumors (7.4%, 2/27; p < ). None of the normal skin specimens showed htert expression. These results indicate that expression of htert, but not TP1 and htr, is highly associated with cutaneous malignancy. (J Formos Med Assoc 2002;101:593 7) Key words: telomerase htert skin diseases It is well established that DNA polymerase cannot fully replicate the linear ends of chromosomal DNA in normal human cells. Telomeres are the chromosomal end regions that consist of a repeating nucleotide sequence (TTAGGG). Possible functions of telomeres include prevention of chromosome degradation, end-to-end fusion, rearrangement and chromosome loss. In each cell division, the incomplete end replication of DNA results in the loss of 50 to 200 telomere nucleotides [1]. This progressive shortening of human telomeres leads to chromosomal instability and eventually ends in cellular senescence [2]. The repression of telomerase activity may be a possible cause of human telomere shortening. Telomerase is a specialized ribonucleoprotein polymerase containing an integral RNA as the template element that directs the synthesis of telomeric repeats at chromosome ends [3]. It ensures chromosomal stability by preserving a constant telomere length and effectively prevents cell aging. It also plays a critical role in maintaining cellular immortality in malignancies [4]. In early experiments, telomerase activity in humans was detected in germ lines and immortal and tumor cells [4]. Normal somatic tissue is usually devoid of detectable telomerase activity. Later experiments also found low levels of telomerase activity in activated lymphocytes, some proliferating normal epithelial cells, and hair bulbs in humans [5, 6]. However, according to a review by Shay and Bacchetti, malignant tumor cells still have the highest expression of this polymerase (> 85%) [7]. Telomerase activation is considered an important step in human carcinogenesis. It may be used as a tumor marker for clinical diagnosis of cancer, as well as a target of anticancer therapy. Three major components of the human telomerase complex have recently been identified. Their function and the regulatory mechanisms by which telomerase is activated have not been fully determined. The RNA component of human telomerase (htr) provides the template for telomere elongation [8]. Disruption of the secondary structure of this functional telomerase RNA may lead to progressive telomere shortening. The gene encoding telomerase-associated Department of Dermatology, Chang Gung Memorial Hospital; and 1 Biostatistics Center and Department of Public Health, 2 Graduate Institute of Clinical Medicine, Chang Gung University, Tao-Yuan. Received: 24 December Revised: 16 January Accepted: 7 May Reprint requests and correspondence to: Dr. Jong-Hwei S. Pang, Graduate Institute of Clinical Medicine, Chang Gung University, Tao-Yuan, Taiwan. J Formos Med Assoc 2002 Vol 101 No 8 593

2 S. Hu, H.L. Chan, M.C. Chen, et al protein (TP1), a mammalian homologue of p80, has been identified, although the actual function of this TP1 remains uncertain [9]. Another human homologue of Est2p and p123, human telomerase reverse transcriptase (htert), has also been cloned [10]. Overexpression of htert has been found in ovarian cancer, cervical cancer and renal cell carcinoma, suggesting that upregulation of htert may play a critical role in development of human malignancy [11 13]. Previous studies have shown enhanced expression of telomerase in malignant skin tumors [14]. Glaessl et al also detected an increase in telomerase activity during the progression of benign melanocytic cells to malignant melanoma [15]. However, the relationship of telomerase activation and the regulation of its subunits in skin cancer is still not fully understood. In the present study, we used the reverse transcription polymerase chain reaction (RT-PCR) to analyze and compare the mrna expression of three telomerase subunits in a variety of skin diseases and cutaneous tumors. 594 Materials and Methods Specimens from a variety of skin tumors and diseases were collected from Taiwanese patients after obtaining informed consent (Table). Each specimen was bisected, with half submitted for routine pathology and half for the isolation of total RNA by lysis in a guanidinium isothiocyanate buffer followed by a one-step phenol-chloroform-isoamyl alcohol extraction (Sigma, St. Louis, MO, USA). Integrity of the RNA preparations was confirmed by routine gel electrophoresis. Total RNA (1 µg) underwent reverse transcription into cdna using M-MLV reverse transcriptase (Gibco BRL, Rockville, MD, USA) followed by PCR amplification using Taq DNA polymerase (Viogene, Taipei, Taiwan) for 30 cycles of denaturation at 94 C for 1 minute, annealing at 55 C for 1 minute, and extension at 72 C for 2 minutes. The primer sequences used for the RT-PCR were as follows: TP1, 5'-TCAAG CCAAA CCTGA ATCTG AG-3' (sense) and 5'-CCCCG AGTGA ATCTT TCTAC GC-3' (antisense), 264 bp; htr, 5'-TCTAA CCCTA ACTGA GAAGG GCGTA G-3' (sense) and 5'-GTTTG CTCTA GAATG AACGG TGGAAG-3' (antisense), 126 bp; htert, 5'-CGGAA GAGTG TCTGG AGCAA- 3' (sense) and 5'-GGATG AAGCG GAGTC TGGA-3' (antisense), 145 bp; and GADPH control, 5'-GAGGG GCCAT CCACA GTCTT-3' (sense) and 5'-TTCAT TGACC TCAAC TACAT-3' (antisense), 469 bp. Data were analyzed using SAS (Statistical Analysis System) version 6.12 (SAS Institute, Durham, NC, USA). A value of p less than 0.05 was considered statistically significant. Chi-square test or Fisher s exact test was used to examine the differences in expression of htr and htert between malignant/ premalignant and benign tumors. Results A total of 62 fresh skin specimens were analyzed for the mrna expression of TP1, htr and htert by RT-PCR (Table). Representative results of the RT-PCR products of these subunits in different samples are shown in the Figure. TP1 was detected in 61 cases (98.4%) and htr in 58 cases (93.6%). Both were expressed in normal skin (5/5). There was no significant difference in the expression of htr between benign (92.6%) and malignant tumors (95.7%; p = 1). htert was observed in more specimens in the malignant and premalignant tumor group (73.9%) than the benign tumor group (7.4%; p < ). Only one specimen from the inflammatory skin disease group and no normal skin specimens expressed htert mrna. Discussion This study demonstrated that the mrna expression of htert was closely related to malignant changes in skin, while TP1 and htr mrna were expressed in both normal and malignant tissues. These results are in agreement with studies of various other cancers including cervical and ovarian cancer, renal cell carcinoma, hepatocellular cancer, and colon and breast carcinoma [10 13, 16]. A similar study by Wu et al in Japan also found that htert expression was strongly correlated with telomerase activation in skin cancers [17]. They analyzed 45 skin tumors and 19 samples of normal skin for the expression of telomerase subunits using RT-PCR and the telomeric repeat amplification protocol assay for telomerase activity, and found a similar rate of htert expression in malignant and premalignant skin tumors (73.7% vs 73.9%). Our study found that six malignant and premalignant cutaneous tumors had no htert expression, including two cases of squamous cell carcinoma (SCC), one of Bowen s disease, two of actinic keratosis, and one of trichilemmal carcinoma. Such discordant results have also been reported in cancers of various organs, which showed a 70 to 80% positive rate for telomerase activity or htert mrna expression. The reason for this discordance remains unclear, but may involve unknown factors substituting for the role of telomerase or htert in the tumorigenesis of skin malignancy [12, 16, 18]. Both our study and that of Wu et al found high expression of TP1 and htr in normal skin (100% and 100%, respectively for TP1; 100% and 94.7% for htr) and skin tumors (98% and 95.6% for TP1; 94% and 93.3% for htr), suggesting that TP1 and htr do not reflect telomerase activity in skin malignancy. In contrast, our study found much lower expression of htert in benign tumors compared to Wu et al (7.4% vs 71.4%). This study analyzed more samples (n = 27) than Wu et al s study, which analyzed only seven benign tumors: five melanocytic nevi, one seborrheic keratosis and one eccrine poroma. The 27 benign skin tumor samples analyzed in this study included intradermal and congenital nevi, seborrheic keratosis, nevus sebaceous, dermatofibroma, keratoacanthoma, acrochordon and lipoma. Among these samples, only one congenital melanocytic nevus and one keratoacanthoma showed expression of htert. Congenital melanocytic nevus carries a 6.3 to 12% risk of developing melanoma [19], while keratoacanthoma is a rapidly growing florid tumor that mimics SCC [20]. Our findings suggest that htert may be responsible for J Formos Med Assoc 2002 Vol 101 No 8

3 Table. Expression of telomerase subunits in specimens of normal skin, cutaneous tumors and various skin diseases Case Age/Sex Site Pathology TP1 htr htert Normal skin 1 22/F Arm (sun-exposed) Normal /M Leg (covered area) Normal /F Trunk (covered area) Normal /M Arm (sun-exposed) Normal /M Sole (covered area) Normal + + Benign tumors 6 28/F Face IDN /F Face IDN /M Neck IDN /M Arm IDN /F Face IDN /M Face IDN /M Trunk IDN /F Face IDN /M Face IDN /F Face IDN /F Forehead IDN /F Arm IDN /F Trunk CMN /M Trunk CMN /F Neck CMN /F Face SK /M Forehead SK /M Face SK /F Face SK /M Scalp NS /M Scalp NS /M Abdomen Lipoma /M Arm Lipoma /M Leg Dermatofibroma /M Leg Dermatofibroma /M Buttock Acrochordon /F Face Keratoacanthoma Malignant tumors 33 63/M Face SCC /F Leg SCC /M Face SCC /F Face SCC /M Face SCC /M Face SCC /F Face SCC /F Face SCC /M Forehead BCC /F Face BCC /M Face BCC /M Face BCC /F Foot MM /M Sole MM /F Thigh MM /F Scalp Trichilemmal carcinoma /M Trunk MF Continued on the next page. J Formos Med Assoc 2002 Vol 101 No 8 595

4 S. Hu, H.L. Chan, M.C. Chen, et al Table. Continued from the previous page. Case Age/Sex Site Pathology TP1 htr htert Premalignant tumors 50 71/F Trunk Bowen s disease /M Leg Bowen s disease /F Trunk Bowen s disease /F Face Actinic keratosis /M Forehead Actinic keratosis /F Face Actinic keratosis + + Skin diseases 56 35/M Leg NV /M Arm DSAP /M Back Ri-Dorf /F Face Pemphigus F /F Foot Granulation T /M Face BLK /F Face BLK + + TP1 = telomerase-associated protein; htr = human telomerase RNA; htert = human telomerase reverse transcriptase; IDN = intradermal melanocytic nevus; CMN = congential melanocytic nevus; SK = seborrheic keratosis; NS = nevus sebaceous; SCC = squamous cell carcinoma; BCC = basal cell carcinoma; MM = malignant melanoma; MF = mycosis fungoides; NV = necrotizing vasculitis; DSAP = disseminated superficial actinic porokeratosis; Ri-Dorf = cutaneous Rosai-Dorfman disease; Pemphigus F = pemphigus foliaceus; Granulation T = granulation tissues; BLK = benign lichenoid keratosis. the malignant transition of the skin in these diseases. Therefore, it is possible that benign tumors that express htert mrna may be associated with a poorer prognosis. Further studies are needed to test this hypothesis. Wu et al s results on the mrna expression of htert in normal skin are slightly different from the findings of this study. They found that 10.5% (2/19) of normal skin specimens GAPDH htert TPI expressed htert mrna, while this study did not detect any expression of htert mrna in normal skin (0/5). This discrepancy may be explained by the different sensitivity of the experimental protocols used in this study or to unknown differences in the nature of skin specimens between Taiwanese and Japanese. Although there was a small discordance with a previous study regarding the findings of htert mrna expression and cutaneous malignancy, most malignant and premalignant tumors showed a higher expression of htert mrna. Since the expression of htert mrna is more specific in malignant skin tumors, exploration of htert-targeting regimens for some life-threatening, unresectable, and metastatic skin cancers is warranted. ACKNOWLEDGMENT: This study was supported by a grant from Chang Gung Memorial Hospital (CMRP929E). We would like to thank Dr. Chien-Hsun Chen for providing fresh tissue samples for this study. htr Figure. Representative results of reverse transcription polymerase chain reaction (RT-PCR) analysis of three telomerase subunits (mrna of human telomerase RNA, htr, telomerase-associated protein, TP1, and human telomerase reverse transcriptase, htert) in different skin samples. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internal control. Lanes 1 and 2: normal skin samples; Lane 3: pemphigus foliaceus; Lane 4: granulation tissue; Lane 5: intradermal melanocytic nevus; Lane 6: nevus sebaceous; Lane 7: basal cell carcinoma; Lane 8: squamous cell carcinoma. Products of both TP1 and htr can be seen in all lanes while htert was only expressed in malignant tumors (Lanes 7 and 8). 596 References 1. Allsopp RC, Chang E, Kashhefi-Aazam M, et al: Telomere shortening is associated with cell division in vitro and in vivo. Exp Cell Res 1995;220: Shay JW: Aging and cancer: are telomeres and telomerase the connection? Mol Med Today 1995;1: Yu GL, Bradley JD, Attardi LD, et al: In vivo alteration of telomerase sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature 1990;344: J Formos Med Assoc 2002 Vol 101 No 8

5 4. Kim NW, Piatyszek MA, Prowse KR, et al: Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266: Broccoli D, Young JW, DeLange T: Telomerase activity in normal and malignant hematopoietic cells. Proc Natl Acad Sci USA 1995;92: Shigeru Y, Chuji K, Keiji K, et al: Telomerase activity in normal human epithelial cells. Oncogene 1996;13: Shay JW, Bacchetti S: A survey of telomerase activity in human cancer. Eur J Cancer 1997;33: Feng J, Funk WD, Wang SS, et al: The RNA component of human telomerase. Science 1995;269: Harrington L, McPhail T, Mar V, et al: A mammalian telomeraseassociated protein. Science 1997;275: Meyerson M, Counter CM, Eaton EN, et al: hest2, the putative human telomerase catalytic subunit gene, is upregulated in tumor cells and during immortalization. Cell 1997;90: Kyo S, Kanaya T, Takakura M, et al: Expression of human telomerase subunits in ovarian malignant, borderline and benign tumors. Int J Cancer 1999;80: Takakura M, Kyo S, Kanaya T, et al: Expression of human telomerase subunits and correlation with telomerase activity in cervical cancer. Cancer Res 1998;58: Kanaya T, Kyo S, Takakura M, et al: htert is a critical determinant of telomerase activity in renal cell carcinoma. Int J Cancer 1998;78: Taylor RS, Ramirez RD, Ogoshi M, et al: Detection of telomerase activity in malignant and nonmalignant skin conditions. J Invest Dermatol 1996;106: Glaessl A, Bosserhoff AK, Buettner R, et al: Increase in telomerase activity during progression of melanocytic cells from melanocytic navei to malignant melanomas. Arch Dermatol Res 1999;291: Nakayama J, Tahara H, Tahara E, et al: Telomerase activation by htert in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 1998;18: Wu 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: Ito H, Kyo S, Kanaya T, et al: Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer. Clin Cancer Res 1998;4: Kopf AW, Bart RS, Hennessey P: Congenital nevocytic nevi and malignant melanomas. J Am Acad Dermatol 1979;1: Chalet MD, Connors RC, Ackerman AB: Squmaous cell carcinoma vs keratoacanthoma: criteria for histologic differentiation. J Dermatol Surg 1975;1:16 7. J Formos Med Assoc 2002 Vol 101 No 8 597