Relative Contribution of Normal and Neoplastic Cells Determines Telomerase Activity and Telomere Length in Primary Cancers of the Prostate, Colon, and Sarcoma1

Telomerase and telomere length are increasingly investigated as potential diagnostic and prognostic markers in human tumors. Among other factors, telomerase and telomere length may be influenced by the degree of tumor cell content in tumor specimens. We studied telomerase activity and telomere length with concomitant integration of histopathological data to determine whether both were influenced by the amount of tumor cells. We measured telomerase in 153 specimens: in 51 solid tumor blocks; in 51 cryostat sections; and in 51 adjacent normal tissues from patients with sarcoma (n 10) and colorectal (n 11) and prostate cancer (n = 30) using the sensitive and rapid detection telomeric repeat amplification protocol assay. Telomere length was determined by telomere restriction fragment Southern blot analysis. From cryostat sections, tumor cell infiltration was assessed. Telomerase activity was detected in all colorectal tumors and sarcomas, as expected. In primary prostate cancer, however, telomerase activity was less frequently observed (14 of 30, 47%). Moreover, a decreased intensity compared to colon cancer and sarcoma was evident (P < 0.001). The median tumor cell infiltration was signifiReceived 3/12/97; revised 6/1 1/97; accepted 7/2/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by NIH Grant U9 CA 67842-01 (to M. A. S. M.), grants from the Gar Rcichman Fund of the Cancer Research Institute (to M. A. S. M.), Byrne Foundation (to M. A. S. M.), Martin Himmel Fund (to H. I. S. and M. A. S. M.), and CaPCurc Foundation (to H. I. S.), Grant 96/5706 from the Fondo de lnvcstigacion Sanitaria (to J. A.), and Grant 95/319/1-I from the Deutsche Forschungsgcmcinschaft (to M. E.). 2 To whom requests for reprints should be addressed. at Laboratory of Developmental Hcmatopoicsis, Memorial Sloan-Kettering Cancer Centen, RRL 717-C, 1275 York Avenue, Mailbox 101, New York. NY 10021 . Phone: (212) 639-8171; Fax: (212) 717-3618: E-mail: m-moore@ ski.mskcc.org. cantly higher in sarcoma (65%) and colon (30%) compared to prostate cancer (5%; P < 0.001). There was a positive correlation between tumor cell infiltration and telomerase activity (r = 0.89; P < 0.001). Telomere restriction fragments in tumors were shorter compared to the normal tissues with peak differences in colon, sarcoma, and prostate of 1.8, 2.8, and 1 kilobase pairs, respectively (P < 0.002). Our data suggest the presence of a positive correlation between the degree of tumor cell content in human solid tumors and the level of telomerase activity detected. We demonstrated that the amount of tumor cells also affects telomere restriction fragment analysis. Therefore, with the predominance of normal cells in tumor specimens, telomerase activity measured may not reflect the malignant phenotype, and telomere loss may be underestimated. This phenomenon was most evident in prostate cancer. Our results will have implications for the future when telomerase activity and telomere lengths may be used for early screening, diagnosis, and prognosis determinations and when telomerase inhibitors are applied to clinical practice. INTRODUCTION Telomeres are specialized chromosomal end structures composed of TT’AGGG repeats in humans. They function to protect chromosomes from degradation, fusion, and recombination events (1). In the absence of compensatory mechanisms, telomeres shorten with each cell division because the termini of linear molecules are not replicated by conventional DNA polymerases (2, 3). To date, telomere shortening has been observed in most dividing somatic cells. This suggests that when a critical telomere length is reached, cell senescence occurs (4, 5). Telomerase has been identified as a ribonucleoprotein enzyme that can compensate for telomene loss by adding TTAGGG nucleotide repeats onto chromosomal ends ( 1-4). Low levels of telomenase activity have recently been detected in human primitive hematopoietic cells (5-7). However, in most other normal somatic cells, telomerase has not been detected, and consequently telomere shortening occurs after a limited number of population doublings (8-10). In contrast, in germ cell tissue, spontaneously immortalized tumor cell lines, and the majority of tumors, high telomenase activity, stable telomere length, and unlimited proliferative potential have been observed (4, 1 1-26). Previous studies have suggested an association of tumor cell infiltration and telomerase activity ( 1 8, 2 1 , 27). but to date no study has conclusively correlated the effect of tumor infiltration on telomenase level and telomere length. In this study. we present a detailed analysis of three different types of solid tumors in an attempt to clarify this phenomenon. We selected sarcoma and colon cancer, both considered to be aggressively proliferating tumors, and prostate cancer, which in comparison to the former two is thought to be more indolent. Our results Research. on July 14, 2017. © 1997 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 1850 Telomerase and Tclomeres in Solid Tumors demonstrate the value of the careful integration of pathological evaluation when interpreting telomerase activity and telomere length measurements. MATERIALS AND METHODS Tissue Samples. Frozen tissues from S 1 solid tumors and 5 1 normal adjacent tissues from patients with sarcoma (ti 10) and colon (ii 1 1 ) and prostate cancer (n = 30) were retrieved from our tissue bank. All cases were reviewed by pathologists. Fifteen prostate tumors were staged T2, 12 were T3, and 3 were 1’4. Twenty-four prostate cancer specimens were moderately differentiated, 4 were poorly differentiated, and 2 were welldifferentiated. Fourteen prostate cancer patients had tumors with Gleason grade 5/6 compared to 16 with Gleason grade 7/8 (n = 12) and 9/10 (n 4). In colon, two specimens were staged T1, three were T,, four were T3, and two were T4. Four colon specimens were moderate, five were poor, and two were well differentiated. Nine high-grade sarcomas (three synovial sarcomas, two fibrosarcomas, two leiomyosarcomas, one liposarcoma, and one neurofibrosarcoma) and one low-grade sarcoma (fibrosarcoma) were evaluated. From each tumor sample, onehalf was used immediately for protein and DNA preparation (solid tumor block preparation); the other half was embedded for cryostat sections using tissue Tek II OCT (Mios Ink; Tissue Tek Diagnostios, Mishawaka, IN). The embedded tissue was completely frozen in liquid nitrogen, and successive 5-, 60and 5-jim cryostat sections were obtained in a “sandwich technique.” Top and bottom 5i.m sections were mounted on glass slides and were stained with H&E for light microscopy interpretation of tumor cell infiltration. The quantitation of the tumor cell infiltration was assessed by visual estimation from both top and bottom 5-jim sections by a pathologist in a blind fashion. The middle 60.tm section was assayed for telomerase. TRAP Assay. Cells from 153 specimens: from 51 solid tumor blocks; from 5 1 cryostat sections; and from 5 1 normal adjacent tissues from sarcoma and colorectal and prostate cancer patients were processed as described previously (4, 28). Tissue samples were thinly sliced, homogenized with ice-cold lysis buffer ( 106 cells/lOO p.!): 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-l-propane-sulfonate, 10 msi Tns-HC1 (pH 7.5), 1 mrsi MgC12. I mM EGTA, 10% glycerol, S msi 3-mercaptoethanol, and 1 ng/ml leupeptin; and disposable pestles notated at 450 rpm by a drill until the tissue was dispersed. Tissues were incubated on ice for 30 mm and centrifuged at 12,000 X g for 30 mm at 4#{176}C. The supernatant was collected, and the protein concentration was determined by the Bradford assay (Bio-Rad Laboratories, Richmond, CA). Aliquots were diluted to 1 ig pnotein/p.l and stoned at -80#{176}C.In brief, the TRAP assay was performed as follows (4, 1 1 , 28). Two ag of protein extract were assayed in a 38.tl reaction mixture containing lOX TRAP buffer [0.2 M Tris-HCI (pH 8.3), 0.5 mrvi MgC1,, 630 mM KCI, 0.05% Tween 20, 10 mrsi EGTA, and 1 mg/mI BSA], 2.5 mM deoxynucleosides, and 0. 1 mg TS primer at 25#{176}C for 20 mm. Ten p.1 of PCR mix containing lOX TRAP buffer, 0.1 mg ACX primer, Taq polymenase, and [32P]dCTP were then added to each sample. M2R3 (5’-GAGCAGAGTFAGGGTfAGGGTfAGGGTTAG-3 ‘), a synthetic oligonucleotide containing the sequences of the TS and RP primers, was included in every TRAP assay to control the efficiency of the PCR reaction. The products were amplified by 30 cycles at 94#{176}C, 60#{176}C, and 72#{176}C for 30 s each and were separated by electrophoresis on a 10% polyacrylamide gel. The gel was dried for I h at 80#{176}C and exposed to an image plate. Using ImageQuant software (Molecular Dynamics, Sunnyvale, CA), we quantified the signal intensity by determining the radioactivity of each repeat ladder corrected for the background and by expressing the total count per reaction (total product generated) as the percentage of activity in a cell line as described previously (5, 1 1 , 18, 29, 30). For consistency, we assayed an extract from a neuroblastoma cell line (SK-N-SH) in parallel on each gel, which shows maximum telomerase activity. The level of specific telomerase activity in SK-N-SH was set to 100%, and the relative specific telomerase activities of each extract were expressed as percentage of the SK-N-SH standard. All results were determined from at least three to six independent TRAP assays, and average activity was calculated. The quantitation method is semiquantitative but sufficient for comparative analysis (5, 1 1 , 29, 30), especially when the results of at least three independent experiments are combined. A sample was scored as telomerase positive when the telomerase-specific, 6-bp DNA ladder was observed. Negative samples were reexposed for 72 h to exclude weak enzyme activity. Specimens showing no telomerase were reassayed at 0.2 and 0.02 i.g of protein, because inhibition of telomerase activity at high protein concentrations has been reported (14, 23, 27, 28). To exclude Taq polymerase inhibitors, which may account for telomerase negativity, negative samples were reassayed using a PCR control TSNT (5’-AATCCGTCGAGCAGAGTFAG[GGTfAG]73’) oligonucleotide and RP plus NT primers under standard TRAP assay conditions.3 In all, the TSNT band presence and lack of telomerase activity were confirmed. Alkaline Phosphatase Activity. The stability of alkaline phosphatase appears to be similar to the stability of telomerase (17, 28). Therefore, alkaline phosphatase activity was analyzed in all 153 samples to assess the possibility of protein degradalion as a cause of negative telomerase activity. Thirty p.g of protein extract, assayed in a 200-p.l reaction mixture [0.75 m i 4-methyl-umbelliferyl phosphate, 1 12 msi 2-amino-2 methyl-I propanol buffer (pH 10.4). 3.75 mrvt MgSO4, and 1 mg/mI polyvinyl pyrrolidone], were incubated at 37#{176}C for 1 h, and the reaction was stopped by adding 20 pi of 1 M NaOH. The fluorescence of the product was read on a fluorescence plate reader (CytoFluor 2350). Alkaline phosphatase activity was found present in all solid tumor and normal adjacent tissue preparations and 60-p.m sections, with no significant difference in tumor and normal tissue control specimens. TRF Assay. Telomene length was determined by TRF” Southern blot analysis as described previously (4, 6, 9, 16). Genomic DNA from tumor and normal adjacent tissues was digested with 400 p.1 of DNA extraction buffer [100 nmi NaCl, 40 ms’i Tris (pH 8.0), 20 msi EDTA (pH 8.0), and 0.5% SDS] and proteinase K (0. 1 mg/ml). Extraction was performed using 3 N. W. Kim and F. Wu. Advances in quantitation and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP), submitted for publication. 4 The abbreviations used arc: TRF, telomere restriction fragment; kbp, kilobase pair(s); PD. population doubling. Research. on July 14, 2017. © 1997 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Table 1 Telomerase activity and tumor cell infiltration in solid tumors (median and ranges)