Tissue quality is an important determinant of telomerase activity as measured by TRAP assay.

Abstract

Telomerase is a ribonucleoproteinwith the function of a DNA pol- ymerase, in which a segment of the RNAcomponent operates as an internal tem-plate. It adds hexameric (TTAGGG) re-peats to the telomeric ends of the chro-mosomes, thus compensating for thecontinued erosion of the telomeres thatoccurs in its absence (3,7). Reactivationof telomerase might be a necessaryevent for the sustained growth of mosthuman tumors (9). The development ofa polymerase chain reaction (PCR)-based telomerase assay, the telomericrepeat amplification protocol (TRAP),has permitted a large number of tumorsamples to be analyzed (5).Telomerase activity has been detect-ed in almost all human immortal celllines and in most human cancer tissues(2,5). However, some human tumor tis-sues seem not to express telomerase. Areview that compiled several studies ofhuman tumors indicated that tel- omerase activity occurred in human can-cer tissues in 75%–100% of cases, withan average of 84.8% (8). Furthermore,different studies on histologically simi-lar tumors revealed different propo- rtions of tumors with telomerase activ-ity. For example, in colorectal cancer,telomerase activity was detected in 32of 35 cases (92%) by Kim et al. (5), in40 of 50 cases (80%) by Li et al. (6)and in 8 of 8 cases (100%) by Yoshidaet al. (11). An explanation for these dis-crepancies could be heterogeneity ofhuman tumor tissues that invariablyconsist of mixtures of tumor cells andsurrounding stromal tissue. However,the TRAP assay has been shown to de-tect telomerase activity in as few as 1positive cell per 104cells (5). We pro-pose that some of the apparently neg-ative tumors yield RNA of insufficientquality and are therefore false negative. A second reason for false-negativeresults might be the presence ofTaq DNA polymerase inhibitors. To reco-gnize this type of problem, the TRAP as-say should be performed using an inter-nal control (10). The TRAP-ez eTelomerase Detection Kit (Oncor,Gaithersburg, MD, USA) includes theamplification of an internal control of36 bp in each assay, and a false-neg-ative result is concluded when the 36-bpamplified product is not observed. Thiscontrol, however, does not account forall apparently negative tumor tissues.Telomerase is a ribonucleoproteinenzyme that uses its RNA as a templatefor the synthesis of TTAGGG repeats atthe ends of the chromosomes (4). Forthis reason, telomerase activity necessi-tates an active protein and a nondegrad-ed RNA. These two conditions indicatethat the quality of the tissue is of vitalimportance for the success of thetelomerase detection assay. Strikingly,RNA quality control has never beenproposed to date. We therefore inclu-ded a control for RNA quality in ourTRAP assay protocol.We measured telomerase activity in50 colorectal cancer tissues derivedfrom 50 patients using the TRAP assay.About 10 mg of frozen tissue were h-omogenized in 150 µL of 1×CHAPS ly-sis buffer (5). The whole-tissue lysatewas rapidly frozen and stored at -80°C.The protein concentrations of the e- xtract were measured using the BC AProtein Assay Kit (Pierce Chemical,Rockford, IL, USA). Approximately0.1 µg of protein extracted from tissuewas used for each telomerase assay (25µL reaction). Forty-two samples (84%)were found to be positive in this assay,while 8 samples were telomerase-nega-tive. All 50 samples were taken fromthe tissue bank of the Institute of Pat-hology (Lausanne, Switzerland). Somesamples were not frozen immediately.This lapse of time that occurred som-etimes between surgery and freezing inliquid nitrogen is relatively important.Warm ischemia might account for tissueautolysis including RNA degradation,which can affect the telomerase activityresults. To test this hypothesis, totalRNA from these 50 colon cancer tissueswas extracted using TR