Telomerase activity in skeletal sarcomas

Abstract

Background: Telomerase is a ribonucleoprotein that adds TTAGGG nucleotide repeats onto the ends of eukaryotic chromosomes to maintain telomere integrity. Somatic cells do not express telomerase and stop dividing when the chromosomal ends are shortened critically after many cell divisions. Immortal cell lines and cancer cells apparently have telomerase activity that contributes to an unlimited number of cell cycles. The purpose of our study is to investigate whether telomerase activity is expressed in primary malignant tumors of the skeletal system when compared to adjacent normal tissue.

Methods: Fresh tumor and normal tissue was collected from 14 patients (10 males, 4 females; age range, 8 to 76 years) and protein extraction performed. The tumors included seven osteosarcomas (three examined before and after chemotherapy), two chondrosarcomas, two spindle cell tumors, one hemangiopericytoma, one chordoma, and one adamantinoma. Telomerase activity was analyzed by using a highly sensitive polymerase chain reaction (PCR)-based assay (telomere repeat amplification protocol [TRAP]).

Results: Telomerase activity was found in 8 of 14 sarcoma patients (57%) using the TRAP assay. Compared to HeLa cell extract (positive control), telomerase activity in the tumor specimen ranged from 0 (in osteosarcoma) to 11.7% (in hemangiopericytoma). There was variation in the number of telomeric repeats generated by telomerase. At least five telomeric bands (e.g. 50, 56, 62, 68, 74 bp) in a ladder pattern had to be present before telomerase activity was considered positive in our analysis.

Conclusions: Telomerase activity may be an oncogenic sustaining event helping to maintain the transformed phenotype seen in malignant tumors of the bone. The degree of telomerase activity varies among skeletal malignancies, but was less than that observed in HeLa cells. The majority of osteosarcomas showed no telomerase activity.

FIG. 1

Polyacrylamide gel (12%) showing evidence of telomerase activity from protein extract of tumor cells from skeletal sarcoma patients and HeLa, a positive control cell line. Lane 1: PCR reaction without protein extract. Lane 2: Hemangiopericytoma (0.03 μg protein). Lane 3: Hemangiopericytoma (0.03 μg protein)—heat treated. Lane 4. Hemangiopericytoma (0.03 μg protein)—RNase treated. Lane 5: muscle (0.03 μg protein). Lane 6: HeLa (0.0006 μg protein). Lane 7: HeLa (0.0006 μg protein)—heat treated. Lane 8: HeLa (0.0006 μg protein)—RNase treated. Lane 9: DNA marker 5 (Boehringer Mannheim, Indianapolis, IN). The numbers represent DNA marker fragment sizes in base pairs. The internal standard (36 bp) is shown at the bottom of the figure.

FIG. 2

Polyacrylamide gel (12%) showing telomerase activity and inhibition in chondrosarcoma (patient 1). HeLa is a positive control cell line. Lane 1: Chondrosarcoma (0.03 μg protein)—heat treated. Lane 2: Chondrosarcoma (0.03 μg protein). Lane 3: Chondrosarcoma (0.3 μg protein)—heat treated. Lane 4: Chondrosarcoma (0.3 μg protein) showing inhibition with an additional band between 36 and 50 bp. Lane 5: HeLa (0.0006 μg protein). Lane 6: HeLa (0.0006 μg protein)—heat treated.