IN SITU DETECTION OF TELOMERASE CATALYTIC SUBUNIT mRNA IN GLIOBLASTOMA MULTIFORME Maria Laura FALCHETTI 1 , Roberto PALLINI 2 , Ettore D’AMBROSIO 1 , Francesco PIERCONTI 3 , Maurizio MARTINI 3 , Graziella CIMINO-REALE 1 , Roberto VERNA 4 , Giulio MAIRA 2 and Luigi Maria LAROCCA 3 1 Istituto di Medicina Sperimentale, CNR, Rome, Italy 2 Istituti di Neurochirurgia, Universita ` Cattolica del Sacro Cuore, Rome, Italy 3 Anatomia Patologica, Universita ` Cattolica del Sacro Cuore, Rome, Italy 4 Dipartimento di Medicina Sperimentale, Universita ` “La Sapienza,” Rome, Italy Activation of telomerase may allow unlimited cell prolif- eration and immortalization. One of the telomerase protein subunits has a reverse transcriptase (hTERT) activity that is essential for telomerase function and regulation. In human gliomas, telomerase is frequently associated with malignant tumor progression. In our study, we investigated the expres- sion of hTERT at the cellular level in 34 primary de novo glioblastoma multiforme (GBM) by in situ hybridization (ISH). The expression of hTERT in tumor tissue was also assessed by RT-PCR. In addition, telomerase activity mea- sured by telomeric repeat amplification protocol (TRAP) and telomere length polymorphism assayed by telomere restric- tion fragment (TRF) Southern blot were investigated. We found that all GBM, including those with negative TRAP reaction, contained abundant amounts of cytoplasmic hTERT mRNA. Interestingly, the ISH analysis revealed that the hTERT mRNA was homogeneously expressed by the whole tumor cell population in about 60% of the GBM. In the re- maining cases, hTERT was absent in subsets of tumor cells. TRF analysis, which shows that both TRAP-positive and TRAP-negative de novo GBM have elongated telomeres, fur- ther supports that telomerase activity is present in all de novo GBM. Correlations with tumor size and extent of necrosis suggest that hTERT reactivation is an early event in GBM development and that telomerase activity may be lost in subpopulations of neoplastic cells during tumor progression. Finally, ISH analysis of hTERT mRNA seems to provide a prognostic parameter for primary de novo GBM. Int. J. Cancer 88:895–901, 2000. © 2000 Wiley-Liss, Inc. Telomerase is a ribonucleoprotein complex that adds telomeric TTAGGG repeats to chromosome ends (Greider and Blackburn, 1989). The enzymatic activity of this complex is present during embryonal development but is usually absent in somatic cells and it remains active in germline cells (Kim et al., 1994). In the absence of telomerase, at each cell division, the telomeres get shorter until a critical length is achieved; this induces the cells to stop proliferation and enter the senescence program (Harley, 1991). Because indefinite proliferation in the absence of telomere maintenance would lead to chromosome abnormalities, cells that re-initiate proliferation generally re-activate telomerase. Thus, te- lomerase is activated in a high percentage of human tumors and is present in most tumor-derived cell lines in culture (Kim et al., 1994; Shay and Bacchetti, 1997). The reverse transcriptase com- ponent of human telomerase, hTERT (telomerase reverse transcrip- tase), has been proposed as the catalytic subunit of telomerase (Counter et al.,1998; Bodnar et al., 1998; Vaziri and Benchimol, 1998). Differently from telomerase RNA (TR), which is expressed even in the absence of telomerase activity, hTERT is tightly reg- ulated and several lines of evidence suggest that its synthesis is induced by the oncogene, myc (Wang et al., 1998; Wu et al., 1999; Falchetti et al., 1999a). It has also been reported that the intro- duction of hTERT into normal cells is per se sufficient to activate telomerase and to allow unlimited cell proliferation (Morales et al., 1999; Jiang et al., 1999). Glioblastoma multiforme (GBM) is the most common malig- nant tumor of neuroepithelial origin in the human brain (Russell and Rubinstein, 1989; Lantos et al., 1996). This neoplasm is highly proliferative and conventional multimodal therapy does not change its poor prognosis (Burger and Green, 1987; Davis et al., 1998; Leenstra et al., 1998; Galanis et al., 1998). In human gliomas, activation of telomerase is frequently associated with malignant tumor progression (Langford et al., 1995; Morii et al., 1999; DeMasters et al.; 1997; Sallinen et al., 1997; Nakatani et al., 1997; Kleinschmidt-DeMasters et al., 1998; Le et al., 1998; Chong et al., 1998; Hiraga et al., 1998; Falchetti et al.,1999b; Huang et al., 1999). It has been suggested that telomerase activity in GBM tumors may be related to tumorigenesis as secondary GBM tu- mors, which progress from low-grade gliomas, constantly exhibit telomerase activity, whereas telomerase activity can be detected only in 63% of primary de novo GBM tumors (Hiraga et al., 1998). Recent studies, however, have shown that when microdissection for selective analysis of tumor cells (Weil et al., 1999) and sam- pling of multiple regions for the same tumor are used (Klein- schmidt-DeMasters et al., 1998), 100% of GBM tumors can be found to display telomerase activity. In our study, we investigated the expression of hTERT at the cellular level by in situ hybridization (ISH) on 34 de novo GBM tumors. This technique, which reportedly provides reliable data on telomerase regulation in normal and neoplastic tissues (Kolquist et al., 1998; Nakano et al., 1998; Liu et al., 1999), has not been used in brain tumor research yet. The expression of hTERT in the tumor tissue was also assessed by RT-PCR. In addition, telomerase activity by telomeric repeat amplification protocol (TRAP) assay and telomere length analysis by telomere restriction fragment (TRF) Southern blot were performed. We report that the TRAP- negative GBM tumors show detectable amounts of apparently intact cytoplasmic RNA for hTERT. However, these tumors dis- play a lower proliferative index compared with TRAP-positive GBM tumors. Furthermore, our results indicate that all GBM tumors have telomerase activity, but the enzymatic activity may be lost in subpopulations of neoplastic cells during tumor progression. MATERIAL AND METHODS Tumor samples Frozen tumor samples and formalin-fixed, paraffin-embedded sections were collected from 34 patients operated on at the Cath- olic University School of Medicine (Rome, Italy; Table I). Taking into account the known heterogeneity of GBM for cell density, cell viability/necrosis and cell cycle-labeling indexes, specimens for different analyses were taken from the same, very closely juxta- posed tissue sample. All the tumors were histologically classified as GBM according to WHO criteria (Kleihues et al., 1993). Our study include only de novo GBM tumor, as defined by clinical data (age, symptom duration, previous surgery; Hiraga et al., 1998; Leon et al., 1994; Kleihues and Ohgaki, 1997; Fujisawa et al., 1999). No patient received radiation therapy before surgery. *Correspondence to: Luigi Maria Larocca, MD, Istituto di Anatomia Patologica Universita ` Cattolica del sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy. Fax: +39-06-3051157. E-mail: llarocca@rm.unicatt.it Received 30 March 2000; Revised 3 August 2000 Int. J. Cancer: 88, 895–901 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer