European Journal of Histochemistry 2012; volume 56:e9 PARP-1 protein expression in glioblastoma multiforme A. Galia,1 A.E. Calogero,2 R.A. Condorelli,2 F. Fraggetta,1 C. La Corte,1 F. Ridolfo,3 P. Bosco,4 R. Castiglione,2 M. Salemi4 1Unit of Pathology, Cannizzaro Hospital, Catania; 2Section of Endocrinology, Andrology and Internal Medicine, Department of Internal Medicine and Systemic Diseases, University of Catania; 3Post-graduate School in Clinical Biochemistry, University of Milan; 4Laboratory of Cytogenetics, Oasi Institute for Research on Mental Retardation and Brain Aging, Troina, Italy Abstract One of the most common type of primary brain tumors in adults is the glioblastoma mul- tiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP- ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 pro- tein is required for the apoptosis-inducing fac- tor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin’s lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplas- mic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further sup- port the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker. Introduction Glioblastoma multiforme (GBM), or World Health Organization (WHO) grade IV astrocy- toma, is the most common type of primary brain tumor in adults. In addition, it is the most malignant and aggressive form of glioma and among the most lethal ones because of its highly invasive potential and least effective treatment available among solid tumors.1 Various genetic alterations lead to the develop- ment of malignant phenotype expression in human GBM.2,3 Heterogeneous pathological features associated with complex molecular heterogeneity are the major problems for the development of effective drug targets.4,5 GBMs can arise either de novo (primary) or progress to GBM from low grade gliomas (sec- ondary) with two distinct molecular pathways.3 Significant indicators of anaplasia in gliomas include: atypia (coarse nuclear chromatin, nuclear pleomorphism, multinucleation), mitotic activity, cellularity, microvascular pro- liferation, and necrosis. Necrosis with the microvascular or endothelial cell proliferations is a histological hallmark of glioblastoma.3-5 Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, is 43 kb-long and divided into 23 exons (OMIM 173870). Grube and Burkle6 suggested that a higher PARP-1 action capacity may contribute to an efficient mainte- nance of genome integrity. Yu et al.7 showed that PARP-1 activation is required for translo- cation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus. PARP-1 pro- tein is then proteolytically cleaved at the onset of apoptosis by caspase-3;8 furthermore, PARP- 1 activity and poly (ADP-ribose) (PAR) poly- mer, mediate PARP-1-induced cell death.8,9 Genetic and pharmacological studies found that overexpression of PARP-1 is a key media- tor of programmed-necrotic cell death in vivo. In addition, PARP-1 appears to be also involved in programmed cell death processes, such as apoptosis or macroautophagocytotic cell death.10 Microarray analysis showed that PARP-1 mRNA expression in histologically nor- mal tissues is low and uniform across a wide variety of tissues.11 Furthermore, microarray analysis of PARP-1 gene expression in more than 8000 samples revealed that PARP-1 is more highly expressed in several types of tumors compared with the equivalent normal tissue. Overall, the most striking differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, skin can- cers, and non-Hodgkin’s lymphoma.11 The present study was undertaken to evalu- ate the expression of PARP-1 protein in normal brain tissues and primary glioblastomas (grade IV WHO) with immunohistochemistry. Correspondence: Prof. Aldo E. Calogero, Section of Endocrinology, Andrology and Internal Medicine, Department of Internal Medicine and Systemic Diseases, University of Catania, via S. Sofia 78, 95123 Catania, Italy. E-mail: acaloger@unict.it Key words: Glioblastoma multiforme, brain, immunohistochemistry, PARP-1 gene. Received for publication: 26 November 2011. Accepted for publication: 26 January 2012. This work is licensed under a Creative Commons Attribution NonCommercial 3.0 License (CC BY- NC 3.0). ©Copyright A. Galia et al., 2012 Licensee PAGEPress, Italy European Journal of Histochemistry 2012; 56:e9 doi:10.4081/ejh.2012.e9 Materials and Methods Patient and control brain samples The comparison has been made between: i) 4 normal tissues brain removed with post mortem autopsy by normal donors (2 males and 2 females); ii) 27 non-tumor peripheral tissues brain removed during surgical resec- tion on patients with GBM and 27 tumor tis- sues WHO grade IV GBM, specifically, by 14 male patients (58.3±10.0 years) and 13 female patients (58.3±12.6 years) (Table 1). Diagnoses of GBM were made on histologi- cal sections stained with hematoxylin-eosin and the sections were evaluated by at least two operators. The protocol was approved by the internal Institutional Review Board and an informed written consent was obtained from each patient with GBM or, if deceased, by his/her relatives. Immunohistochemical staining Brain sections (4 μm thick) were obtained from all normal donors and GMB patients. Brain sections from GBM patients included both GBM and normal tissue (Table 1). All sec- tions were formalin-fixed and paraffin-embed- ded following standard methods. PARP-(F-2), a mouse monoclonal antibody raised against PARP-1 protein, was used for immunohistochemistry (Santa Cruz Biotechnology, Inc., Heidelberg, Germany) PARP-1 (F-2): sc-8007). As indicated by the manufacturer instructions, this antibody, at the dilution of 1:300, has been shown to reli- ably recognize PARP-1 proteins in glioblas- tomas and normal brain by immunohistochem- istry. Slides were deparaffinized, rehydrated, subjected to three 5-min cycles in a microwave at 360 W in citrate buffer, preincubated in 3% [European Journal of Histochemistry 2012; 56:e9] [page 45]