p73 Expression and Function in Vestibular Schwannoma FREE

The molecular biological processes in the development of vestibular schwannoma (VS) is poorly understood. For a subset of patients with VS, one of the more well-defined mechanisms responsible for tumor development is the functional loss of the tumor suppressor neurofibromatosis 2 (NF2) (GenBank AF069751), which results in an autosomal dominant disorder clinically characterized by bilateral VS and meningiomas.^1- 4 Other tumor suppressors have been studied to investigate other potential mechanisms in the development of this tumor. Currently, it is unclear what the role of p53 (GenBank AF210308 S1-3), the quintessential tumor suppressor that is mutated in 50% of all cancers, has in the genesis of VS. While earlier studies have reported that p53 is unlikely to contribute to the development of VS (as investigators found no mutation), deletion, or loss of heterozygosity,^5- 6 a recent report by Dayalan et al⁷ indicates that loss of heterozygosity occurs at the first intron of the p53 gene locus in a significant fraction of the cases examined. Furthermore, these investigators have observed age-dependent phosphorylation of Ser 392 of p53 in younger patients with VS. Together with the loss of heterozygosity, the authors suggest that deregulation of the p53 gene has a role in the development of VS. In addition, the tumor suppressor p27 (GenBank AF480891), a cyclin-dependent kinase inhibitor, has recently been demonstrated to have decreased expression in a subset of VS tumors that grew aggressively postoperatively.⁸

To further understand the role of tumor suppressors in the pathogenesis of VS, our laboratory has been studying p73 (GenBank AF077616-AF077628), a gene that maps to 1p36.3, a region often deleted in human carcinomas, including childhood neuroblastoma.^9- 11p73 Shares strong homology with p53 in terms of domain structure and conformation.^11- 12 Despite homology, in more than 60% of amino acid sequencing of their DNA binding regions,^13- 14p73 is more complex than its cousin p53.¹⁴ The p73 gene can give rise to multiple protein isoforms generated by alternative promoters and alternative splicing. The alternative promoters generate either a full length, transcriptionally active p73 (TAp73) or a truncated, transcriptionally inactive isoform, deltaN p73 (ΔNp73), which have competing and opposing functions.¹² The ΔNp73 isoform suppresses both TAp73 and p53, indicating its oncogenic potential,^12,15 while TAp73 mimics the suppressor function of p53 and induces apoptosis.^12,14 We, as well as other groups, have demonstrated that TAp73 determines sensitivity to various chemotherapeutic agents in head and neck squamous cell carcinoma as well as in thyroid cancer cells (W.M.O., unpublished data, 2005). However, ΔNp73 has been demonstrated to transform cell lines, giving more evidence that it functions as an oncogene. ΔNp73 blocks the transactivation activity of p53 and TAp73, hindering their apoptotic capacity, while p53 and TAp73 reciprocally induce the expression of ΔNp73 in a complex feedback loop.¹²

Although overexpressed in several epithelial cancers,^14,16p73 is rarely mutated.^9,12 Unlike mice lacking p53, p73-null mice do not appear to have spontaneous tumor development.¹⁷ In contrast, overexpression of p73 in human breast, urinary, bladder, and hepatocellular carcinomas and B-chronic lymphocytic leukemias correlates with a poor patient prognosis.^18- 21

The records of all patients with a diagnosis of VS at the Servicio de Otorrinolaringología, Hospital Donostia, San Sebastián, Spain, between January 1991 and December 2002 were reviewed. Demographic data recorded for all patients included age, sex, and symptoms.

The appropriate formalin-fixed, paraffin-embedded VS tumors were obtained from the archives of the Department of Pathology in accordance with the institutional review board of Hospital Donostia. Four-micrometer sections were cut and placed on 3-aminopropyltriethoxysilane–coated slides. Immunohistochemical studies were performed using mouse monoclonal antibody against p73 (Imgenex, San Diego, California). Negative control slides were from the same tissue sample but without the addition of the primary antibody. Formalin-fixed, paraffin-embedded head and neck squamous cell carcinoma tissue samples were used as positive controls. The samples were heated for 1 hour at 60°C, deparaffinized in xylene, and rehydrated in a graded series of ethanol. Antigen retrieval was performed by steam heating with DAKO Target Retrieval solution (DAKO, Carpinteria, California). The endogenous peroxidase was quenched by 3% hydrogen peroxide. Nonspecific binding of biotin and avidin was blocked by blocking solution (Protein Block Serum-Free; DAKO). The background staining was reduced by incubation with goat serum (1:20) for 60 minutes. Primary antibodies were placed on slides and incubated for 1 hour at room temperature. Secondary antibodies conjugated with streptadivine/horseradish peroxidase (LSAB2; DAKO) were used. The slides were washed and the antibody complex visualized by 3,3′-diamino-benzidine (DAB; DAKO). The nuclei were counterstained by Gill's II hematoxylin (Protocol; Fisher-HealthCare, Houston, Texas). Cases were considered positive if the staining scored at least a 2+ on a scale of 1 to 4+ and more than 10% of the tumor cells were staining above that of the background and negative control. Slides were scored as positive or negative by 2 of us (X.A. and M.J.A.), one of whom is a board-certified pathologist.

HEI193 is an HPV E6-E7 immortalized human vestibular schwannoma cell line, which was a gift from David Lim, PhD (House Ear Institute, Los Angeles, California), and normal human Schwann cells were purchased from ScienCell Research Laboratories, San Diego. Both cell lines were cultured in Dulbecco modified Eagle medium, supplemented with 10% fetal bovine serum, 10 000-U/mL penicillin G sodium, 10-mg/mL streptomycin sulfate (Invitrogen Co, Carlsbad, California), and L-glutamine (Invitrogen Co) at 37°C in 5% carbon dioxide.

The full-length p73 plasmid (TAp73α) was transfected using Lipofectamine 2000 (Invitrogen Co) following the manufacturer's instructions, and expression of the genes was verified by Western blot analysis. Membranes were probed with primary antibodies to p73 and p21, followed by incubation with the appropriate secondary antibodies. Membranes were then visualized with an enhanced chemiluminescence detection system (Pierce, Rockford, Illinois). To demonstrate standardized loading, the membranes were probed with polyclonal antibody against β-actin (Sigma-Aldrich Co, St Louis, Missouri).

Cells were plated on glass cover slides until they reached the desired confluence and transfected with a range of concentrations of p73. After 24 or 48 hours, cells were fixed with 4% paraformaldehyde. After washing with phosphate-buffered saline (PBS), paraformaldehyde fixed cells were permeabilized with 0.2% Triton X-100. Cells were then incubated in primary antibodies for 1 hour at room temperature or overnight at 4°C. Primary antibodies were removed and cells were washed with PBS and incubated in Alexa Fluor fluorescein isothiocyanate (FITC)-conjugated goat antimouse or goat antirabbit secondary antibodies (Molecular Probes; Invitrogen Co) for 1 hour at room temperature. Slides were counterstained with 4",6"-diamidino-2-phenylindole dihydrochloride (DAPI) for 1 hour before final embedding. Fluorescent images were obtained using a Leica DMIRE2 inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The computer program Simple PCI (Compix Inc, Sewickley, Pennsylvania) was used for image capture.

Flow cytometry was used to assess the extent to which p73 could induce cell death and modulate the response to ionizing radiation (IR) in HEI193 and human Schwann cells. The assay was performed with a 2-color analysis of FITC-labeled annexin V binding and propidium iodide (PI) uptake using the Annexin V–FITC Apoptosis Detection kit (Calbiochem, San Diego). Positioning of quadrants on annexin V/PI dot plots was performed and live cells (annexin V⁻/PI⁻ [F3]), early/primary apoptotic cells (annexin V⁺/PI⁻ [F4]), late/secondary apoptotic cells (annexin V⁺/PI⁺ [F2]), and necrotic cells (annexin V⁻/PI⁺ [F1]) were distinguished. The Annexin V–FITC Apoptosis Detection kit was used following the manufacturer's instructions.

To determine the role of p73 alone in HEI193 cell death, cell cycle analysis and apoptosis assays were performed 24 and 48 hours after transfection of the HEI193 cells with 0, 2, or 10 μg of p73 plasmid. To determine the role of p73 in modulating the response of HEI193 and human Schwann cells to IR, the cells were exposed to 0, 1, or 5 Gy of IR 24 hours after the cells were transfected with p73. Cell cycle analyses and cell death assays were also performed subsequently.

The second assay used for measuring apoptosis was the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay. For this assay, cells were cultured on 10-cm diameter dishes, treated with agents as indicated, and harvested by trypsinization. Cells were fixed with 1% paraformaldehyde, and cytoplasmic DNA fragments with 3′-hydroxyl ends were detected with an APO-Direct TUNEL kit (Phoenix Flow Systems, San Diego).

To determine the cell cycle distribution and levels of apoptosis as an effect of p73 overexpression, and the response of the cells to IR in the presence or absence of p73, cells were harvested after performing the designated treatment and fixed in 50% cold ethanol overnight. The cells were then washed once with PBS and resuspended in a solution of PBS plus 0.1% Triton-X100, 2-mg/mL DNAase-free RNaseA, and 0.02-mg/mL propidium iodide. Cells were analyzed by flow cytometry on a FACScan flowcytometer (Becton, Dickinson, and Company, San Jose, California). Multicycle AV Cell Cycle software (Phoenix Flow Systems) was used to calculate the fraction of cells in each phase of the cell cycle.

For the association between tumor size and p73 expression, the Mann-Whitney test was used. The 2-way analysis of variance test was used to evaluate the statistical significance of cell death due to p73 expression and IR. A paired t test was used to evaluate the statistical significance of the effect of knocking down p73 on cell death due to IR. P < .05 was considered statistically significant.

A total of 34 patients ranging in age from 25 to 72 years were studied (19 male and 15 female). The tumors ranged in size from 3 to 40 mm, with the mean size being 17 mm (Table 1).

In the 34 VS tissues examined, p73 was expressed in 14 (41%) (Table 1). The mean dimension of the tumor was 15 mm for p73-negative samples and 20 mm for p73-positive tissues. No statistically significant difference in p73 positivity was related to tumor size (P = .12). Figure 1A is a representative example of p73 staining in this tissue, with Figure 1B as negative control. HEI193 cells have barely detectable p73 protein as measured by Western blot analysis, but transfection of 2 and 10 μg of p73 plasmid resulted in a dose-dependent expression of p73 protein expression (Figure 2A).

Immunofluorescent staining after transfection of HEI193 cells with p73 plasmid revealed that p73 is localized primarily in the nucleus, similar to that seen in most other cancer cell lines such as head and neck squamous cell carcinoma and thyroid cancer (Figure 2B).

At 48 hours after transfection of p73 plasmid, there was an increase in G₁ from 51.3% to 56.5% and 56.8% at 2 and 10 μg of p73 plasmid, respectively (Figure 3 and Table 2). There was also a modest decrease of cells in S-phase from 26.8% to 23.2% and 24.7% with treatment with 2 and 10 μg of p73, respectively. Likewise, there was a slight decrease in the proportion of cells in G₂ with increasing levels of p73. A concomitant induction of p21, the cyclin-dependent kinase inhibitor, was also observed, which corresponded with the G₁ accumulation (Figure 2A). Cell death, as measured by sub-G₁ DNA, increased from 2.78% to 3.98% and 16.74% when HEI193 cells were transfected with 2 and 10 μg of p73 plasmid, respectively (Figure 3A). Apoptotic bodies and fragmented DNA were present in cells that were successfully transfected with p73, especially in the plates in which 10 μg of p73 was used (Figure 2B). By TUNEL assay, on transfection of 2 μg of p73 plasmid, apoptosis increased from 2.95% to 11.47% (Figure 4).

At 24 hours after exposure to IR, there was a significant increase in early apoptosis (F4) with increasing amounts of p73 transfected (Figure 5 and Table 3) (P = .046). There was also a marked dose-dependent increase in late apoptosis (F2) in the presence of p73 (Figure 5 and Table 3), with late apoptosis increasing from 0.69% to 4.01% in cells transfected with 10 μg of p73 and exposed to 5 Gy of IR (P < .001). In the presence of p73, a significant increase in levels of necrosis/very late apoptosis was also observed 24 hours after exposure to IR. Cells transfected with an empty vector and exposed to 1 Gy of IR resulted in 2.31% necrosis/very late apoptosis, but when transfected with 10 μg of p73, the necrosis/very late apoptosis increased to 19.93% (Table 3). Even if the numbers are adjusted for the necrosis/very late apoptosis due to the presence of p73 alone, there is still approximately an 800% increase in necrosis/very late apoptosis between control and p73-transfected cells at 1 Gy of IR. By TUNEL assay, transfection of 2 μg of p73 increased cell death from 6.6% to 18.3% in cells exposed to 1 Gy of IR (Figure 4).

To determine if the presence of p73 would also regulate the response to IR in an untransformed cell line, normal, nonimmortalized, human Schwann cells were used. Western blot analysis showed the presence of p73 in these cells (Figure 6A). Cells were then exposed to IR 72 hours after transfection with small-interfering (si)-RNA p73 or scrambled si-RNA (Figure 6B). Knock-down of p73 protein resulted in decreased levels of necrotic/very late apoptotic cells compared with control (P < .02) (Table 4).

Inactivation of tumor suppressors is one of the hallmarks of tumor formation. In this study, we have demonstrated that p73, a close cousin of p53, is expressed in only 41% of tissue samples studied. This is particularly important because it has been determined that p73 is necessary for the full apoptotic potential of p53.²² Other investigators have studied the p73 gene in meningiomas with deletion at 1p,²³ and there is a report that p73 is not mutated in meningiomas but that p73 expression increases with tumor grade.²⁴ To our knowledge, this is the first time that the protein expression of p73 has been investigated in VS. At present, the only other study on this topic is on the methylation of p73 in schwanommas^25- 27 that could explain in part the absence of expression of this gene in more than half of the VS tissues studied. Unfortunately, due to unavailability of sufficient amounts of protein to perform primary tissue Western blot analysis as well as unavailability of p73 antibodies that can discriminate between the TAp73 and ΔNp73 by immunohistochemical analysis, we are unable to determine which isoform is expressed in the VS tissue samples. Given the method by which the indicated tissue samples were archived and the relative age of some of these samples, we believed that it was most appropriate that this be primarily a retrospective study because obtaining sufficient protein from archival tissue was unlikely. Thus, we are left with the fundamental question of whether the full length, transcriptionally active form of p73 is deleted, methylated, or inactivated, or whether the expression of ΔNp73, functioning as an oncogene, had a role in the development of the tumor. What is clear, though, as demonstrated by our experiments, is that TAp73α can regulate the proliferation of a cell line derived from this tumor. We demonstrate in this study that p73 can alter the cell-cycle distribution, induce cell death, and has an additive effect of IR-induced cell death.

The effect of overexpression of p73 alone in a cell line with no detectable levels of p73 is very dramatic and may be sufficient to control tumor growth. In combination with IR, the effect of p73 is additive, with most of the cell death attributed to p73. At 24 hours after radiation, there is approximately a 472% and 277% increase in total cell death due to the presence of 10-μg p73 at the 1- and 5-Gy exposures of IR, respectively, doses that are clinically relevant in the treatment of VS. This increased cell death due to the proapoptotic effects of p73 in HEI193 cells may be critical in better control of the tumor or would allow the use of lower doses of radiation, thus reducing morbidity from this treatment. These experiments demonstrate that p73 may have a role in the clinical setting by either its direct administration via gene therapy or by using it in combination with IR during stereotactic radiosurgery.

Correspondence: Weg M. Ongkeko, MD, PhD, University of California, San Diego, Basic Science Building, Room 1202, Mail Code 0612, 9500 Gilman Dr, La Jolla, CA 92093-0612 (wongkeko@ucsd.edu).

Submitted for Publication: January 10, 2008; final revision received August 29, 2008; accepted October 6, 2008.

Author Contributions: Dr Ongkeko had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Mr Ahmad, Dr Altuna, and Mr Lopez contributed equally to this work. Study concept and design: Altuna, Wang-Rodriguez, Harris, and Ongkeko. Acquisition of data: Ahmad, Altuna, Lopez, An, Chen, Arandazi, Aguilera, and Ongkeko. Analysis and interpretation of data: Ahmad, Altuna, Lopez, An, Wang-Rodriguez, Juneja, Arandazi, and Ongkeko. Drafting of the manuscript: Ahmad, Altuna, Lopez, Wang-Rodriguez, Chen, Arandazi, and Ongkeko. Critical revision of the manuscript for important intellectual content: Ahmad, An, Wang-Rodriguez, Juneja, Aguilera, Harris, and Ongkeko. Statistical analysis: An and Juneja. Obtained funding: Wang-Rodriguez and Ongkeko. Administrative, technical, and material support: Altuna, Wang-Rodriguez, Chen, Arandazi, Aguilera, Harris, and Ongkeko. Study supervision: Ahmad, Lopez, Wang-Rodriguez, and Ongkeko.

Financial Disclosure: None reported.

Funding/Support: This work was supported by the Bell Foundation.