Affiliations of authors: L. Del Valle, J. Gordon, S. Enam, S. Delbue, S. Croul, S. Abraham, S. Radhakrishnan, M. Assimakopoulou, K. Khalili, Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, PA; C. D. Katsetos, Department of Pediatrics, MCP-Hahnemann University, and Department of Pediatrics and Pathology and Laboratory Medicine, St. Christopher's Hospital for Children, Philadelphia.
Correspondence to: Kamel Khalili, Ph.D., Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, 1900 North 12th St., 015-96, Rm. 203, Philadelphia, PA 19122 (e-mail: kkhalili{at}astro.temple.edu).
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ABSTRACT |
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INTRODUCTION |
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The biologic role of the auxiliary agnoprotein in the viral lytic infection cycle and its effect on various cellular regulatory pathways remain unknown. The most recent studies from our laboratory (18), however, demonstrate that the interaction of the JCV T antigen with agnoprotein may have a functional consequence on the ability of the T antigen to enhance transcription and replication of the viral genome. We investigated the presence of the Agno gene and the expression of the agnoprotein in human medulloblastomas and in several PML samples.
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MATERIALS AND METHODS |
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A total of 20 formalin-fixed, paraffin-embedded biopsy and autopsy samples of medulloblastomas were collected from the pathology archives of St. Christopher's Hospital for Children and from MCP-Hahnemann University, in Philadelphia, PA. Three samples of PML cases (two from human immunodeficiency virus-1 [HIV-1]-infected patients with AIDS and one from a patient without AIDS) were obtained from the Manhattan HIV-1 Brain Bank at Mount Sinai Medical Center in New York, NY. All of the samples were obtained in accordance with the guidelines from the institutional review boards of the participating universities.
Development of Anti-agnoprotein Antibody
Three peptides, as shown below, that correspond to various regions of the agnoprotein were synthesized by Macromolecular Resources (Colorado State University, Fort Collins, CO). The sequences of the peptides are as follows: JAG-38MVLRQLSRKASVKVSKTWSGTKKRAQRILIFLLEFLLDC; JAG-36LLDFCTGEDSVDGKKRQRHSGLTEQTYSALPEPKATC; and JAG-34GTKKRAQRILIFLLEFLLDFCTGEDSVDGKKRQRC.
Approximately 2 mg of each peptide was pooled and used for injection of rabbits (Lampire Biological Laboratories, Pipersville, PA). The final sera were obtained 98 days after the initial injection and used in these studies.
Infection and Western Blot Analysis
SVG-A cells, which were subcloned from the parental line SVG that was derived from human primary fetal glial cells transformed with origin-defective simian virus 40 (SV40), were provided by Dr. Walter Atwood (Brown University, Providence, RI) for use in infection experiments. These cells express the SV40 T antigen but do not express the viral late genes. Approximately 5 x 106 SVG-A cells were infected with approximately 500 hemagglutination units of the Mad-4 strain of JCV in Dulbecco's modified Eagle medium containing fetal calf serum. After 4 hours, fetal calf serum was added to the culture medium (final concentration = 10%), and the cells were harvested 5, 10, and 15 days after infection. The protein extracts from the uninfected or infected cells (50 µg) were analyzed directly by western blot or were first reacted with preimmune or anti-agnoprotein antibody, and the immune complex was analyzed by western blot according to the standard procedures described previously (18).
Histologic and Immunohistochemical Analyses
Formalin-fixed, paraffin-embedded tissue was sectioned at 4-µm thickness and stained with hematoxylineosin for histologic studies. The classification of the tumors was based on the latest World Health Organization Classification of Tumors (19). Immunohistochemical analysis was performed with the use of an avidinbiotinperoxidase complex system according to the manufacturer's instructions (Vectastain Elite ABC Peroxidase Kit; Vector Laboratories Inc., Burlingame, CA). The protocol includes deparaffinization in xylenes and rehydration of the tissue through descending grades of alcohols up to water. Nonenzymatic antigen retrieval was performed by heating the sections to 95 °C in 0.01 M sodium citrate buffer (pH 6.0) for 40 minutes in a vacuum oven. After cooling for 30 minutes, the slides were rinsed in phosphate-buffered saline (PBS) and incubated in methanol3% H2O2 for 20 minutes to quench the endogenous peroxidase. The sections were then rinsed with PBS and blocked with 5% normal horse or goat serum in 0.1% PBSbovine serum albumin for 2 hours at room temperature. Primary antibodies, including those reactive against viral proteins and cellular markers, were incubated overnight at room temperature in a humidified chamber. Primary antibodies used in this study included rabbit polyclonal antibody against agnoprotein (1 : 500 dilution), rabbit polyclonal antibody against JCV capsid proteins (1 : 1000; provided by Dr. Walter Atwood), mouse monoclonal antibody for the detection of SV40 T antigen that cross-reacts with JCV T antigen (clone pAb416, 1 : 100 dilution; Oncogene Science, Boston, MA), mouse monoclonal antibody against p53 (clone DO-7, 1 : 100 dilution; Dako, Carpinteria, CA), mouse monoclonal antibody for GFAP (i.e., glial fibrillary acidic protein; clone 62F, 1 : 100 dilution; Dako), and mouse monoclonal antibody against the 38-kd form of synaptophysin (clone MAB332, 1 : 500 dilution; Chemicon International, Temecula, CA). After the sections were rinsed in PBS, biotinylated anti-mouse or anti-rabbit secondary antibodies were incubated for 1 hour at room temperature and rinsed in PBS. The tissue was subsequently incubated with avidinbiotinperoxidase complexes for 1 hour at room temperature. Finally, the sections were developed with a diaminobenzidine substrate counterstained with hematoxylin and mounted under coverslips with Permount (Fisher, Pittsburgh, PA).
DNA Extraction and Polymerase Chain Reaction Amplification
DNA was prepared from 10 sections of 10-µm thickness from each of the tissue samples by use of the QIAamp DNA Tissue Kit, according to the manufacturer's instructions (Qiagen, Valencia, CA).
For amplification of the Agno gene, a set of primers that specifically recognize JCV Agno gene was used, and the amplified DNA was hybridized with the use of JCV-specific DNA probe as depicted in Fig. 1. Southern blot analysis was performed according to a procedure that has been established in our laboratory and described previously (20,21). Amplification of the JCV early sequence was performed with the use of a set of primers that recognize the N-terminal region of the JCV T antigen, and hybridization was performed with the use of JCV-specific probes as described earlier (21).
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RESULTS |
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In the first series of experiments, a pair of primers that specifically recognize JCV DNA corresponding to the Agno gene was used for gene amplification, followed by Southern blot analysis. Fig. 1, A, illustrates the position of the Agno gene in the JCV genome (left panel), a representative gene amplification/Southern blot (top, right panel), and the DNA sequence highlighting the Agno gene sequence. Results from DNA analysis revealed that 11 (69%) of the 16 samples had sequences corresponding to the Agno gene (Table 1
). It is interesting that some, but not all, of the tumors positive for the Agno gene also contained DNA sequences corresponding to the early gene of JCV, T antigen (data not shown). Examination of the T-antigen sequences was performed according to our earlier procedure using a set of primers that recognize the N-terminal region of the JCV T antigen followed by hybridization using a JCV-specific DNA probe (21). Seven (44%) of 16 samples that were examined contained DNA sequences corresponding to the T antigen and the Agno gene, whereas three were positive for the T antigen and negative for the Agno gene, and four contained the Agno gene but not the T antigen.
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In the next series of experiments, to examine the expression of agnoprotein in tumor cells and to determine its subcellular localization, we conducted an immunohistochemical analysis on the samples. Fig. 2 illustrates a desmoplastic medulloblastoma containing small, polar cells with neuritic processes (panel A), which exhibited a strong immunoreactivity for synaptophysin (panel B). Nine of the tumor samples showed nuclear immunoreactivity when they were tested with a monoclonal antibody against the T antigen, corroborating our earlier observations on the detection of the T antigen in human medulloblastomas. Immunoreactivity for the T antigen was observed in the nuclei of neoplastic cells in nine samples. By immunohistochemistry, p53 (a protein that, upon association with the T antigen, remains nonfunctional but in a stable form) was localized in the nuclei of the neoplastic cells, which corresponded to the same cellular compartment in which T antigen was detected (Fig. 2
, C and D, respectively). Again, agnoprotein was found in cytoplasmic perinuclear regions of the neoplastic cells (Fig. 2
, E). No evidence for the production of the viral late protein VP1 was observed in the neoplastic cells (Fig. 2
, F). Examination of brain samples from patients with PML revealed an extremely weak immunoreactivity of the infected cells with an anti-T-antigen antibody, which is a typical feature of cells under lytic infection by polyomavirus due to the fact that viral early gene expression is reduced during later stages of infection when the capsid proteins are expressed (Fig. 2
, G). In contrast, robust cytoplasmic perinuclear and nuclear localizations were detected, respectively, for agnoprotein and VP1 in JCV-infected, enlarged oligodendrocytes (Fig. 2
, H and I, respectively). Table 1
summarizes the results from immunohistochemical and polymerase chain reaction analyses of medulloblastoma and PML samples. Immunohistochemical analysis showed cytoplasmic localization and widespread distribution of agnoprotein in the neoplastic cells in 11 (55%) of 20 samples.
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DISCUSSION |
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The etiology and pathogenesis of medulloblastomas are not fully understood. Risk factors associated with the development of medulloblastomas are radiation exposure, certain genetic heritable syndromes, which include neurofibromatosis type I, Turcot's syndrome (27,28), and the nevoid basal cell carcinoma syndrome or Gorlin's syndrome (29,30). Although other genetic alterations, including p53 germline mutations (31,32), unbalanced translocations, deletions and duplications of chromosomes 1 and 10q, and isochromosome 17q, have been detected in approximately 50% of the cases (33,34), the majority of these malignant embryonal tumors are sporadic and their etiology remains unknown.
The development of medulloblastoma in transgenic mice containing the JCV early genome provided the rationale for earlier efforts by our group and other groups (3537) to investigate the association of JCV with human medulloblastoma. In that setting, the major emphasis was on the viral early gene and on the expression of the T antigen in tumor cells. Detection of the JCV DNA sequence in surprisingly high numbers of cases prompted us to increase the scope of the study and to examine the expression of viral proteins that are encoded by the late regions in human medulloblastoma. Although we found no evidence for the expression of the late capsid proteins VP1, VP2, and VP3 (data not shown) in these samples, results from immunostaining showed the detection of agnoprotein in the tumor cells. It is interesting that, in some tumor samples, DNA sequences corresponding to the Agno gene and the expression of agnoprotein were found in the absence of the JCV early gene sequence and its oncogenic product, T antigen. This observation may imply a role for agnoprotein in the development of medulloblastoma.
Infection with JCV occurs in early childhood, with 65% of the population infected by 14 years of age (38), most likely through spread from parent to child (39). Reviews (40) have discussed a role for B lymphocytes in the circulation of the virus throughout the body because virus has been detected in B cells from healthy individuals. Although the specific mechanism by which JCV may enter the brain is unknown, one can envision a scenario in which initial infection with JCV in children results in virus entry into the brain by the infected B cells during cerebellar development, resulting in infection of the cells that are thought to give rise to medulloblastoma.
The detection of agnoprotein in the perinuclear cytoplasmic region of both tumor cells as well as in infected oligodendrocytes from patients with PML indicates that subcellular localization of the protein remains unchanged during the course of these two distinct pathologic events. Although the function of agnoprotein remains to be investigated, one may envision a role for this protein in enhancing various cellular processes, such as transcription and replication, which are essential for the viral lytic cycle. In the absence of viral lytic infection, stimulation of cellular events by agnoprotein may lead to rapid and perhaps uncontrolled growth of the cells and to the development of neoplasm. Indeed, not mutually exclusive, one may also assume an anti-apoptotic role for agnoprotein, which is beneficial for virus replication in the infection pathway and the maintenance of tumor cells and growth in the neoplastic pathway.
The detection of JCV in human medulloblastoma may indicate a role for the virus in a subset of medulloblastoma. We have demonstrated viral DNA sequences in tumors that do not express T antigen or agnoprotein, as well as the presence of only one of the viral proteins. Chromosomal instability has been attributed to the T antigen leading to loss of viral genes during the course of tumorigenesis, which can explain why the whole genome of JCV may not be detected in every tumor sample (41). In addition, studies have indicated that the T antigen may not be required to maintain a transformed phenotype, leading to loss of its expression (42). Thus, the absence of the T antigen in some tumor cells with or without agnoprotein is not surprising. Cooperation between the agnoprotein and the T antigen in early stages of tumor development, however, remains to be investigated.
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NOTES |
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Supported by Public Health Service grants NS36466 and NS30916 (to K. Khalili) from the National Institute of Neurological Disorders and Stroke, National Institutes of Health, Department of Health and Human Services.
We thank Dr. Susan Morgello (Director of the Manhattan HIV Brain Bank [R-24-MH-59724] at Mount Sinai School of Medicine, New York, NY) for providing the cases of progressive multifocal leukoencephalopathy and Dr. Walter Atwood (Brown University, Providence, RI) for providing antibodies and the SVG-A cell line. We express our gratitude to Dr. Mahmut Safak (Center for Neurovirology and Cancer Virology, Temple University, Philadelphia, PA) for his time in designing the peptides that were used for peptide synthesis and antibody production. We also thank the present and past members of the Center for Neurovirology and Cancer Biology for their support, insightful discussion, and sharing of ideas and reagents. We are grateful to Cynthia Schriver of the Center for Neurovirology and Cancer Biology for her editorial assistance and preparation of this manuscript.
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Manuscript received July 25, 2001; revised October 12, 2001; accepted December 18, 2001.
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