Abstract
Allelic losses of the q13.3 region of chromosome 19 have been documented in all major types of diffuse gliomas, strongly suggesting the presence of a 19q13.3 tumor suppressor gene responsible for these malignancies. The P78 gene precisely maps to 19q13.3, the glioma candidate region, and encodes a recently identified novel protein (P78). The purpose of this study was to determine P78 protein expression in gliomas. Serial analysis of gene expression (SAGE) reveals P78 mRNA expression to be significantly reduced in high-grade gliomas such as glioblastoma (GB), as compared with the low-grade tumors including astrocytomas, oligodendrogliomas, and ependymomas. We observed the distribution of staining of P78 protein was concentrated on the cell membranes of the luminal epithelial cells, not cytoplasm. In contrast, the pre-immune serum controls demonstrated no staining. These results demonstrate that P78 protein is highly expressed in the cytoplasmic membranes of low but not high-grade astrocytomas, and correlates with grade of malignancy. In these double immunostaining experiments, the anti-Map-2 and anti-NeuN antibodies did not stain round cells that were stained with the anti-P78 carboxyl-terminal peptide antibodies, demonstrating that these round cells were not neurons, and likely protoplasmic astrocytes. Current results also suggest that the astrocytes stained with the anti-P78 carboxyl-terminal peptide antibody are likely protoplasmic astrocytes. We also observed preincubation of anti-P78 carboxyl-terminal antibodies with immunizing peptides abolished immunostaining in gliomas. These results suggest a role for the P78 protein in the process of abnormal growth in glial tumors.
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Acknowledgments
We thank Lew Pointer and Marsha Wright for the assistance in the preparation of the rabbit anti-P78 amino-terminal peptide antibody, and Sigma Genosys for the generation of the rabbit anti-P78 carboxyl-terminal peptide antibody. We thank Chiang Wang, Ph.D., for assisting Dr. Bruce M. Frankel. This study was supported by a translational research grant from the American Brain Tumor Association and NIH-sponsored grants to Bruce M. Frankel (1R01FD003542-01).
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Conceived and designed the experiments: BF. Analyzed the data: BF. Wrote the draft of the manuscript: BMF, AD, and DC. Contributed to the writing of the manuscript: BMF, AD, SJP, and DC (all authors). Agree with manuscript results and conclusions: all authors. Jointly developed the structure and arguments for the paper: all authors. Made critical revisions and approved final version: BMF. All authors reviewed and approved of the final manuscript.
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This research was approved by the Institutional Review Board (IRB) for Human Research in the Office of Research Integrity at the Medical University of South Carolina (MUSC). GB samples were obtained and processed using a standard IRB procedure. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Frankel, B.M., Cachia, D., Patel, S.J. et al. Analysis of P78: A Novel Cytoplasmic Membrane-Associated Protein Encoded on Chromosome 19q13.3 in Glioma Specimens. J Mol Neurosci 70, 1415–1424 (2020). https://doi.org/10.1007/s12031-020-01562-3
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DOI: https://doi.org/10.1007/s12031-020-01562-3