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[Cancer Research 60, 1353-1360, March 1, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

Identification of a Human Glioma-associated Growth Factor Gene, granulin, Using Differential Immuno-absorption1

Linda M. Liau2, Roger L. Lallone, Robert S. Seitz, Alexei Buznikov, Jeff P. Gregg, Harley I. Kornblum, Stanley F. Nelson and Jeff M. Bronstein

Division of Neurosurgery [L. M. L.], Departments of Neurology [A. B., J. M. B.], Pathology [J. P. G.], Pharmacology [H. I. K.], and Human Genetics [S. F. N.], and the Jonsson Comprehensive Cancer Center [L. M. L., S. F. N.], University of California at Los Angeles School of Medicine, Los Angeles, California 90095; Brookwood Biomedical, Birmingham, Alabama 35209 [R. L. L.]; and Research Genetics Inc., Huntsville, Alabama 35801 [R. S. S.]

Identification of the genes that are differentially expressed in brain tumor cells but not in normal brain cells is important for understanding the molecular basis of these neurological cancers and for defining possible targets for therapeutic intervention. In an effort to discover potentially antigenic proteins that may be involved in the malignant transformation and progression of human glioblastomas, a novel antibody-based approach was developed to identify and isolate gene products that are expressed in brain tumors versus normal brain tissue. Using this method, whereby tumor-specific antibodies were isolated and used to screen a glioblastoma cDNA expression library, 28 gene products were identified. Nine of these clones had homology to known gene products, and 19 were novel. The expression of these genes in multiple different human gliomas was then evaluated by cDNA microarray hybridization. One of the isolated clones had consistently higher levels of expression (3–30-fold) in brain tumors compared with normal brain. Northern blot analysis and in situ hybridization confirmed this differential overexpression. cDNA sequence analysis revealed that this gene was identical to a relatively new class of growth regulators known as granulins, which have tertiary structures resembling the epidermal growth factor-like proteins. The 2.1-kb granulin mRNA was expressed predominantly in glial tumors, with lower levels in spleen, kidney, and testes, whereas expression was not detected in non-tumor brain tissues. Functional assays using [3H]thymidine incorporation indicated that granulin may be a glial mitogen, as addition of synthetic granulin peptide to primary rat astrocytes and three different early-passage human glioblastoma cultures increased cell proliferation in vitro, whereas increasing concentrations of granulin antibody inhibited cell growth in a dose-dependent manner. The differential expression pattern, tissue distribution, and implication of this glioma-associated molecule in growth regulation suggest a potentially important role for granulin in the pathogenesis and/or malignant progression of primary brain neoplasms.




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