[CANCERRESEARCH55,2746—2751, July 1, 1995] Advances in Brief Loss of Wild-Type p53 Bestows a Growth Advantage on Primary Cortical Astrocytes and Facilitates Their in Vitro Transformation1 Oliver Bogler,2 H-J. Su Huang, and Webster K. Cavenee Ludwig institute for Cancer Research (0. B., H-f. S. H., W. K. C.], Department ofMedicine [H-i. S. H., W. K. C.], and Centerfor Molecular Genetics 1W. K. CI, University of California San Diego, La Jolla. California 92093-0660 Abstract Primary cortical astrocytes were belated from normal (+1+), heterozy gous (+1—), or homozygous(—/—)p53-knockout mice.The normal astro cytes grew slowly and underwent crisis after limited division, while the homozygously defective cells grew rapidly and without contact inhibition. These —I— cells could not initially form colonies in soft agarose but acquired this capability after 10 passages in FCS or basic fibroblast growth factor but not epidermal growth factor. Almost all —I— astrocytes wealdy expressed glial fibrillary acidic protein at passage 10 and were also A2B5@ when cultured in basic fibroblast growth factor. Most heterozy gous cells resembled normal ones; however, some survived crisis, grew rapidly, and formed colonies. Outgrowing cells had all lost the wild-type p53 allele. These molecular and cellular events manic the early stages of human brain tumors, suggest a role forp53 in the earliest stages of disease progression, and provide an experimental system to analyze the effects of other tumor-specificmutations in the disease process. Introduction Loss or mutation of the p53 tumor suppressor gene is common in astrocytomas and is the earliest detected genetic event in their devel opment (1). The importance of this somatically acquired event is underlined by patients with the Li-Fraumeni syndrome, who harbor p53 mutations in theirgermline,andarepredisposed to thedevelop ment of tumors of the brain and other tissues early in life (2). Similarly, mice lacking wild-type p.53 genes develop brain tumors among a range of other cancers (3, 4). Here we report a system allowing the direct assessment of the role of p.53 in the growth properties of primary cortical astrocytes, the apparent precursors of astrocytomas. We isolated primary astrocytes from mice whose p53 genes were disrupted by homologous recombination (3) and found that early passage cells divided more rapidly than normal control cells, were immortalized, and did not exhibit contact inhibition but were not transformed. Continued passage of p53-null astrocytes under specific growth conditions resulted in cells able to form colonies in soft agarose, consistent with the acquisition of a transformed phenotype in culture. Most astrocytes, derived from mice bearing one disrupted and one wild-type p53 allele, behaved like normal astrocytes in that they grew slowly and underwent crisis. However rare clones, which mor phologically resembled —I— p53 astrocytes, grew out from these cultures, and these cells had lost the remaining wild-type p53 allele. These results suggest that astrocytes lacking p53 may represent an experimentally malleable system with which the functional role of the Received 5/3195; accepted 5/19/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Supported by the California Division of the American Cancer Society, Fellowship 1-62-95 (to 0. B.). 2 To whom requests for reprints should be addressed, at Ludwig Institute for Cancer Research, University of California San Diego, 9500 Oilman Drive, La Jolla, CA 92093-0660. many genetic lesions described for human brain tumors (5) can be assessed. Materials and Methods A pair of heterozygousTSG—p53 mice (Genpharm, Mountain View, CA), each bearing one wild-type and one disrupted p53 allele (designated +1—; Ref. 3) were mated to generate a litter containing all three genotypes: +1+ , +1—, and —I— . Cortical astrocyte cultures were prepared and purified as described previously (6, 7), except that separate cultures were established from individ ual cortices in 25-cm2 tissue culture flasks, and the treatment with 1-g3-D- arabinofuranosylcytosine was extended to 6 days. A portion of the scalp from each mouse was reserved during dissection for the preparation of genomic DNA that was used for genotyping by the PCR. Cells were passaged after purification, the initial passage number was designated as 0, and one culture representing each of the three genotypes was selected for further study. After an additional passage that was used to generate stocks of cells for storage, the astrocytes were plated in triplicate at a density of 50,000 cells/10-cm Petri dish and were grown in three conditions; they either remained in DMEM containing 10% FCE or were switched gradually and stepwise to DMEM containing 0.5% FCS, which was supplemented with either 10 ng/ml recombinant bovine bFGF3or 20 ng/ml recombinant human EGF (growth factors from Boehringer Mannheim, Mannheim, Germany). The media was changed, and growth factors were supplied every other day. After 14 days in vitro, one set of dishes was stained with crystal violet. After this initial period of culture, —I—astrocytes were subjected to sequential passage, with 100,000 cells/10-cm Petri dish being plated in tripli cate every 8 days. The cell number was assessed by counting after each passage, and their antigenic phenotype was assessed periodically by immuno cytochemistry using anti-GFAP and A2B5 antibodies as described previously (8, 9). Labeling with Ran-2 was also attempted, but labeling was too weak to be of value, as is often the case for rat cells (10). The growth of cells in soft agarose was monitored periodically using a standard method (11), except that when astrocytes that had been grown in DMEM-bFGF or DMEM-EGF were transferred into soft agarose (SeaPlaque, Rockland, ME), they received 20-fold less serum than called for by the standard protocol, and continued to receive growth factors every other day. The rare clones that grew out from the +1—dishes were ring-clonedand expanded; then genomic DNA was isolated, restricted with BamHI, and used for analysis by standard Southern blotting, using a full-length murine p53 cDNA as a probe (3). Results Purified cortical astrocytes (of which >99% expressed the astrocyte marker GFAP) were cultured from individual newborn mice that were litter mates from a mating between heterozygous (+1—)p53 knock out mice. Cultures of each genotype, [wild-type homozygote (+1+), knock out homozygote (—I—), and heterozygote (+1—)]were char acterized with respect to their growth rate, antigenic phenotype, and transformation. An indicationof the impact of p53 loss on the growth characteris tics of early passage astrocytes was obtained when second passage 3 The abbreviations used are: bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; GFAP, glial fibrillary acidic protein. 2746 on August 4, 2015. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from