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Reduction of Caveolin and Caveolae in Oncogenically Transformed Cells (Cancer/Growth Regulation/Oncogene/Potocytosis)

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Reduction of Caveolin and Caveolae in Oncogenically Transformed Cells (Cancer/Growth Regulation/Oncogene/Potocytosis) Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1381-1385, February 1995 Cell Biology Reduction of caveolin and caveolae in oncogenically transformed cells (cancer/growth regulation/oncogene/potocytosis) ANThoNY J. KOLESKE*, DAVID BALTIMORE*, AND MICHAEL P. LISANTIt *Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139; and tWhitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142 Contributed by David Baltimore, November 28, 1994 ABSTRACT Caveolae are flask-shaped non-clathrin- Caveolae may regulate the proliferation of normal cells in coated invaginations of the plasma membrane. In addition to vitro. We have recently observed that NIH 3T3 cell lines the demonstrated roles for caveolae in potocytosis and trans- transformed by several different oncogenes express greatly cytosis, caveolae may regulate the transduction of signals reduced levels of caveolin. Furthermore, the reduction of from the plasma membrane. Transformation of NIH 3T3 cells caveolin in these cell lines correlates with the size of colonies by various oncogenes- leads to reductions in cellular levels of they form upon growth in soft agar. Electron microscopy caveolin, a principal component of the protein coat of caveo- reveals that caveolae are missing from the transformed cells lae. The reduction in caveolin correlates very well with the size that express reduced levels ofcaveolin. The reduction in caveo- of colonies formed by these transformed cells when grown in lin does not correlate with serum requirements for growth of soft agar. Electron microscopy reveals that caveolae are mor- these cell lines in culture. The loss of caveolin could contribute phologically absent from these transformed cell lines. These to a loss of contact inhibition of motility or mitosis in trans- observations suggest that functional alterations in caveolae formed cells that may be critical to allow cells to proliferate in may play a critical role in oncogenic transformation, perhaps soft agar. by disrupting contact inhibition in transformed cells. MATERIALS AND METHODS Oncogenic transformation leads to many changes in cultured cells. These changes include the loss of a requirement for Cell Culture. Normal and transformed NIH 3T3 cell lines growth factors (1), alterations in cell-surface molecules and were cultured on 90- or 150-mm tissue culture plates in Dul- membrane fluidity (2-4), the loss of contact inhibition of cell becco's modified Eagle's medium (DMEM) containing 10% division and motility (5, 6), and the loss of anchorage depen- (vol/vol) donor calf serum (DCS) (GIBCO). The v-abl, poly- dence for growth (7). Measurements of these parameters are oma virus middle-sized tumor antigen (mTAg)-, bcr-abl used to evaluate the oncogenic potential of a gene or its mutant (p210)-, and crkl-transformed NIH 3T3 cell lines were the gifts derivatives. Despite the utility ofthese assays in scoring oncogenic ofWarren Pear, Ruibao Ren, and Martin Scott (Massachusetts potential in vitro, the molecular mechanisms that regulate these Institute of Technology). The H-rasGl2V-transformed cell line changes are poorly understood. A dissection of the molecular was the gift of David Sanders (Whitehead Institute). The mechanisms behind these changes should lead to enhanced un- v-src-transformed NIH 3T3 cell line was the gift of David derstanding of tumor initiation and progression in vivo. Shalloway (Cornell University, Ithaca, NY). To assay serum Caveolae are 50- to 100-nm flask-shaped invaginations of requirements for growth and survival, 1000 cells of each cell the plasma membrane Caveolae have been in line were plated onto multiple 60-mm plates in DMEM con- (8, 9). implicated taining 10% DCS. The medium was replaced the following day potocytosis and transcytosis of small molecules and ions in with DMEM containing 10%, 5%, 2%, or 0.5% DCS. Cells endothelial cells and sorting of surface proteins in polarized were fed every 3 days. After 10 days, colonies arising from the epithelial cells (8, 10-13). Several lines ofevidence suggest that plated cells were visualized by staining with 0.1% crystal violet caveolae may also participate in signal transduction. For ex- in 50% (vol/vol) MeOH. Colony numbers were normalized to ample, caveolin, a protein found on the inner striated coat of numbers observed on plates grown in 10% DCS. To assay caveolae, is a major v-src substrate in Rous sarcoma virus- growth in soft agar, 1000 cells of each line were suspended in transformed chicken embryo fibroblasts (14, 15). Both the 3 ml of DMEM containing 5% and 10% DCS and 0.33% phosphorylation of caveolin and transformation are depen- SeaPlaque low-melting-temperature agarose (American Bio- dent on the myristoylation of v-src (16). Furthermore, several analytical, Natick, MA). These cells were plated over a 2-ml G-protein-coupled receptors and bacterial toxins that modify layer of solidified DMEM containing 5% or 10% DCS and G proteins are localized to or internalized by caveolae (for 0.5% agarose, and cells were allowed to settle to the interface review, see ref. 17). Glycosyl-phosphtidylinositol (GPI)-linked between these layers at 37°C. After 20 min, plates were allowed proteins and the muscarinic acetylcholine receptor cluster to to harden at room temperature for 30 min before returning to caveolae in response to cross-linking antibodies or receptor 37°C. The plates were fed every 3-4 days by overlaying with 2 agonists, respectively (18, 19). Finally, purified caveolar mem- ml of medium containing 0.33% agarose. After 2 weeks, the brane domains are enriched in signaling proteins: Src-like plates were stained with 0.1% crystal violet in 50% MeOH at nonreceptor tyrosine kinases, heterotrimeric G proteins, the 4°C. Plates were destained with cold water. Colonies were ras-related GTPase Rap 1A, mitogen-activated protein ki- photographed under low (X4) magnification. nases, other serine/threonine kinases, SH2-SH3 adaptor pro- Western Blot Analysis. Cells were disrupted in RIPA buffer teins, and other proteins with roles in signal transduction (20 mM Mes, pH 6.5/150 mM NaCl/1% Triton X-100/0.1% (20-22). SDS/1 mM phenylmethylsulfonyl fluoride/2 mM benzamidine hydrochloride). Protein was quantitated with Bio-Rad protein The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: DCS, donor calf serum; mTAg, middle-sized tumor accordance with 18 U.S.C. §1734 solely to indicate this fact. antigen. 1381 Downloaded by guest on September 27, 2021 1382 Cell Biology: Koleske et al. Proc. NatL Acad Sci. USA 92 (1995) assay kit and bovine serum albumin as a standard. Blots were sion of the v-abl, bcr-abl,H-rasG12V, polyoma virus mTAg, or prepared by standard procedures (23). Blots were probed with crkl oncogenes. The relative amount of caveolin in the trans- anti-caveolin monoclonal antibodies (Transduction Laborato- formed cells was estimated by Western blot analysis with ries, Lexington, KY) or anti-Hsp7O polyclonal antiserum (a gift dilutions of NIH 3T3 protein extract as a standard (data not of Peter Murray, Whitehead Institute). The proteins were shown). Similar analysis was performed for the cytosolic heat visualized with horseradish peroxidase-conjugated secondary shock protein Hsp7O as a control for loading. The amount of antibodies and the ECL system (Amersham). Amounts of caveolin in transformed cells varied from about 30% of levels caveolin in transformed cells were determined by comparison found in normal NIH 3T3 cells (in crkl-transformed cells) to to standard dilutions of NIH 3T3 cell extract. less than 1% of normal levels (in v-abl-transformed cells) (see Northern Blot Analysis. Total RNA was extracted with Fig. 2). TriReagent LS (Molecular Research Center, Cincinnati) by Northern blot analysis of total cellular RNA revealed that the manufacturer's instructions. Fifteen micrograms of total caveolin mRNA was reduced in the transformed NIH 3T3 cell cellular RNA was denatured with glyoxal and subjected to lines in parallel with the amount of caveolin protein found in Northern blot analysis with 32P-labeled probes for the mouse these cell lines (Fig. 1B). In the one noteworthy exception, caveolin mRNA (2.4 kb) and human 28S RNA (a gift of Pat there appeared to be a slight induction of caveolin mRNA in Guilfoile, Whitehead Institute), as a control for loading. the NIH 3T3 cell line transformed by expression of polyoma Transmission Electron Microscopy. Cells were fixed with mTAg. Western blot analysis revealed that these cells had only glutaraldehyde, postfixed with OS04, and stained with uranyl 4% the level of caveolin found in normal NIH 3T3 cells, acetate and lead citrate as described (20). Samples were ex- suggesting that caveolin is either less stable or translated at a amined under the Philips 410 transmission electron microscope. reduce-d rate in these cells. Identical results were obtained by Cells were at a x S1 nuclease analysis with an oligonucleotide complementary photographed magnification of 115,500. Caveo- to caveolin mRNA (data not shown). laewere identified by their characteristic flask shape, size (50-100 Correlation with the Size of Colonies in Soft Agar. Onco- nm), and location at or near the plasma membrane. genically transformed cells are often able to grow under con- ditions that limit growth of normal cells. The normal or trans- RESULTS formed NIH 3T3 cell lines analyzed above were assayed for growth under restrictive conditions to gain insight into possible Caveolin Is Reduced in Transformed NIH 3T3 Cell Lines. roles of caveolin in regulating cell growth. When plated in soft Caveolin, a principal component of the protein coat of caveo- agar, normal cells will cease division whereas transformed cells lae, is phosphorylated on tyrosine in v-src-transformed fibro- will continue to proliferate. The NIH 3T3 cell lines were plated blasts, suggesting a relationship between alterations in the at low density in soft agar and incubated for 2 weeks.
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