Lack of Replicative Senescence in Cultured Rat Oligodendrocyte Precursor Cells Dean G. Tang, Yasuhito M. Tokumoto, James A. Apperly, Alison C. Lloyd, Martin C. Raff

Reagents and antibodies. All reagents were from Sigma unless indicated otherwise. PDGF-AA and neurotrophin-3 (NT-3) were purchased from Peprotech (Rocky Hill, NJ) and bFGF from Promega (Madison, WI). The following antibodies were used: goat anti-p15 (sc-1429; Santa Cruz Biotechnology, Inc); rabbit anti-p16 (sc-1207); monoclonal anti-p16 (2 different clones, JC8 and 16P07, NeoMarkers Inc., Union City, CA); rabbit anti-p18 (Upstate Biotech., Lake Placid, NY); monoclonal anti-p19 (sc-1665); goat anti-p19^ARF (sc-7403); rabbit anti-p19^ARF (R562, AbCam); monoclonal anti-p21 (sc-6246); monoclonal anti-p27 (sc-1641); goat anti-p57 (sc-1037); monoclonal anti-p53 (Pab246, which recognizes only wild-type p53, sc-100); goat anti-Rb (sc-50-G); monoclonal anti-actin (clone C4, ICN, Aurora, OH); monoclonal anti-cyclin A (E23); rabbit anti-cyclin B (sc-595); mouse mAb to cyclin D1 (sc-450); rabbit anti-cyclin D2 (sc-593); rabbit anti-cyclin D3 (sc-182); rabbit anti-cyclin E (sc-481); rabbit anti-Cdk-2 (sc-163); rabbit anti-Cdk-4 (sc-260); and rabbit anti-Cdk-6 (sc-177).

Purification and culture of OPCs. OPCs were purified from optic nerves of P7 rats by sequential immunopanning as previously described (1, 2) and cultured in serum-free, modified Bottenstein-Sato medium (3) containing PDGF (10 ng/ml) but no thyroid hormones. The medium also contained bovine insulin (5 mg/ml), NT-3 (5 ng/ml), human transferrin (100 mg/ml), BSA (100 mg/ml), progesterone (60 ng/ml), sodium selenite (40 ng/ml), N-acetyl-cysteine (63 mg/ml), putrescine (16 mg/ml), forskolin (2.5 mM), trace minerals, penicillin, and streptomycin. Culture maintenance and clonal analyses for survival, differentiation, and death were performed as previously described (2). To obtain enough cells for western blotting analysis of young OPCs, generally 100-120 P7 rats were required.

Western blotting. Whole cell lysates were made in complete RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 10 mM ETDA and a cocktail of protease inhibitors). Generally, 50-100 mg of protein was loaded in each lane of a 15% SDS-PAGE gel. Western blotting was performed as previously described, using ECL (4).

p53 checkpoint responses. OPCs were either used uninfected, or they were infected with either pBabepuro control vector or pBabepuro-p53¹⁷⁵ (dn-p53). Viruses were harvested from supernatants of transfected Phoenix packaging cells. About 48 h post infection, cells were treated with ALLN (100 mM x 12 h), adriamycin (10 nM x 12 h), or were x-irradiated (4 Gy; analysis was done 18 h after irridiation). Cells were then harvested, stained in suspension with propidium iodide (1 mg/ml), and analyzed by flow cytometry (5) to measure apoptosis. For p21 western blotting, cells were harvested 24 h post treatment, and 30 mg of protein was used for the analysis. Blots were probed for p21 and then reprobed for actin.

Response to oncogenic Ras (Ras^V12). OPCs were infected with either pBird or pBird-Ras^V12, using supernatants from transfected Phoenix packaging cells. Two types of experiments were subsequently performed. In the first, the effect of Ras^V12 overexpression on cell-cycle progression was evaluated 5 days post infection: BrdU (2.5 mM final concentration) was added to the culture medium for the last 4 h of culture, and the cells were then stained for BrdU as previously described (2). The % of BrdU⁺ cells in both GFP⁺ and GFP^- populations was determined in a fluorescence microscope. In the second type of experiment, OPCs were infected with the viral vectors and after 24 h were dissociated, plated at clonal density (100 cells/Nunc slide flask), and cultured for another 7 days. Then the number of cells per clone was counted in an inverted fluorescence microscope for multiple GFP⁺ and GFP^- clones. Thereafter, the cells were cultured for an additional 7 days and then assessed for SA-b-gal activity (6).

Rb-dependent checkpoint. 540 day OPCs were grown to confluence. Thirty micrograms of protein from low-density or confluent cultures was used in western blotting with antibodies that recognize both hypo- and hyperphosphorylated forms of RB. In a different set of experiments, OPCs were infected with a retroviral vector encoding either wild-type p16 (pBabepuro-2HA-p16) or a mutant form of p16 (pBabepuro-2HA-p16P48L)(7). Retroviruses were harvested from Phoenix packaging cells. Four days after infection, the OPCs were tested for BrdU incorporation as described above. In preliminary double-labeling experiments, we could not label the HA tag using either polyclonal or monoclonal antibodies, probably because the treatment for BrdU labeling inactivated the HA epitope. We therefore performed side-by-side experiments, in which the monoclonal anti-HA antibody was used to assess the overall infection efficiency in parallel cultures.

TRAP assay. OPCs cultured in PDGF without TH for 5, 120 or 480 days were solubilized in extraction buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, 5 mM 2-ME, 0.5% CHAPS and 10% glycerol). One hundred nanograms of protein for each sample was used in the TRAP assay, which contained a mixture of TRAP buffer (20 mM Tris-HCl, pH 8.0, 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween-20, 1 mM EGTA and 0.1 mg/ml BSA), 50 mM dNTP, 2 mg/ml of ³²P-end-labeled TS primer (5´-AATCCGTCGAGCAGAGTT-3´), 2 mg/ml of reverse (ACT) primer (5´-GCGCGGCTAACCCTAACCCTAACC-3´), and 40U/ml Taq polymerase (Promega). For one set of controls, 0.1 mg/ml DNase-free RNase was added. For another, cell lysates were preincubated at 94°C for 10 min before being added to the reaction mixture. All the samples were first incubated at 23°C for 30 min, followed by 94°C for 4 min. The samples were then amplified by PCR (94°C x 15", 60°C x 30" and 72°C x30" for 40 cycles), and the products were resolved on a 12.5% non-denaturing PAGE gel, followed by autoradiography.

Supplemental Figure 1. Telomerase activity in OPCs cultured for different times in PDGF was assayed as described above. Note that telomerase activity is not detectable when the samples are heat-inactivated (the right 3 lanes) or treated with RNase (not shown) before PCR amplification.

Medium version

References

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