[Frontiers in Bioscience 2, a37-45, November 1, 1997]

	[Frontiers in Bioscience 2, a37-45, November 1, 1997] Reprints PubMed CAVEAT LECTOR
	A p53 GROWTH ARREST PROTECTS FIBROBLASTS FROM ANTICANCER AGENTS E. Siobhan McCormack, Arthur M. Bruskin, Gary V. Borzillo OSI Pharmaceuticals Inc., 106 Charles Lindbergh Blvd., Uniondale, NY 11553-3649 Received 10/27/97 Accepted October 31, 1997 3. MATERIALS AND METHODS 3.1 Cell lines Low passage rat embryo fibroblasts (REFs) were seeded into 10 cm plastic dishes in Dulbecco's Modified Eagle Medium (DMEM) from GibcoBRL (Grand Island, NY) supplemented with L-glutamine and 10% fetal bovine serum from Sigma Chemical Company (St. Louis, MO). Cells were grown to 60-80% confluency, and were then transfected to generate stable transformed cell lines using a CellPhect transfection kit from Pharmacia (Piscataway, NJ). Plasmid constructs for the expression of C-MYC and activated H-RAS were kindly provided by Dr. John Haley of OSI Pharmaceuticals. Plasmids for the expression of human p53 proteins in mammalian cells were as follows: pC53-C1N₃ (for wild-type [wt] p53), pC53-CX3₃ (for mutation 143A), pC53-Cx22AN₃ (for mutation 175H), pC53-248₃ (for mutation 248W) and plasmid pC53-4.2N₃ (for mutation 273H). p53 constructs have been previously described (7). REF lines were cotransfected with the H-RAS plasmid, plus either the C-MYC plasmid or one of the p53 mutation plasmids, and selected in 800 microgram/ml G418 (Gibco/BRL). After 10-14 days of selection, G418-resistant colonies with a transformed morphology were picked using cloning cylinders from Bellco Glass Company (Vineland, NJ), and were seeded into 12-well plates for further expansion. A1-5 cells, containing murine p53 mutation 135V (8), were kindly provided by Dr. Arnold Levine. Levels of p53 expression in the lines were examined using a commercial p53 mutant-selective ELISA assay from Oncogene Research Products/Calbiochem (Cambridge, San Diego, CA), following the manufacturer's instructions. Results on select lines were corroborated by western blots or immunoprecipitations of [³⁵S]-labeled lysates (below). Cell division times at 37^°C and 31^°C were determined by seeding cells into 6-well plates followed by allowing cells to attach at 37^°C for 3 hours, at which time half of the plates were shifted to 31^°C. Every day thereafter, cells counts were made in duplicate, from which division times at both temperatures were calculated. 3.2 Protein analyses For the analyses of p53 and p21 expression by western blotting, REF cells were seeded into multiple 10 cm dishes (1-3 million cells/dish). Following an overnight incubation at 37^°C, half of the plates were shifted to 31^°C. Plates were incubated at the indicated temperatures for 24 hours, then washed twice in cold PBS and lysed in situ in 1 ml of a buffer composed of 150 mM NaCl, 50 mM Tris-Cl pH 8, 5 mM EDTA, 1% NP-40, 100 microgram/ml phenylmethylsulfonylfluoride (PMSF), 0.2% aprotinin and 1 microgram/ml leupeptin. Plates were kept on ice for 30 minutes, followed by scraping and transfer of the lysate to eppendorf tubes. Cellular debris was pelleted by a 15 minute spin in a microcentrifuge at 4^°C, and protein concentrations in the supernatant were determined using a Bradford assay from Biorad (Hercules, CA). Proteins were resolved by electrophoresis in 8-12% polyacrylamide gels, and transferred to nitrocellulose (Trans-Blot transfer medium, Bio-Rad). Filters were washed, blocked and probed as described in the ECL detection manual from Amersham (Arlington Heights, IL). p53 was detected using a combination of PAb 421 and DO-1 antibodies (both from Calbiochem), each at 1 microgram/ml. Rat p21/WAF1 was detected with culture supernatant mp21-22AA, generously provided by Marilee Burrell and Dr. David Hill from Oncogene Research Products/Calbiochem For analyses of [³⁵S]-radiolabeled proteins by immunoprecipitation, cells were seeded into 10 cm dishes and temperature shifted as before, then washed in PBS, and incubated with 5 mls of DMEM medium lacking methionine and cysteine from ICN (Costa Mesa, CA) containing 10% dialyzed fetal calf serum for two hours. The medium was then replaced with two mls of the same medium, containing 100 microCi Tran³⁵S-label (ICN; 800-1000 Ci/mmol) and incubated for 2-4 hours. Cells were lysed as above, and desired proteins were immunoprecipitated using antibodies preadsorbed to protein G sepharose beads from Pharmacia. Antibody 2A10 (9) was used to detect MDM2. Proteins were resolved by electrophoresis in 8-12% polyacrylamide gels and detected by autoradiography. For studies of the effect of temperature shifts on the expression of an integrated p53-responsive luciferase gene, plasmid pLH3 was transfected into the REF cells. Plasmid pLH3 encodes resistance to hygromycin-B, and contains a DNA sequence spanning four ribosomal gene cluster (RGC) sites (10) located upstream of the SV40 minimal promoter, which drives expression of the firefly luciferase gene. After 10-14 days of selection in 200 U/ml hygromycin-B from Calbiochem, 30-100 surviving colonies were trypsinized and pooled. The cells were expanded and seeded into quadruplicate wells of white 96-well plates from Becton Dickinson (Franklin Lakes, NJ). After overnight incubation at 37^°C, 6 of the plates were shifted to 31^°C. At 2-3 hour time-points, one plate at each temperature was processed for luciferase activity using an assay from Promega (Madison, WI). Light production was quantitated by reading the plates in a ML1000 microplate luminometer from Dynatech Laboratories, set to "Medium Gain." Readings from a single plate were averaged, standard deviations were calculated, and 31^°C/37^°C induction ratios were determined. 3.3 Chemoprotection colony assays To study the effects of a p53 growth arrest on cell survival, it was first necessary to generate IC₅₀ cytotoxicity plots for the REF lines exposed to different chemotherapeutic agents. The drugs chosen for study were cytosine beta-D-arabino-furanoside hydrochloride (Ara-c), etoposide, 5-FU, taxol, cisplatin and vinblastine (sulfate salt). The drugs were suspended in 100% DMSO at 3-25 mg/ml, aliquoted, and stored at –70^°C until use, except for cisplatin, which was freshly dissolved in DMSO prior to each experiment. All drugs were purchased from Sigma, except for taxol (ICN). First, cells were seeded into 96-well plates (250-1,500 cells/well) in 100 microliter of medium. Twenty-four hours later, two-fold serial dilutions of the drugs in medium were added to each REF line in triplicate wells, to give a final volume of 200 microliter. Cells were exposed to the drugs for 24 hours, followed by one PBS wash, the addition of 200 microliter fresh medium without drugs, and incubation at 37^°C for an additional 48 hours. More stringent washing resulted in sloughing and unacceptable levels of cell loss. The agent 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, from Sigma) was used as a measure of cell viability. Fifty microliter of a 0.2% solution of MTT in PBS was added directly to the medium of the wells; the plates were then returned to 37^°C for two hours. The media was aspirated, and the metabolized MTT formazan product was solubilized with 100 microliter of ethanol:acetone (50:50). Plates were read at 540 nm in a microplate reader from Bio-Tek Instruments (Winooski, VT), background absorbance from wells without MTT was subtracted, and cytotoxicity curves were plotted. For colony assays to assess chemoprotection, each cell line was seeded into four 6-well plates at 10-30K cells/well. The majority of experiments assayed five different lines. All plates were left at 37^°C for three hours for cell attachment to occur, at which time 2 of the plates were moved to 31^°C incubators. Since all drug and control additions were done at both temperatures, and in duplicates, this format allowed for a maximum of 5 drug concentrations plus the no drug (DMSO-only) controls to be tested. After preincubation of plates at the designated temperatures for 20 hours (to allow for growth arrests), the cells were exposed to the agents at the chosen concentrations for 24 hours at the same temperatures. The wells were washed once with PBS, re-fed with fresh medium without drugs, and returned to the 37^°C or 31^°C incubators for a final 18 hour incubation. The cells were then washed, trypsinized, and a known fraction were seeded into 10 cm dishes. All 10 cm dishes were incubated at 37^°C for 10-14 days, to allow colony formation to occur, at which time the dishes were stained with crystal violet or MTT, and the colonies were counted in duplicates. Final colony counts reflect normalization for the fraction of cells seeded. Results are shown in two ways: first, as a percentage of the colonies formed after drug exposure, relative to the same line incubated without drug on the y axis, versus drug concentration on the x axis. Separate plots were generated for cells exposed at 37^°C vs 31^°C, and the slopes were compared. Secondly, the IC₅₀values were calculated for the differentcell lines exposed to the various drugs at 37°C and 31°C and placed into a table.

[Frontiers in Bioscience 2, a37-45, November 1, 1997]
Reprints
PubMed
CAVEAT LECTOR

A p53 GROWTH ARREST PROTECTS FIBROBLASTS FROM ANTICANCER AGENTS

E. Siobhan McCormack, Arthur M. Bruskin, Gary V. Borzillo

OSI Pharmaceuticals Inc., 106 Charles Lindbergh Blvd., Uniondale, NY 11553-3649

Received 10/27/97 Accepted October 31, 1997

3. MATERIALS AND METHODS

3.1 Cell lines

Low passage rat embryo fibroblasts (REFs) were seeded into 10 cm plastic dishes in Dulbecco's Modified Eagle Medium (DMEM) from GibcoBRL (Grand Island, NY) supplemented with L-glutamine and 10% fetal bovine serum from Sigma Chemical Company (St. Louis, MO). Cells were grown to 60-80% confluency, and were then transfected to generate stable transformed cell lines using a CellPhect transfection kit from Pharmacia (Piscataway, NJ). Plasmid constructs for the expression of C-MYC and activated H-RAS were kindly provided by Dr. John Haley of OSI Pharmaceuticals. Plasmids for the expression of human p53 proteins in mammalian cells were as follows: pC53-C1N₃ (for wild-type [wt] p53), pC53-CX3₃ (for mutation 143A), pC53-Cx22AN₃ (for mutation 175H), pC53-248₃ (for mutation 248W) and plasmid pC53-4.2N₃ (for mutation 273H). p53 constructs have been previously described (7). REF lines were cotransfected with the H-RAS plasmid, plus either the C-MYC plasmid or one of the p53 mutation plasmids, and selected in 800 microgram/ml G418 (Gibco/BRL). After 10-14 days of selection, G418-resistant colonies with a transformed morphology were picked using cloning cylinders from Bellco Glass Company (Vineland, NJ), and were seeded into 12-well plates for further expansion. A1-5 cells, containing murine p53 mutation 135V (8), were kindly provided by Dr. Arnold Levine. Levels of p53 expression in the lines were examined using a commercial p53 mutant-selective ELISA assay from Oncogene Research Products/Calbiochem (Cambridge, San Diego, CA), following the manufacturer's instructions. Results on select lines were corroborated by western blots or immunoprecipitations of [³⁵S]-labeled lysates (below). Cell division times at 37^°C and 31^°C were determined by seeding cells into 6-well plates followed by allowing cells to attach at 37^°C for 3 hours, at which time half of the plates were shifted to 31^°C. Every day thereafter, cells counts were made in duplicate, from which division times at both temperatures were calculated.

3.2 Protein analyses

For the analyses of p53 and p21 expression by western blotting, REF cells were seeded into multiple 10 cm dishes (1-3 million cells/dish). Following an overnight incubation at 37^°C, half of the plates were shifted to 31^°C. Plates were incubated at the indicated temperatures for 24 hours, then washed twice in cold PBS and lysed in situ in 1 ml of a buffer composed of 150 mM NaCl, 50 mM Tris-Cl pH 8, 5 mM EDTA, 1% NP-40, 100 microgram/ml phenylmethylsulfonylfluoride (PMSF), 0.2% aprotinin and 1 microgram/ml leupeptin. Plates were kept on ice for 30 minutes, followed by scraping and transfer of the lysate to eppendorf tubes. Cellular debris was pelleted by a 15 minute spin in a microcentrifuge at 4^°C, and protein concentrations in the supernatant were determined using a Bradford assay from Biorad (Hercules, CA). Proteins were resolved by electrophoresis in 8-12% polyacrylamide gels, and transferred to nitrocellulose (Trans-Blot transfer medium, Bio-Rad). Filters were washed, blocked and probed as described in the ECL detection manual from Amersham (Arlington Heights, IL). p53 was detected using a combination of PAb 421 and DO-1 antibodies (both from Calbiochem), each at 1 microgram/ml. Rat p21/WAF1 was detected with culture supernatant mp21-22AA, generously provided by Marilee Burrell and Dr. David Hill from Oncogene Research Products/Calbiochem

For analyses of [³⁵S]-radiolabeled proteins by immunoprecipitation, cells were seeded into 10 cm dishes and temperature shifted as before, then washed in PBS, and incubated with 5 mls of DMEM medium lacking methionine and cysteine from ICN (Costa Mesa, CA) containing 10% dialyzed fetal calf serum for two hours. The medium was then replaced with two mls of the same medium, containing 100 microCi Tran³⁵S-label (ICN; 800-1000 Ci/mmol) and incubated for 2-4 hours. Cells were lysed as above, and desired proteins were immunoprecipitated using antibodies preadsorbed to protein G sepharose beads from Pharmacia. Antibody 2A10 (9) was used to detect MDM2. Proteins were resolved by electrophoresis in 8-12% polyacrylamide gels and detected by autoradiography.

For studies of the effect of temperature shifts on the expression of an integrated p53-responsive luciferase gene, plasmid pLH3 was transfected into the REF cells. Plasmid pLH3 encodes resistance to hygromycin-B, and contains a DNA sequence spanning four ribosomal gene cluster (RGC) sites (10) located upstream of the SV40 minimal promoter, which drives expression of the firefly luciferase gene. After 10-14 days of selection in 200 U/ml hygromycin-B from Calbiochem, 30-100 surviving colonies were trypsinized and pooled. The cells were expanded and seeded into quadruplicate wells of white 96-well plates from Becton Dickinson (Franklin Lakes, NJ). After overnight incubation at 37^°C, 6 of the plates were shifted to 31^°C. At 2-3 hour time-points, one plate at each temperature was processed for luciferase activity using an assay from Promega (Madison, WI). Light production was quantitated by reading the plates in a ML1000 microplate luminometer from Dynatech Laboratories, set to "Medium Gain." Readings from a single plate were averaged, standard deviations were calculated, and 31^°C/37^°C induction ratios were determined.

3.3 Chemoprotection colony assays

To study the effects of a p53 growth arrest on cell survival, it was first necessary to generate IC₅₀ cytotoxicity plots for the REF lines exposed to different chemotherapeutic agents. The drugs chosen for study were cytosine beta-D-arabino-furanoside hydrochloride (Ara-c), etoposide, 5-FU, taxol, cisplatin and vinblastine (sulfate salt). The drugs were suspended in 100% DMSO at 3-25 mg/ml, aliquoted, and stored at –70^°C until use, except for cisplatin, which was freshly dissolved in DMSO prior to each experiment. All drugs were purchased from Sigma, except for taxol (ICN). First, cells were seeded into 96-well plates (250-1,500 cells/well) in 100 microliter of medium. Twenty-four hours later, two-fold serial dilutions of the drugs in medium were added to each REF line in triplicate wells, to give a final volume of 200 microliter. Cells were exposed to the drugs for 24 hours, followed by one PBS wash, the addition of 200 microliter fresh medium without drugs, and incubation at 37^°C for an additional 48 hours. More stringent washing resulted in sloughing and unacceptable levels of cell loss. The agent 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, from Sigma) was used as a measure of cell viability. Fifty microliter of a 0.2% solution of MTT in PBS was added directly to the medium of the wells; the plates were then returned to 37^°C for two hours. The media was aspirated, and the metabolized MTT formazan product was solubilized with 100 microliter of ethanol:acetone (50:50). Plates were read at 540 nm in a microplate reader from Bio-Tek Instruments (Winooski, VT), background absorbance from wells without MTT was subtracted, and cytotoxicity curves were plotted.

For colony assays to assess chemoprotection, each cell line was seeded into four 6-well plates at 10-30K cells/well. The majority of experiments assayed five different lines. All plates were left at 37^°C for three hours for cell attachment to occur, at which time 2 of the plates were moved to 31^°C incubators. Since all drug and control additions were done at both temperatures, and in duplicates, this format allowed for a maximum of 5 drug concentrations plus the no drug (DMSO-only) controls to be tested. After preincubation of plates at the designated temperatures for 20 hours (to allow for growth arrests), the cells were exposed to the agents at the chosen concentrations for 24 hours at the same temperatures. The wells were washed once with PBS, re-fed with fresh medium without drugs, and returned to the 37^°C or 31^°C incubators for a final 18 hour incubation. The cells were then washed, trypsinized, and a known fraction were seeded into 10 cm dishes. All 10 cm dishes were incubated at 37^°C for 10-14 days, to allow colony formation to occur, at which time the dishes were stained with crystal violet or MTT, and the colonies were counted in duplicates. Final colony counts reflect normalization for the fraction of cells seeded. Results are shown in two ways: first, as a percentage of the colonies formed after drug exposure, relative to the same line incubated without drug on the y axis, versus drug concentration on the x axis. Separate plots were generated for cells exposed at 37^°C vs 31^°C, and the slopes were compared. Secondly, the IC₅₀values were calculated for the differentcell lines exposed to the various drugs at 37°C and 31°C and placed into a table.