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

4. RESULTS

The two most commonly studied temperature-sensitive (ts) p53 mutations, mouse 135V (8) and human 143A (11), were used in these studies. Constitutive "hot-spot" p53 mutations 175H and 248W served as negative controls, as did C-MYC, which (like mutations in p53) can collaborate with H-RAS to immortalize primary REF cells. The 135V and 143A mutations are ts for conformation, with incubation at 37-39.5^°C (the nonpermissive temperature) resulting in a mutant (mt) conformation, and incubation at 31-32.5^°C (the permissive temperature) resulting in a predominantly wild-type (wt) conformation. In cells bearing such mutations, growth at the permissive temperature correlates with increased localization of p53 to the nucleus, enhanced binding of p53 to DNA oligonucleotides containing p53-consensus sites, and increased transactivation of p53-responsive genes (8). Mutation 143A has been identified in a human gastric tumor line (12).

Immortalized REF lines were derived expressing activated H-RAS, plus either C-MYC, or one of four p53 mutations (135V, 143A, 175H and 248W). For simplicity, cloned lines are designated with either C-MYC, or one of the p53 mutations, with the clone designation in parentheses. All cell lines exhibited a transformed morphology with doubling times at 37^°C of 13-22 hours (table 1), although the 143A-expressing cells were flatter and less refractile than the other lines. Cells transfected with wt p53 constructs became growth arrested and permanent cell lines could not be derived. Temperature downshifts to 31^°C resulted in increased doubling times for all of the lines, although cells expressing C-MYC, 175H or 248W continued to grow rapidly and acidify the medium at this temperature. By contrast, the lines expressing p53 mutations 135V or 143A either failed to divide noticeably, or grew with division times of 70 hours or greater. Similar results for K562 cells bearing 143A have been reported previously (11). The growth inhibition for lines with ts p53 mutations could be reversed by returning the cultures to 37^°C (see below). Incubation at 31^°C did not result in detectable levels of apoptosis in any of the lines, based on studies of cell viability, and a comparison of DNA samples from cells maintained at either temperature (data not shown).

Activation of wt p53 results in increased transcription of a set of p53-responsive genes (9, 14), leading to increased levels of the corresponding proteins. When metabolically-labeled lysates of REF lines were immunoprecipitated with an antibody (PAb 421) to p53, significantly higher levels of p53 were observed for the cells transformed with p53 mutations, relative to the cell lines transformed with C-MYC and expressing only endogenous rat p53 (figure 1A, top). A coprecipitating band in the 70 kDa range, presumably hsc70 (7), was seen for cells expressing 135V, 143A or 175H at 37^°C; when incubated at 31^°C, this band was reduced in intensity only for cells expressing 135V or 143A. By contrast, a protein in the 90 kDa range was observed to coprecipitate preferentially with cells expressing either 135V or 143A at 31^°C. The size of the protein and its expression pattern was highly suggestive of MDM2, which increases in quantity in response to p53 activation (9). MDM2 has been shown to form a physical complex with p53, and to inhibit p53 transactivation functions in a manner suggestive of a negative feedback mechanism. Accordingly, precipitation of the lysates with antibody 2A10, which recognizes rat MDM2 (9), revealed increased MDM2 levels in the 135V and 143A lines at 31^°C (figure 1A, middle). As expected, the MDM2-coprecipitating proteins in the 50 kDa range were identified as p53, based upon western blots of anti-MDM2 precipitates probed with horseradish peroxidase-conjugated antibodies to p53 (data not shown).

Figure 1. Activation of wild-type p53 responsive genes in REF cells bearing the ts p53 mutations 135V and 143A. A (Top), Five REF lines were labeled at either 37°C (- lanes) or 31°C (+ lanes) with [³⁵S]-methionine, lysed, and an aliquot was immunoprecipitated with antibody 421 to detect p53. The numbers on the right represent molecular weight markers in kDa. (Center) A second aliquot of lysates was precipitated with antibody 2A10 to detect endogenous MDM2. (Bottom), A western blot of unlabeled lysates was probed to detect endogenous p21^Waf1. B, p53 mutations 135V and 143A are ts for the expression of integrated wt p53-responsive luciferase genes. REF cells expressing the p53 mutations shown were transfected with plasmid pLH3, containing a firefly luciferase gene expressed from a minimal SV40 promoter containing four p53 consensus sites (10). After selection in hygromycin, cells were seeded into white 96-well microtiter plates and incubated at 37°C. Sixteen hours later (time 0), half of the plates were placed at 31°C. At 2 to 3 hour timepoints thereafter, one plate at each temperature was processed for luciferase activity. The results are plotted as fold induction, defined as light signal (31°C/37°C). Each point is the mean of quadruplicate wells, with all coefficients of variation < 20.

The cell doubling times and MDM2 expression patterns were consistent with the temperature-dependent expression of wt p53 functions in the lines bearing p53 mutations 135V or 143A. Western blots of whole cell lysates, probed with an antibody to p21/WAF1, revealed increased p21 expression in the cells presumed to express wt p53 (figure 1A, bottom). Unlike the results with MDM2, detectable baseline levels of p21 were observed for all the REF lines, consistent with previous observations that wt p53 signaling is not required for the expression of p21 (15).

p53-responsive reporter genes have proven useful for studies of chemicals, proteins and mutations which impinge on p53 functions, and could conceivably be used to identify novel p53-activating mechanisms. Here, a wt p53-responsive luciferase gene (see Materials and Methods) was used to confirm the ts nature of mutations 135V and 143A, and to follow the acquisition of wt p53 transactivation properties as a function of time at 31^°C. Such reporter genes generally provide variable p53-independent baseline signals, which can be increased from 10 to >1000-fold by the presence of wt p53 ([11], and data not shown). Incubation at 31^°C for five hours was sufficient to trigger increased levels of luciferase expression in the 135V and 143A populations, which peaked at nine hours (figure 1B). At later timepoints, the 31^°C/37^°C values decreased, due to the continued growth of the cells at 37^°C and consequent increases in the luciferase baselines. In the chemoprotection assays below, cells were preincubated at 31^°C for 20 hours prior to the addition of the chemotherapy agents.

4.1 Protection from taxol

Many chemotherapy drugs inhibit tumor growth by interfering, either directly or indirectly, with DNA replication or mitosis. Irrespective of whether the killing of these normal populations is via necrosis or apoptosis, the induction of a temporary growth arrest (outside of S or M phase) during the period of drug exposure might result in enhanced survival. Briefly, a 20 hours pre-incubation at 31^°C was used to arrest the cells with ts p53 mutations, prior to co-incubation with chemotherapy drugs for 24 hours, also at 31^°C. After the drugs were washed out of the cultures, the designated plates were returned to 31^°C for an additional 18 hours. The reasoning behind extending the protection "window" beyond the point of drug removal was to allow for the intracellular drug concentrations to decrease before allowing the cells to resume cell division at 37^°C. The cells were trypsinized, diluted and replated, such that individual cells gave rise to colonies. The assay thus requires that a protected cell will retain not only viability, but be able undergo a sufficient number of rounds of replication (> 6) to be counted.

Results in figure 2 are shown for taxol, an agent that acts at the level of mitosis, through an enhancement of tubulin polymerization and consequent stabilization of microtubules (16). At 31^°C, ts p53 mutations mediated a growth arrest that clearly protected the 135V and 143A-expressing lines from this drug. All REF lines exposed to taxol at 37^°C exhibited an inhibition of colony formation that was >90%, even at the lowest concentration tested (0.5 microgram/ml; figure 2, left). When the MYC, 175H and 248W-expressing clones were exposed at 31^°C, plating efficiency in response to 0.5 microgram/ml improved slightly (figure 2, right), but virtually no colonies formed from cells exposed to 2 or 10 microgram/ml. Similar results were observed with two additional clones, 175H (D), and 248W (B) (not shown). By contrast, the 135V and 143A-expressing cells retained >37% colony forming efficiency when exposed to drug at 31°C. An additional clone, 143A (D) was similar to the 135V (A1-5) line in its survival profile (not shown). Visual inspection of the stained dishes revealed that colony sizes derived from the treated versus untreated cells were not different (not shown).

Figure 2. Transient induction of wt p53 activity by temperature shift correlates with chemoprotection from high doses of taxol. Left, the REF clones shown were exposed to different doses of taxol for 24 hours at 37°C (no temperature shift), washed and processed for colony assays as described in Methods. Right, the same lines were processed similarly, except that drug addition occurred after temperature shift to 31°C. After trypsinization and seeding, colonies were stained and counted ten to fourteen days later. The points shown represent the mean (with standard deviation [SD]) from triplicate plates from one of two independent experiments with similar results.

4.2 Protection from vinblastine, etoposide and Ara-C

Vinblastine acts to prevent the polymerization of tubulin dimers, disrupting the formation of microtubules. While the primary cytotoxicities of vinblastine and other vinca alkaloids occur in metaphase, the agents may also act in other phases of the cell cycle (16). Vinblastine was a potent inhibitor of colony formation, generating >80% inhibition at 15 ng/ml, for all REF lines exposed at 37^°C (figure 3, top). REF lines expressing MYC, 175H or 248W were similarly inhibited when exposed at 31^°C. By contrast, the 143A line retained >85% of its clonogenicity at 31^°C, after drug exposures of up to 125 ng/ml, whereas protection of the 135V (A1-5) line plateaued at about 40%. Thus, enhanced colony formation after drug exposure correlated with wt p53-mediated growth arrests, and was observed over a range of vinblastine concentrations (12.5 to 125 ng/ml). Similar results using vinblastine were seen in 2 subsequent experiments, in which the concentrations of drug tested were 0, 2, 10 and 150 ng/ml (summarized in table 2).

Figure 3. Induction of wt p53 activity correlates with chemoprotection from vinblastine and etoposide. Top, the REF clones shown were exposed to vinblastine at 37°C or 31°C, and processed for colony assays. Data points represent the mean (with SD) from triplicate plates from one of three independent experiments with similar outcomes. Bottom, Lines were exposed to the indicated doses of etoposide at both temperatures and processed similarly. The data points represent the mean (with SD) from triplicate plates from one of two independent experiments with similar outcomes.

The p53-mediated growth arrest also correlated with chemoprotection from etoposide, an epipodophyllotoxin that is thought to exert its major cytotoxic effects by inducing DNA strand breaks consequent to interactions with topoisomerase II (16). The highest concentration of etoposide tested (3 microgram/ml) was sufficient to inhibit 100% colony formation in all of the lines exposed at 37^°C, as well as for the control lines exposed at 31^°C (figure 3, bottom). By contrast, the 143A and 135V-expressing lines retained significant clonogenicity (70% and 38%, respectively) when exposed to 3 microgram/ml drug at 31^°C. Relative to the DMSO-only control plates, a slight increase in clonogenicity following drug exposures of 0.5 and 1 microgram/ml was observed for the 143A-expressing line. The mechanism underlying this repeatable observation is unknown, and has been observed previously using TGFB3 as the arresting agent on CCL64 epithelial cells (5).

The main toxicity of Ara-C, an antimetabolite, arises from the ability of its active form (Ara-CTP) to be incorporated into DNA, and to act as a competitive inhibitor of DNA polymerase. These effects are consistent with the drug's selective toxicity for cells in S phase (16). Ara-C was tested at five concentrations ranging from 25-500 ng/ml, against six cell lines (in triplicate) from table I: MYC (C), 135V (A1-5), 143A (C), 143A (D), 175H (D) and 248W (B). For all of the lines exposed at 37^°C, >75% inhibition of colony formation was seen for the two highest concentrations (250 and 500 ng/ml) of Ara-C tested (table 2). When added to cells at 31^°C, the same two concentrations inhibited colony formation by >95%, but only for the control cell lines. As was observed with the previous three agents, the 135V and 143A mutations enhanced viability when cells were exposed at the permissive temperature.

4.3 p53 enhances survival weakly for cells treated with 5-FU, and does not protect from cisplatin

The compounds shown in figures 2 and 3 are known to exert their maximum cytotoxicities in the S or M phases of the cell cycle (16). Since p53 has been shown to arrest cells in G₁ (8), the results are consistent with the idea that a transient arrest of the cell cycle, outside of S or M phase, can be exploited to protect cells from a subsequent exposure to chemotherapy. However, not all anticancer drugs are restricted in their actions to particular phases of the cell cycle. We reasoned that a p53-mediated growth arrest might fail to protect from such agents, and might even exacerbate toxicity. Two agents that were not significantly affected in their cytotoxicity profiles by wt p53 function were cisplatin and 5-FU.

The antitumor activity of cisplatin involves the crosslinking of intracellular molecules, primarily DNA. Cells in all stages of the cell cycle are susceptible, although cells in G₁ may be the most affected (16). Virtually no protection was conferred by wt p53 for this agent. Five concentrations of cisplatin (0, 0.1, 1, 5, 10 and 20 microgram/ml) were tested figure 4, as well as lower concentrations in subsequent experiments, and the cytotoxicity plots at both temperatures were virtually superimposable.

Figure 4. Induction of wt p53 activity fails to chemoprotect from an agent (cisplatin) that can damage cells in G₁ phase. The REF clones indicated were exposed to cisplatin at the doses shown at 37°C or 31°C, and processed in colony forming assays. Data points represent the mean (with SD) from triplicate plates from one of two experiments with similar outcomes.

Active metabolites of 5-fluorouracil (5-FU), a pyrimidine analog, are incorporated into both RNA and DNA (16). Incorporation into RNA causes defects in nuclear processing and polyadenylation, whereas incorporation into DNA causes strand breakage and fragmentation. Based on the initial cytotoxicity curves, three concentrations of 5-FU (0.5, 5 and 50 microgram/ml) were chosen for testing in colony assays against the five REF lines in figure 4. Concentrations of 5-FU >5 microgram/ml were sufficient to inhibit colony formation by >90% for all the lines exposed at 37^°C; the same concentrations at 31^°C inhibited colony formation 100% for the cells lacking ts mutations (summarized in table 2). At 31^°C, colony-forming efficiency was reduced to 35% and 20% for the 143A and 135V-expressing lines, respectively, but only at 5 microgram/ml. Chemoprotection was not observed at 50 microgram/ml.