p53 mutations and tetraploids under r- and K-selection

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Abstract

Cotransfection of rat embryo fibroblasts with c-myc and activated H-ras oncogenes is one experimental model of the multistep oncogenesis associated with p53 mutations and aneuploidy. Using the model, we found that selection processes, e.g., r- and K-selection, affect emergence of p53 mutants and tetraploids. Culture optimum for logarithmic growth (r-selection) selected p53 mutants as they proliferated rapidly, while in confluent culture (K-selection) tetraploids emerged regardless of the p53 status. Transfection of the mutated p53 gene with dominant negative functions eradicated untransfected cells under both r- and K-selection. However, these p53 mutants can be eradicated under K-selection by cells with normal p53 function and that had been selected under prolonged K-selection. The presence of competitors and the type of selection should determine whether or not p53 mutants and/or tetraploids predominate. These observations strengthen the importance of selection processes in case of cancer.

Introduction

The p53 tumor suppressor gene has multiple functions and is mutated in half the number of human tumors. The maintenance of genetic integrity is one of the functions of p53 and dysfunctions cause genetic instability culminating in aneuploidy, a hallmark of cancers (Galipeau et al., 1996; Ramel et al., 1995; Yin et al., 1999). However, genetic instability per se is not a growth advantage unless p53 mutants gain a certain population in which a variety of genotypes for clonal evolution can be generated in the presence of genetic instability. Among the consequences of p53 mutations, there are resistance to hypoxia (Graeber et al., 1996), ribonucleotide deficiency (Linke et al., 1996) and an acidified microenvironment (Williams et al., 1999). Thus a reduced apoptosis may be advantageous for mutant cells in a corresponding microenvironment. Therefore, p53 mutants may be selected under unfavorable conditions.

In evolutionary biology, r- and K-selection was initially described by MacArthur and Wilson (1967) for species evolution in isolated islands: r is an intrinsic growth rate and K is carrying capacity of the environment. r-selection favors a higher population growth rate, higher productivity and thus a small body size. This form of selection will come to the fore during the colonizing episode and hence must frequently build back up to K. K-selection favors a more efficient utilization of resources and a larger body size. This form of selection will be more pronounced when the species is at or near K (MacArthur and Wilson, 1967; Pianka, 1970). We made use of r- and K-selection to study rat embryo fibroblasts (REFs) transfected with c-myc and activated H-ras (EJ-ras) oncogenes. Cotransfection of REFs with these two oncogenes lead to in vitro focus formation and in vivo tumor development (Land et al., 1983). Such a tumorigenic model has accompanying additional genetic events involving the p53 gene and others, e.g., point mutation, allele loss, gene amplification and/or aneuploidy, recapitulating a multistep nature of cancer development (Thompson et al., 1989). Using this classical tumorigenic model, we asked whether r- and K-selection would affect cancer development. Initially, we speculated the emergence of p53 mutants under unfavorable K-selection. However, p53 mutants have growth advantages even in r-selection. On the contrary, we observed emergence of tetraploids only under conditions of K-selection, which is reminiscent of the hypothesis described by MacArthur and Wilson (1967). We discuss the significance of r- and K-selection in cancer development.

Results

Emergence of p53 mutants and tetraploids under r- and K-selection

We cotransfected human c-myc and EJ-ras expression plasmids in REFs, using the calcium phosphate method. Resultant transformants expressed these transfected genes, formed foci on the monolayer of untransformed cells and tumors formed in nude mice, as reported (data not shown) (Motokura et al., 1995; Uchimaru et al., 1996). We cloned six diploid c-myc and EJ-ras transformants, MR5, 6, 8, 9, 11 and 12, from well-isolated foci and passaged them under conditions of r- and K-selection. For r-selection, cells were plated sparsely and passaged well before confluence in order to maintain logarithmic growth. For K-selection, cells were passaged densely and growth was constrained because of overcrowding. The diploid DNA contents of these six clones were fairly constant under r-selection (Figure 1a,b). Cells were fusiform and overlapped and morphologies were largely unchanged. Under K-selection, cells were initially arrested at the G0/G1 phase, then tetraploid cells emerged and remained in substantial proportion (Figure 1a,b). Meanwhile, a large proportion underwent apoptosis, as evidenced by the presence of a sub-G1 peak in flow-cytometric analysis and the presence of DNA ladders (data not shown). After the ploidy shift, tetraploids were readily isolated: numbers of tetraploid subclones/numbers of subclones established were 3/5 in MR5, 3/7 in MR8 and 2/8 in MR9 (Figure 1b). Usually these tetraploids were flat cells forming a monolayer, which differed from r-selected diploids (Figure 1c). Cytogenetic analysis revealed their tetraploidy (data not shown). These tetraploids were derived from diploid transformants because integration of the transfected plasmid was the same as determined using Southern blot analysis (Figure 1d).

Figure 1
figure4

Six c-myc and EJ-ras cotransfected clones, MR5, 6, 8, 9, 11, 12 cells, under r- and K-selection. (a) Cell numbers are plotted on a semilogarithmic scale (upper part) and polyploid cells are indicated in per cent (lower part). Open symbols denote r-selection and closed ones K-selection. (b) Cell-cycle distribution of MR5 cells was determined by flow cytometry at the indicated time under r (left side)- and K (right side)-selection. A tetraploid subclone derived from K-selection is at the top. (c) Morphologies by phase-contrast microscopy of MR5 and MR8 cells under r- and K-selection and a K-selected tetraploid subclone, magnification ×50. Bars denote 50 μm. (d) Southern blot analysis of MR5, MR8 and MR9 parent cells (p), r-selected cells (r) and K-selected tetraploids (t). Conventional techniques were used for DNA extraction and for Southern blot analysis of HindIII digests (10 μg/lane) of genomic DNAs using the ClaI/HindIII fragment of pGEMmycB (Halazonetis and Kandil, 1991) as a radioactive probe. DNA sizes are shown on the right (kilobases). Ethidium bromide-stained gel shows loading of DNA (lower part). (e) Part of silver-stained gel of RT–PCR–SSCP analysis shows p53 mutation and INK4A/ARF deletion before (p) and after r (r)- and K (K)-selection and in tetraploid subclones (t). W, wild-type allele; M, mutant allele; *, double-strand DNAs

After c-myc immortalization, p53 gene mutation or the INK4A/ARF locus deletion occurs frequently (Zindy et al., 1998). Therefore, we examined these gene aberrations in clones using RT–PCR and SSCP analyses followed by direct sequencing in case of aberrant signals. The REFs we used had a point mutation of the INK4A/ARF locus, CTT to TTT, leading to substitution of Phe for Leu 151 in p16INK4A protein. It is located in the noncoding region of the ARF gene and may be a polymorphism. Two clones had the INK4A/ARF deletions and these did not correlate with ploidy shift nor with selection process (Figure 1e). One more clone, MR12, might have one deleted allele or might be homozygous for the locus. MR5, one of six clones, carried the p53 gene missense mutation, CTG to ATG, leading to substitution of Met for Leu 109 in the DNA binding domain. A tetraploid subclone derived from K-selected MR5 had a deleted wild-type allele of the p53 gene resulting in loss of heterozygosity (LOH) (Figure 1e), a finding that we initially speculated might be causally associated with ploidy shift.

Emergence of p53LOH and not tetraploids under r-selection

To determine if the LOH is involved in ploidy shift and whether K-selection induces LOH, we repeated MR5 culture under r- and K-selection in quadruplicate and monitored the occurrence of ploidy shift and LOH. Although ploidy shift was reproducible under K-selection (Figure 2a), it was not associated with LOH that occurred under r-selection in two of four cultures, despite an incomplete LOH under K-selection (Figure 2b). We also subcloned and subjected 10 diploid MR5 cells to r- and K-selection to examine the mode of emergence of de novo LOH. Under K-selection, seven of 10 subclones became tetraploid although p53 LOH was newly obtained only in three of nine and preceded ploidy shift in a single case alone (Figure 2c). Conversely, no ploidy shift occurred under r-selection (data not shown) and wild-type alleles were continually deleted in parallel with shortening of doubling times: the mean doubling times±s.d. of parent MR5 cells and resultant cells with p53 LOH were 16.1 h±1.1 h (n=4) and 9.8 h±0.3 h (n=7), respectively (Figure 2a,d,e). Deletion of the wild-type allele should confer a selective advantage under r-selection, i.e., accelerated growth, as noted by Harvey et al. (1993) who used fibroblasts from p53-knockout mice. In addition, the sigmoid curve of LOH emergence shown in Figure 2e reflects a logistic curve of the selection process and strongly suggests the clonal evolution of LOH (Crow and Kimura, 1970). Delayed and incomplete emergence of LOH shown as squares in Figure 2e can be explained as a result of competition with rapid growers with mutations other than p53 LOH because the doubling times were already shortened at the beginning of r-selection. Our findings suggest that prevalent p53 mutations in human cancers can be attributed in part to r-selection.

Figure 2
figure3

MR5 cells under r- and K-selection. (a) Culture conditions were renewed after 6 weeks. Open circles denote r- and then K-selection. Closed circles denote K- and then r-selection. (b) Ethidium bromide-stained gel of PCR–SSCP analysis for the p53 zygosity after r- and K-selection of 6 weeks. W, wild-type allele; M, mutant allele. (ce) Ten subclones derived from MR5 cells were subjected to r- and K-selection and analysed for p53 zygosity, DNA ploidy and doubling times at the indicated time. (c) Ploidy shift and p53 LOH under K-selection. Open circles denote cells in a heterozygous state and closed circles denote cells with LOH. (d) Weekly PCR–SSCP analysis of subclones under r-selection. Alleles are shown as in (b). Subclones after 8 weeks of K-selection are at the very bottom. (e) Emergence of cells with p53 LOH under r-selection. Time is plotted from the point when MR5 parent cells were plated sparsely for subcloning. The proportion of cells with p53 LOH was calculated from densitometrical analysis, using ethidium bromide-stained gel, given that simple deletion of the wild-type allele occurred, according to the formula as follows: (mw)/m, m is a signal density of the mutant allele and w is that of the wild-type. Doubling times were calculated from a fold increase during the previous week. Circles denote subclones acquiring complete LOH and squares denote subclones with incomplete LOH during the experiment. Closed circles correspond to more than 80% of cells with LOH

After 6 weeks of r-selection, we found that ploidy shift was not reproducible under K-selection (Figure 2a). The potential for ploidy shift in culture appeared to be lost during r-selection although estimated cell numbers reached under the r-selection were far more numerous than 6×1013 cells present in a human body, thus an unrealistic situation. On the contrary, when K-selection was changed to r-selection, tetraploid cells were rapidly replaced with diploids. The doubling times±s.d. of diploids and tetraploids subcloned from K-selected culture were 10.7 h (n=2) and 18.1±0.3 h (n=3), respectively, a difference largely due to different lengths of G1 phases (4.7 h vs 9.0±0.4 h, calculated using flow-cytometric analysis and the RFIT model). Therefore, diploids grew more rapidly than tetraploids. Lower ploidy correlates with a higher growth rate in yeast cells (Galitski et al., 1999) and, according to our findings, even in mammalian cells. Therefore, r-selection prefers diploids as well as p53 mutants due to a simple competition for growth rates.

p53 dysfunction is not required for ploidy shift under K-selection

To confirm that p53 dysfunction is unnecessary for ploidy shift, we examined MR8 cells. These cells had no p53 mutation and expressed p21CIP1 protein in response to doxorubicin (DXR) treatment, thereby suggesting normal p53 functions in sharp contrast with MR5 cells with p53 LOH that had an almost abolished response (Figure 3a). Tetraploids emerged again in three of four cultures under K-selection (Figure 3b). A prolonged culture resulted in different proportions of tetraploids under K-selection but not under r-selection. An isolated tetraploid subclone retained the p53 and p21CIP1 response to DXR treatment, as shown in Figure 3a. We also subjected 12 subclones derived from MR8 cells to r- and K-selection and observed one apparent ploidy shift and two other slight increases in a proportion of tetraploids only under K-selection (date not shown). We found no emergence of p53 mutation or INK4A/ARF deletion in these cultures, except for one subclone which had one allele of the INK4A/ARF locus deleted under r-selection (data not shown). Therefore, ploidy shift is not dependent on dysfunction of the p53 pathway rather it depends on K-selection.

Figure 3
figure2

MR8 cells under r- and K-selection. (a) Western blot analysis of p53 and p21CIP1 proteins in MR5 parent cells (p), a subclone with p53 LOH (L), MR8 parent cells (p) and a tetraploid subclone derived from K-selection (t) before (−) and 24 h after (+) 2 h treatment with doxorubicin (DXR 0.5 μM). Ink-stained membrane shows loading of whole cell lyzates at the bottom. (b) MR8 cells were subjected to r- and K-selection in quadruplicate and DNA contents were measured. Open circles denote r-selection and closed ones K-selection

Predominance of transfected p53 mutants under r- and K-selection

Because p53 mutations spontaneously developed under r- and K-selection, we speculated that transfection with the p53 dominant negative mutant would lead to the predominance of transfectants under r- and K-selection without any selection for drug resistance. Two days after electroporation of the mutated p53-expressing plasmid, p53KH215, to MR8 cells, cells were subjected to r- and K-selection for 6 weeks. At the beginning of selection, expression of a murine p53 sequence was not detected using RT–PCR. Weeks later, the p53KH215 sequence was readily detectable both under r- and K-selection, indicating that p53 dysfunction is advantageous for both r- and K-selection (Figure 4, lanes 4–9). All 10 subclones established after 6 weeks' r-selection expressed the p53KH215 sequence thereby indicating the predominance of p53KH215-transfected cells (data not shown). Under r-selection, emergence of p53 transfectants was associated with more rapid growth rates compared to mock transfection and under K-selection, increased cell density was readily achieved along with expression of the transfected p53KH215 sequence (data not shown). We also used another mutated p53, p53val135 (Michalovitz et al., 1990), and found similar results (data not shown). Therefore, p53 mutations are selected for under both r- and K-selection.

Figure 4
figure1

Emergence of p53 mutants under r- and K-selection and competition between p53 mutants vs K-selected cells. Two days after electroporation with the murine p53 mutant-expressing plasmid, p53 KH215, to MR8 parent cells (p, lane 2), cells were subjected to r- and K-selection for 6 weeks (lanes 3–9). After 6 weeks' r-selection, cells were admixed in equal cell numbers with MR8 cells which had been selected under prolonged K-selection and were subjected to r- and K-selection (lanes 10–14). RNAs extracted at the indicated time were analysed using p53 RT–PCR and PvuII digests of PCR products discriminated transfected murine from endogenous rat p53 sequences. Lane 1 denotes DNA size markers (M) and DNA sizes (bp) are on the left; lane 15, negative control with just water instead of cDNA

Eradication of p53 mutants

To determine if p53 dysfunction is part of an inevitable process and to observe effects of the presence of competitors, co-culture was done. After 6 weeks' r-selection, transfectants expressing the p53KH215 sequence were mixed in the equal number with cells that had been derived from prolonged K-selection of MR8 cells and were again subjected to r- and K-selection. Under r-selection, these transfectants readily predominated in the culture while under K-selection, these cells were rapidly eradicated from the culture in the presence of K-selected cells (Figure 4, lanes 13 and 14). Eradication of p53 mutants was evidenced not only by disappearance of murine p53 expression but also by predominance of a flat cell morphology seen by phase-contrast microscopy (data not shown). We used four independently K-selected cells as competitors and we obtained similar results. Three of these four cells expressed p21CIP1 in response to DXR treatment and thus appeared to have normal p53 function (data not shown). These observations suggested that emergence of p53 mutants is not necessarily inevitable but does depend on the type of selection and on the absence of competitors.

Discussion

Experimental culture conditions usually correspond to r-selection adopted in the present study. r-selected cells tend to be homogeneous because only the fastest wins the competition. To ensure reproducible results in any experiment it is reasonable to use cell culture systems that adopt r-conditions and thus maintains homogeneous populations. Conversely, under conditions of K-selection different strategies, e.g., adherence to matrix, a decreased tendency toward apoptosis or increased contact inhibition, may contribute to survival. Therefore, K-selection should foster a variety of tumor cell characteristics, e.g., different ploidies and morphologies, and thus provide a variety of genotypes for cancer development (Cavalier-Smith, 1980). Furthermore, r-conditions should be transient in vivo because exponential proliferation of cells would readily exceed the tolerable cell number in a human body. Therefore, it is reasonable that cancer development is largely governed by K-selection in vivo.

Deregulated growth of cancer cells initially leads to r-selection but then reaches K-selection as cells proliferate. Such selection sequence favors the predominance of p53 mutants that have advantages in both r- and K-selection, at least in the background of c-myc and H-ras activation, and the prevalent p53 mutations in human cancers can be explained. However, it is noteworthy that competitors do exist to suppress emergence of p53 mutants, as shown in Figure 4. The low frequency of p53 mutations in some tumors may be associated with rapid emergence of competitors in a tumor. Successful selection depends on the absence of intratumor competitors as well as on the type of selection. In addition, the growth advantage conferred by p53 mutations is probably affected by the genetic background of the tumor cells and the type of selection depends on interactions between a tumor and the microenvironment.

The INK4A/ARF locus deletion frequently occurred after myc oncogene activation (Zindy et al., 1998) and was repeatedly detected in our present study. Even one allele deletion appeared to be advantageous under both r- and K-selection. Because of a functional link between ARF and p53 genes, the deletion may well be selected in the same manner, as are p53 mutations.

Several investigators suggest a direct link between p53 mutations and aneuploidy. In their experimental models, emergence of tetraploids and then aneuploids was explained by genetic instability conferred by p53 mutations (Galipeau et al., 1996; Ramel et al., 1995; Yin et al., 1999). Even though p53 mutations are selected for under r-selection, rapid growth readily leads to a higher density (Harvey et al., 1993). Therefore, the culture with p53 mutants tends to be confluent and then become under K-selection, which favors survivors rather than rapid growers and thus facilitates ploidy shift. Genetic instability confered by p53 mutations may increase the frequency of transition from a diploid to tetraploid state at a single cell level under both r- and K-selection. However, at least in our model, p53 mutation did not lead to a ploidy shift at a population level under r-selection. Therefore, ploidy shift from diploids to tetraploids at a population level was not deterministic rather it depended on the type of selection. Tetraploid predominance required K-selection and was not necessarily associated with p53 dysfunction. In yeast cells, ploidy-dependent repression of G1 cyclins can explain the greater cell size and the slower growth (Galitski et al., 1999). Although underlying molecular mechanisms for slow growth of tetraploids remain to be investigated, it seems reasonable that slow growing tetraploids never predominate over parent diploids under r-selection.

According to our findings, frequent polyploid cells in a tumor suggest that K-selection is underway. Clinically, a higher population density is an early-warning change in a preinvasive state (Backman et al., 2000). Therefore, tetraploidization can occur even in a relatively early phase of cancer development such as in the case of Barret's esophagus (Galipeau et al., 1996). On the contrary, ploidy shift from diploid to polyploid was rarely seen after a cancer had fully developed. Consistently, in our model the frequency of the ploidy shift decreased after prolonged culture. There appeared to be a time window when tetraploids can predominate over parent diploids. We speculate that if a ploidy shift occurs it is early in cancer development probably because a growth advantage of tetraploids over diploids is readily abrogated by genetic aberrations accumulated in diploids during prolonged r- and K-selection.

Summary, cancer cells with a deregulated growth are initially under r-selection. As a tumor grows, K-selection begins. Both types of selection prefer p53 mutations in case of c-myc and H-ras activation while ploidy shift requires K-selection. Furthermore, a competitor with different mutations may suppress the emergence of p53 mutants. Our findings strengthen the importance of selection process that leads to cancer (Tomlinson and Bodmer, 1999) and underscore the significance of r- and K-selection in cancer development.

Materials and methods

Expression plasmids

Expression plasmids used in this study were pucEJ for an activated allele of human H-ras (EJ-ras) (Land et al., 1983; Motokura et al., 1995), pSVmycB for human c-myc, p53KH215 for a murine p53 mutant with dominant negative functions (Finlay et al., 1988; Uchimaru et al., 1996), and pcDneo for the neo resistance gene (Chen and Okayama, 1987; Uchimaru et al., 1996). pSVmycB was constructed by replacing the c-jun insert of pSV2humjun (Zhang et al., 1990) with the EcoRI/HindIII fragment of pGEMmycB (Halazonetis and Kandil, 1991).

Rat embryo fibroblasts (REFs) and transfection

REFs were prepared from 14-day pregnant Wistar rats (Tokyo Laboratory Animals Science, Tokyo) as described (Motokura et al., 1995; Uchimaru et al., 1996). Transfection in REFs was done using the calcium phosphate method described (Chen and Okayama, 1987) but with the slight modification described elsewhere (Motokura et al., 1995; Uchimaru et al., 1996). Focus-forming cells were cloned 10 to 12 days after transfection. Transfection of p53KH215 in c-myc- and EJ-ras-transformants was done using electroporation. Ten micrograms of the plasmids were electroporated into 2×106 cells suspended in 200 μl medium at 0.5 kV with 25 μF with Gene Pulse II (Bio-Rad). Transfection efficiency was at least 2×10−4, as determined by drug resistance.

Cell culture and r- and K-selection

Cells were initially plated at 2×104 and were incubated with Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% fetal bovine serum (Bio Whittaker) and kanamycin 60 mg/l (Meiji Seika Kaisha, Ltd, Tokyo) at 37°C in a humidified atmosphere with 5% CO2. To maintain cells in the logarithmic phase of growth (r-selection), cells were passaged at about 1–8 : 500 (around 1×104 cells) per 100-mm dish with 10 ml medium every 3–4 days. For K-selection, half the number of cells was passaged and incubated with 4 ml medium per 60-mm dish every 3–4 days or weekly. Cells were counted using a hemocytometer and Trypan blue exclusion.

Flow cytometry

After trypsinization, nuclei prepared from cells were stained with propidium iodide (Sigma) according to Vindeløv et al. (1983). DNA contents of at least 2×104 nuclei per sample were analysed using a FACScan and CellFIT software (Becton Dickinson). Polyploid cells were defined as cells with more than or equal to 4n DNA contents. Ploidy shift was defined as an increase of polyploid cells over 50%.

Reverse transcription (RT)-polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analyses

RT was done as previously described (Uchimaru et al., 1997). PCR was done as described (Uchimaru et al., 1997) with primer pairs (0.5 μM each) and conditions in a volume of 20 μl were as follows: for p53 cDNA in the DNA binding region (684 bp), 5′-CTGTCATCTTCTGTCCCTTC-3′, 5′-TTTCTTTTGCTGGGGAGAGG-3′, 94°C (1 min), 60°C (1 min), 72°C (2 min), 35 cycles; INK4A/ARF cDNA (404 bp), 5′-CAGGTGATGATGATGGG-3′, 5′-TGCCAGAAGTGAAGCCAAGG-3′, 94°C (1 min), 58°C (1 min), 72°C (1 min), 35 cycles; for the exon 4 of the p53 gene (125 bp), 5′-CTGTCATCTTCTGTCCCTTC-3′, 5′-AAGCAACTCTTCAGGCCCAC-3′, 94°C (1 min), 60°C (1 min), 72°C (1 min), 35 cycles. After restriction enzyme treatment (HpaII and KpnI for p53 cDNA and SmaI for INK4A/ARF), SSCP was done using a non-radioactive method with silver staining or ethidium bromide staining, as described (Sugano et al., 1993).

Western blot analysis

Primary monoclonal antibodies were Pab240 for p53 (Santa Cruz Biotechnology) and SX118 for p21CIP1 (PharMingen) used at 0.2 μg/ml. A secondary antibody was alkaline phosphatase-conjugated rabbit anti-mouse IgG1 antibody (Zymed Laboratories) used at 1 : 1000 dilution. Western blot analysis was done as described (Uchimaru et al., 1998).

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Acknowledgements

We thank T Iiri for helpful comments and M Ohara for language assistance. This work was supported in part by grants (to T Motokura) from the Ministry of Education, Science, Technology, Sports and Culture of Japan.

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Correspondence to Toru Motokura.

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Keywords

  • p53
  • r-selection
  • K-selection
  • multistep tumorigenesis

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