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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Oncogene. 2010 Jun 21;29(33):4715–4724. doi: 10.1038/onc.2010.220

Deletion of p53 in human mammary epithelial cells causes chromosomal instability and altered therapeutic response

MB Weiss 1,2, MI Vitolo 1, M Mohseni 3, DM Rosen 4, SR Denmeade 4, BH Park 4, DJ Weber 1,5, KE Bachman 1,5
PMCID: PMC3164558  NIHMSID: NIHMS313786  PMID: 20562907

Abstract

The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. To evaluate the biological and clinical relevance of p53 loss, human somatic cell gene targeting was used to delete the TP53 gene in the non-tumorigenic epithelial cell line, MCF-10A. In all four p53−/− clones generated, cells acquired the capability for epidermal growth factor-independent growth and were defective in appropriate downstream signaling and cell cycle checkpoints in response to DNA damage. Interestingly, p53 loss induced chromosomal instability leading to features of transformation and the selection of clones with varying phenotypes. For example, p53-deficient clones were heterogeneous in their capacity for anchorage-independent growth and invasion. In addition, and of clinical importance, the cohort of p53-null clones showed sensitivity to chemotherapeutic interventions that varied depending not only on the type of chemotherapeutic agent, but also on the treatment schedule. In conclusion, deletion of the TP53 gene from MCF-10A cells eliminated p53 functions, as well as produced p53−/− clones with varying phenotypes possibly stemming from the distinct chromosomal changes observed. Such a model system will be useful to further understand the cancer-specific phenotypic changes that accompany p53 loss, as well as help to provide future treatment strategies for human malignancies that harbor aberrant p53.

Keywords: p53, TP53, chromosomal instability, Doxorubicin, MCF-10A

Introduction

Human cancers arise because of the combinatorial effects of tumor suppressor gene loss and aberrant activation of oncogenes. The TP53 tumor suppressor gene is the most commonly mutated gene in human malignancy with mutations present in as many as 50% of all cancers (Hollstein et al., 1991; Greenblatt et al., 1994). Germline mutations in TP53 lead to Li Fraumeni syndrome, associated with a high incidence of tumor-igenesis in many tissues (Malkin et al., 1990). Similarly, p53 knockout mice are susceptible to spontaneous neoplasms of different origins that present early in life, leading to increased mortality before ten months of age (Harvey et al., 1993).

p53 is a 53 kDa nuclear phosphoprotein that is expressed at low levels in all cells. Upon DNA damage or viral infection, wild-type p53 is upregulated rapidly and functions as a sequence-specific transcription factor to activate a vast number of genes, including GADD45, Bax, MDM2 and p21WAF1 (p21) (el-Deiry, 1998; Horn and Vousden, 2007; Riley et al., 2008). The main function of p53 is to maintain a cell's genomic integrity by a dual mechanism. First, p53 governs the G1/S phase checkpoint of the cell cycle through induction of the cyclin-dependent kinase inhibitor p21. This allows the cell time for DNA repair and prevents the cell from replicating damaged chromosomes (Kuerbitz et al., 1992; Agarwal et al., 1995). Alternatively, if the damage incurred is too severe, p53 triggers apoptosis (Janus et al., 1999). Owing to the anti-proliferative nature of wild-type p53, its action must be strongly regulated to allow normal cellular processes to proceed in the absence of stress. MDM2, a main transcriptional target of p53, participates in a negative-feedback loop in which an increase in p53 induces expression of MDM2, an E3 ligase, which in turn causes ubiquitination and degradation of p53 (Horn and Vousden, 2007).

On the basis of the laboratory and preclinical studies, several groups have attempted to assign prognostic and predictive value to TP53 mutations in terms of cancer progression and treatment outcomes with, at times, very contradictory conclusions. Examination of a p53 knockout colorectal cancer cell line model showed no chromosomal instability as compared with p53 wild-type cells (Bunz et al., 2002). However, these cells are microsatellite unstable whereas p53 mutations have been found significantly more often in colorectal adenocarcinomas that are microsatellite stable and aneuploid (Campomenosi et al., 1998; Bunz et al., 2002). When considering outcomes to standard cancer treatments, the results become more ambiguous. Some studies of breast cancer patients found that TP53 mutation was associated with resistance to therapeutics, such as doxorubicin, but sensitive to others, such as 5-fluorouracil or γ-irradiation (Aas et al., 1996; Formenti et al., 1997). Yet other groups considering breast tumors found no significant correlation between p53 mutation and a patient's response to treatments (Rozan et al., 1998). Also, depending on the laboratory and tumor type examined, p53 status may or may not correlate with a worse prognosis and decreased survival (Thorlacius et al., 1993; Sengelov et al., 1997; Rozan et al., 1998). Hence, the direct causal connections between p53 inactivation/loss and the development, progression and treatment response of human malignancies have remained elusive.

To evaluate the biological and potential clinical relevance of p53 loss, we have generated a novel in vitro model system using human somatic-cell gene targeting. By `knocking out' the TP53 gene in the non-tumorigenic epithelial cell line, MCF-10A, we have found that p53−/− clones are capable of epidermal growth factor (EGF)-independent proliferation and acquire a defective DNA damage response. Genomic analysis revealed that loss of p53 led to chromosomal instability with marked heterogeneity between different p53 deficient clones. Coinciding with heterogeneous chromosomal aberrations were varying degrees of more aggressive tumorigenic phenotypes, including anchorage-independent growth and invasion. In addition, cell proliferation and clonogenic survival assays showed that doxorubicin treatment of p53−/− clones resulted in decreased cell growth but increased clonogenic survival relative to control cell lines. Thus, our study strongly suggests that loss of p53 leads to genetic instability allowing for the clonal expansion of human epithelial cells, which may recapitulate the carcinogenic process observed in human malignancies.

Results

Creation and confirmation of TP53 gene knockout in MCF-10A cells

The non-tumorigenic MCF-10A (10A) breast epithelial cell line was spontaneously immortalized from a woman who underwent reduction mammoplasty (Soule et al., 1990). The cells are near diploid, relatively genetically stable, estrogen receptor negative and contain wild-type p53. A vector containing a neomycin resistance cassette flanked by LoxP recombination sites, as well as homologous sequences to the intronic regions 5′ and 3′ of TP53 exon 2, was used to target both alleles of TP53 (Figure 1a). The construct, which functions to delete TP53 exon 2, has been shown previously in the HCT116 colorectal cancer cell line to yield clones that are devoid of p53 protein expression (Bunz et al., 1998). After viral introduction of the target construct, homologous recombination and neomycin selection, several 10A-p53+/− clones were identified. Next, a virus expressing the Cre recombinase protein was used to facilitate the removal of the neomycin resistance cassette between LoxP sites, leaving a 34 bp `scar' as previously described (Park et al., 2001). A second round of gene targeting was performed as before to target the second allele. A PCR strategy was used to distinguish non-targeted p53 heterozygotes from p53 homozygous knockouts (Figure 1b). Four independently derived 10A-p53−/− clones were acquired and underwent Cre-recombinase-mediated neomycin excision. At least two Cre-recombinase-mediated neomycin excision clones were obtained for each p53 knockout, one of which will be shown for all further experiments (10A-p53−/− clones 1A, 2B, 3B, 4B). In addition, several non-targeted heterozygote clones were obtained in the process of generating the homozygous knockout, one of which is shown for all further experiments (10A-p53 +/− clone 1).

Figure 1.

Figure 1

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Creation and confirmation of TP53 gene knockout in MCF-10A cells. (a) A schematic of the targeting vector containing a neomycin resistance cassette flanked by LoxP sites and homologous sequences to the intronic regions 5′ and 3′ of TP53 exon 2. Dashed lines represent areas of homology between vector and endogenous sequences used for recombination. Black triangles represent LoxP sites, which are targets of the Cre recombinase. (b) PCR analysis was performed to differentiate between wild-type and knockout TP53 alleles that had undergone Cre recombinase-mediated excision. Shown are four independently derived clones that were successfully targeted at the TP53 locus (clones 1A, 2B, 3B and 4B). (c) Western blot analysis of total cell lysates (25 μg) of all cell lines was performed to confirm gene knockouts. Shown are MCF-10A parental cells and four p53−/− clones, as well as 10A-p53+/− clone 1 and 10A-p21−/− clone 1, which will be used as additional controls.

All TP53 homozygous gene knockouts were devoid of p53 protein expression as predicted by PCR (Figure 1c). A previously described MCF-10A p21 knockout was used to determine which p53-null phenotypes were due to loss of downstream p21 activity (Bachman et al., 2004). Similar to 10A-p53+/− clone 1 and 10A-p21−/− clone 1, all other p53 heterozygous clones and p21 knockout clones showed increased levels of p53 over basal levels seen in parental MCF-10A cells. We attribute this to a possible compensatory response of these cells against the loss of one TP53 allele or the complete loss of p53-dependent p21 activity.

A main facet of a functional, p53-dependent DNA damage response is the enactment, through downstream trans-activation, of a G1/S phase checkpoint to prevent cells from replicating through a damaged genome (Kuerbitz et al., 1992; Agarwal et al., 1995). In response to DNA damage, both induction of the p53-responsive genes p21 and MDM2 and the p21-dependent G1/S arrest were completely abrogated in p53−/− clones, as expected (Supplementary Figures 1 and 2). Similar analysis of exponentially growing cells without DNA damage revealed no such differences (data not shown).

p53 knockout cells show EGF-independent growth

Studies have shown that p53−/− mouse neonatal astrocytes show significantly increased proliferation over that of p53+/+ astrocytes (Yahanda et al., 1995). To assess alterations in normal proliferation associated with the loss of p53, proliferation in complete MCF-10A growth media was analyzed using a standard MTT colorimetric assay. We found no substantial difference in proliferation between the parental MCF-10A cells and those that are p53+/−, p53−/− or p21−/− at all time points examined (Figure 2a).

Figure 2.

Figure 2

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p53 knockout cells show EGF-independent growth. (a) Cells were examined to determine any changes in normal proliferation rates through a standard MTT colorimetric assay. Shown is a representative experiment carried out in triplicate. (b) Cell proliferation in assay media (growth media without EGF and supplemented with 2% charcoal-dextran stripped serum) was also assessed. Shown is a representative experiment carried out in triplicate (*P<0.03; **P<0.002; ***P<0.0008).

As the MCF-10A cell line is non-tumorigenic, it requires the addition of EGF to maintain normal growth and avoid G1 cell cycle arrest (Soule et al., 1990). Assessing proliferation without EGF would thus yield important information regarding the effect that p53 loss has on driving these cells toward a more oncogenic phenotype. Therefore, cells were grown in assay media comprised of growth media without EGF and supplemented with 2% charcoal–dextran stripped serum instead of 5% horse serum. All cells with at least one intact p53 allele showed similar growth profiles throughout the time course, with very little proliferation taking place (Figure 2b). However, all p53 knockout clones showed significantly increased proliferation compared with control when grown in assay medium (*P<0.03, **P<0.002, ***P<0.0008). The capability of p53-null cells for EGF-free proliferation is attributable to a p21-independent mechanism, as the p21−/− cells were unable to accomplish this proliferation.

Copy number analysis of p53-knockout genome reveals chromosome instability

Previous evidence suggests that p53 loss correlates with the loss or gain of entire chromosomes, polyploidy, gene amplification and centrosome hyperamplification (Bischoff et al., 1990; Fukasawa et al., 1996, 1997; Campomenosi et al., 1998; Chiba et al., 2000). To examine whether chromosomal aberrations had occurred following targeted disruption of p53, we used a single-nucleotide polymorphism (SNP) array to identify copy-number changes in p53-knockout cells as they reached higher passages in culture (Dutt and Beroukhim, 2007). For this experiment, we used genomic DNA from MCF-10A cells at passage 3 and 30 to function as controls for normal chromosomal changes that may take place during prolonged culture.

The examination of copy number from parental MCF-10A cells revealed that the genomic profile was unchanged during 30 passages in culture (data not shown). Full genome copy number profiles of all isogenic clones indicated that p53−/− cells had specific, often unique DNA copy number alterations after thirty passages in culture that distinguished them from MCF-10A cells (Figure 3). For example, only clone 4B at passage 30 showed large regions (>100 megabases) of single copy loss on chromosome 4, as well as regions of gain and loss of chromosomes 11 and 20. Similarly, clone 3B had gains in chromosome 1q and losses in chromosome 10p not evidenced in the rest of the p53−/− clones. Conversely, some chromosomes showed common copy number changes across all p53 knockout clones that were not observed in the parental cells. For example, all 10A-p53−/− clones showed minor or major gains in both chromosomes 13 and 19. These data lend support to establish a link between loss of p53 function and the development of chromosomal instability.

Figure 3.

Figure 3

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Copy number analysis of p53 knockout genome reveals chromosome instability. All 10A-p53−/− clones at culture passage 30 were compared against a baseline of MCF-10A cells at culture passage 3 and culture passage 30. Shown are the results from examination of copy number alterations in which a black box signifies copy number loss and a light grey box signifies copy number gain.

p53−/− clones show heterogeneous capabilities for anchorage-independent growth and invasion

As growth factor-independent growth and chromosomal instability is often associated with a transformed phenotype, we wanted to examine other features of transformation in p53−/− cells. It has been previously shown in fibroblasts extracted from Li Fraumeni patients that cells of high passage number contain chromosomal aberrations and gain the ability to form colonies in soft agar at low efficiency (Bischoff et al., 1990). As MCF-10A cells are non-tumorigenic, they do not have the capacity for anchorage-independent growth (Soule et al., 1990; Moon et al., 2000; Narayan et al., 2004). To determine whether the loss of p53 can confer anchorage-independent growth, all clones were tested for their ability to form colonies in soft agar. MCF7, a breast carcinoma cell line known to form colonies in soft agar, was used as a positive control. As expected, parental MCF-10A cells, as well as 10A-p53+/− and 10A-p21−/− cells (data not shown), were unable to form colonies in soft agar (Figure 4a). After 21 days, only clone 4B was able to form colonies at low efficiency as compared with the MCF7 control (photographed at day 14). Unlike the EGF-independent growth phenotype, these results suggest that loss of p53 does not by itself lead to anchorage-independent growth, and that clonal variability may allow for phenotypic selection in p53−/− clone 4B.

Figure 4.

Figure 4

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p53−/− clones show heterogeneous capabilities for anchorage-independent growth and invasion. (a) All cell lines were plated in semi-solid agar medium to assess the ability of those clones to form colonies in an anchorage-independent manner. All MCF-10A derivatives were photographed after 21 days incubation. MCF7s, a positive control for colony formation, were photographed after 14 days incubation due to the colonies growing too large if photographed after that time point. Shown are duplicates of a representative experiment. (b) All cell lines were assessed for their ability to invade through a matrigel-type extracellular matrix. Cells were allowed to invade toward no attractant, 0.1% fetal bovine serum or 1.0% fetal bovine serum for 24 h before being stained and photographed. Shown are photographs of a representative experiment.

Another characteristic of cancer cells is their ability to invade surrounding tissue and metastasize to distant sites. MCF-10A cells are unable to invade through matrigel, a basement membrane matrix (Moon et al., 2000; Willmarth and Ethier, 2006). To determine whether p53 loss allows for an invasive phenotype, all clones were subjected to a matrigel invasion assay. Cells that invade through the matrigel to the underside of the chamber are darkly stained and spread out in comparison with the punctuate pattern of crystal violet that highlight the filter pores (Figure 4b). After 24 h, MCF-10A cells showed only minor invasion through matrigel toward a high level (1.0%) of the fetal bovine serum attractant. A similar amount of invasion was also seen from the p53+/−, p21−/− and three out of four p53−/− clones (data not shown). One of the p53−/− clones, 3B, was able to invade with marked increased efficiency compared with control. With 1.0% fetal bovine serum attractant, approximately 30% of the filter field of view was obscured by crystal violet stained cells. As was the case with anchorage-independent growth, these results strongly suggest that the lack of p53 and secondary clonal changes that occur in clone 3B allow for MCF-10A cells to invade through matrigel.

p53 knockout cells are not tumorigenic in nude mouse xenograft assays

To assess the in vivo tumorigenicity of p53-null clones, xenograft assays in athymic nude mice were performed. Thirty days after inoculation, no tumors developed from any cells regardless of p53 status compared with those mice injected with MDA-MB-231 cells as a positive control, which formed tumors in that time frame (data not shown). Similar to our previous work (Konishi et al., 2007; Gustin et al., 2009), these results show that p53 can impart features of transformation in vitro, but the loss of p53 is not sufficient for in vivo tumorigenesis.

p53 knockout cells show differential sensitivity to the DNA damaging drug, doxorubicin

Defining the mechanisms that lead to differing responses to chemotherapeutics is an active area of clinical investigation and cancer research. Tumor-specific alterations, such as the loss of p53, may confer alternate outcomes to established treatment regimens. Along these lines, disruption of p53 in colorectal cancer cells sensitizes the cells to apoptosis induced by the chemotherapeutic doxorubicin, a topoisomerase II inhibitor. However, these cells are less sensitive to the apoptotic effects of the antimetabolite 5-fluorouracil (Bunz et al., 1999). Other groups using different systems have shown unique and at times contradictory results when examining p53 loss and response to chemotherapeutics (Aas et al., 1996; Formenti et al., 1997; Rozan et al., 1998; Wallace-Brodeur and Lowe, 1999). Owing to the high frequency of p53 mutation and loss in human cancers, the isogenic MCF-10A p53-knockout cell lines provide a novel system to examine the efficacy of various agents in a defined genetic background.

To determine whether p53 status correlates with response to various drugs, cells were assayed using a standard MTT protocol following the addition of chemotherapeutics. All p53−/− clones showed approximately 10-fold increased sensitivity to a seven-day doxorubicin treatment, with concentrations ranging from 0.02 to 0.16 μM (*P<0.02; Figure 5a). The p21−/− cells showed similar sensitivity to doxorubicin. Clone 3B showed a slightly larger range of increased sensitivity, with the lower boundary reaching 0.01 μM. In addition, p53−/− cells were found to have increased sensitivity to the DNA-damaging drug bleomycin, but there was no substantive difference relative to parental MCF-10A cells in response to paclitaxel, an anti-microtubule agent, or 5-fluorouracil (data not shown).

Figure 5.

Figure 5

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p53 knockout cells show differential sensitivity to the DNA damaging drug, doxorubicin. (a) Cells were plated in triplicate (set at 0%) and were subsequently allowed to grow in either drug-free media or media containing doxorubicin for seven days. Cell growth was analyzed by a standard MTT colorimetric assay. Results from a representative experiment are shown as the percentages of cell growth compared with cells that had grown without any drug treatment (100%). (*P<0.02). (b) A clonogenical survival assay was performed on all cells after short-term (6 h) doxorubicin exposure (0.08 μM). Results from three independent experiments carried out in triplicate are shown as the percentages of colonies that were formed after treatment in comparison with those colonies formed after no drug treatment (100%) (*P< 0.004).

We further examined the response to doxorubicin using a clonogenic survival assay. In this assay, cells were treated for six hours with 0.08 μM doxorubicin, as this concentration was approximately the half maximal inhibitory concentration for the 10A-p53−/− clones as compared with parental MCF-10A (see Figure 5a). Surprisingly, ten days after drug removal, the p53−/− and p21−/− clones were able to persist and form colonies significantly more than p53+/+ or +/− cells (*P<0.004; Figure 5b). Taken together, the MCF-10A p53−/− cells proved resistant to short-term doxorubicin treatment when tested by a clonogenic survival assay, but sensitive to long-term exposure to doxorubicin analyzed by MTT. These responses to different doxorubicin treatment schedules can be linked to a downstream p21 mechanism, as the 10A-p21−/− cells behaved in the same manner as the p53 knockouts, and may further correlate with checkpoint deficiencies.

Discussion

We have successfully performed targeted deletion of p53 in the non-tumorigenic epithelial cell line, MCF-10A. Using targeted homologous recombination, we established several independently derived 10A p53−/− clones that were void of p53-dependent downstream damage signaling events.

Using this isogenic system, we investigated the long-debated link between p53 loss and chromosomal instability. In the HCT116 p53−/− model, no difference in chromosome stability was seen between cells with or without p53. In addition, this group examined p53-null human fibroblasts and found no increase in the rate of aneuploidy but did observe an increased frequency of tetraploidy (Bunz et al., 2002). However, examination of some primary tumors, as well as fibroblasts taken from Li Fraumeni patients or p53 knockout mice, suggests that p53 loss correlates with chromosome loss/gain, gene amplification and polyploidy (Bischoff et al., 1990; Fukasawa et al., 1996, 1997; Campomenosi et al., 1998; Chiba et al., 2000). In our MCF-10A studies, a correlation between p53 loss and chromosomal instability was revealed through an elevated incidence and variability of gene copy number aberrations. The p53−/− clones 3B and 4B were found to have highest levels of copy number aberrations and were able to gain more aggressive phenotypes involving invasion and anchorage-independent growth, respectively. We hypothesize that the increase and variability in transformation phenotypes in p53-null MCF-10A cells are attributable to genome instability. Using this isogenic model, future studies that focus on these regions of gain or loss in the 10A-p53−/− clones could uncover specific genetic changes that drive aggressive phenotypes.

Interestingly, areas in both chromosomes 13 and 19 showed gains in copy number in each of the four p53−/− clones. Although copy number losses of chromosome 13 are much more common in human malignancy, copy number gains have been found to occur often in colorectal adenomas (44%) (Bomme et al., 2001), colorectal carcinomas (32%) (Lothe et al., 1992) and liver metastases of colorectal cancer (48%) (Korn et al., 1999). This may correlate with data suggesting that cancer of colorectal origin is one of the most common tumor sites for p53 mutation and loss (Calistri et al., 2006). Conversely, copy number gains in chromosome 19 are more widespread across the spectrum of human malignancy, occurring most notably in primary gastric tumors (68%) (Varis et al., 2003), breast tumors (45%) (Papa et al., 1997) and ovarian high-grade serous carcinomas (19.5%) (Park et al., 2006). It has previously been established that positive and negative alterations in gene copy number have a significant correlation with gene expression changes in tumors (Hyman et al., 2002; Pollack et al., 2002; Tsafrir et al., 2006). These areas of commonality, therefore, may contain key genomic regions necessary for tumorigenic development and so may be early targets for chromosomal instability upon loss of p53. This model system can also allow us to ascribe and separate functions of p53 that are p21 dependent versus those that are independent by comparing the p53 knockout lines to the paired p21 knockout cell lines which do not show chromosomal instability. Thus, the cell lines described in this paper add greatly to our existing knowledge regarding p53 function, but will also serve as useful tools for further deciphering p53 mechanisms.

Importantly, our p53 isogenic cell system also allowed us to begin exploring the possible clinical implications resulting from p53 loss. In a proliferation assay with continuous chemotherapeutic exposure, we have found that p53-null MCF-10A cells showed marked sensitivity to the DNA damaging drug doxorubicin as compared with p53 +/+ and +/− cells. Although p53 −/− clones showed increased sensitivity to longer courses of treatment, a clonogenic survival assay with cells treated for only 6 h suggested that the p53 −/− cells were able to persist and form colonies significantly better than p53 +/+ and +/− controls. These results parallel those seen in clinical trials that compared continuous infusion with bolus administration of the chemotherapeutic 5-fluorouracil in the treatment of colorectal cancer, with the majority of studies showing superiority of the continuous infusion regimen (Citron et al., 2003). Similarly, trials of adjuvant chemotherapy in breast cancer patients have shown increased benefit when the interval between chemotherapy cycles is reduced (1998), confirming the notion that sustained and/or repeated exposure of cancer cells to cytotoxic agents is necessary for optimal benefit.

In summary, these findings suggest that exploration of the consequences of tumor-specific gene alterations, and how these alterations link to chemotherapeutic sensitivity or resistance, may be beneficial for tailoring new patient-specific clinical interventions. In addition, the clinical efficacy of chemotherapeutic treatment of cancers, specifically with p53 mutation/loss, may depend on the length or frequency of treatment, as well as the type of treatment itself. Our results, however, suggest that loss of p53 leads to random genetic events and subsequent phenotypic differences depending on the predominant clone. These observations may underlie the many issues surrounding the use of p53 as a prognostic marker. The genetic heterogeneity observed among p53-null clones supports this postulation as the clones showing invasive and soft agar growth were not preselected to show these phenotypic characteristics. Therefore, each individual p53-null cell could possess the capability to evolve differently compared with others depending on the type and duration of selective pressure (that is, chemotherapy). This highlights the importance of further studies involving downstream changes following p53 loss and also increases the complexity of developing efficient targeted therapies and use of p53 as a diagnostic marker or therapeutic target.

Materials and methods

Cell culture

MCF-10A cells (American Type Culture Collection, Manassas, VA, USA) were cultured in phenol red-free Duibecco's modified eagle medium-F12 media (Mediatech, Herndon, VA, USA) with 5% heat-inactivated horse serum (Invitrogen, Carlsbad, CA, USA), 0.1 μg/ml cholera toxin (Sigma, St Louis, MO, USA), 0.5 μg/ml hydrocortisone (Sigma), 0.02 μg/ml EGF (Sigma), 10 μg/ml insulin (Sigma), 100 units/ml penicillin and 100 units/ml streptomycin (Invitrogen). Where indicated, cells were cultured in assay media composed of phenol red-free Duibecco's modified eagle medium-F12, 2% charcoal-stripped dextran-treated fetal bovine serum (Hyclone, Logan, UT, USA), 0.1 μg/ml cholera toxin, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 100 units/ml penicillin and 100 units/ml streptomycin. MCF7 cells (American Type Culture Collection) were grown in Duibecco's modified eagle medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 units/ml penicillin and 100 units/ml streptomycin. Cells were maintained in a 37 °C incubator with 5% CO2.

Targeted deletion of TP53 in MCF-10A cells

To remove each allele of TP53, a vector targeting exon 2 of TP53 was obtained as a gift from the laboratory of Dr Fred Bunz and targeted clones were created as previously described (Bunz et al., 1998; Park et al., 2001).

Western blot analysis

Western blot analyses were performed as previously described (Vitolo et al., 2009). Primary antibodies for p53 (Calbiochem, La Jolla, CA, USA), p21WAF1 (Calbiochem) and MDM2 (Calbiochem) were used at manufacturers' recommended dilutions. Immunoreactive proteins were detected using Amersham ECL western blot detection reagents (GE Healthcare, Piscataway, NJ, USA).

Proliferation assays

Cells were seeded in triplicate at 1.5 × 103 per well in 96-well plates in assay media overnight. The media was then changed to either fresh growth media or assay media (day 0). At appropriate time points, MTT (Sigma) was added to a final concentration of 0.5 mg/ml for 3 h. Formazan crystals were solubilized by the addition of 10% 0.1 M glycine (pH 10.5) in DMSO overnight at room temperature. Absorbance was measured at 450 nm. Absorbance readings at day 0 (0%) were used to calculate percent cell growth over cells plated. Proliferation assays in the presence of chemotherapeutic agents were performed similarly with 2.5 × 103 cells per well being plated. Doxorubicin (Calbiochem), 5-fluorouracil (Calbiochem), bleomycin (Calbiochem) and paclitaxel (Invitrogen) were added in full MCF-10A growth media on day 0. The MTT protocol was carried out on day 0 (control = 0%) and 7 of the experiment. Results are showed as the percentages of cell growth in drug compared with the growth of untreated cells (100%).

Clonogenic survival assay

1.0 × 105 cells were plated on assay media overnight at 37 °C. The following day, cells were treated with 0.08 μm doxorubicin or left untreated in fresh growth media for six hours. After treatment, 1.0 × 103 cells were moved to 100 mm tissue culture dishes in triplicate and allowed to form colonies in regular growth media for ten days. The cells were then fixed and stained with 2 mg/ml crystal violet in buffered formalin. All colonies visible by eye were counted. Results of triplicate experiments carried out in triplicate are shown as the percentages of colonies that were formed after treatment in comparison with those colonies formed after no treatment.

Polymerase chain reaction

Cells were harvested and genomic DNA was extracted using the QIAmp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). Genomic DNA was PCR amplified using the following primers: Cre screen—forward 5′-AGCTGTCTCAGACACTGGC-3′ and reverse 5′-CCTTGTCCTTACCAGAACG-3′.

Matrigel invasion assay

Exponentially growing cells were starved overnight in assay media. Millicell culture plate inserts (8.0 μm pore size; Millipore Corp., Billerica, MA, USA) were coated inside and outside with gelatin (Sigma) at 2 mg/ml. The inside of the chamber was then coated with 0.5 mg/ml matrigel (BD Biosciences, Bedford, MA, USA). Starved cells were trypsinized and prepared in invasion media (phenol red-free Duibecco's modified eagle medium-F12 media with 0.5% bovine serum albumin only (Sigma)). 1.0 × 105 cells were allowed to invade through the chamber filters toward invasion media only (no attractant) or invasion media containing 0.1% or 1.0% heat-inactivated fetal bovine serum (Invitrogen) for 24 h at 37 °C. The chamber filters were fixed in buffered formalin and stained with 0.5% crystal violet in 25% methanol. The cells in the inner chamber were scraped off using sterile cotton swabs. Images at × 10 magnification were taken with a Nikon eclipse TE300 inverted microscope (Nikon, Melville, NY, USA) with a Cooke sensicam high performance camera (Cooke Corp., Auburn Hills, MI, USA).

Colony formation in soft agar

7.5 × 103 cells were cast in top layer medium composed of full growth media and 0.6% SeaPlaque agarose (Cambrex, Rockland, ME, USA). The cell mixture was plated on 6-well plates on top of a solidified layer of 0.8% agarose and 2 × growth media and allowed to solidify. The next day, 2 mls of growth media was added to the top of each well. Images were taken as before at × 4 magnification. MCF7 colonies were photographed after 14 days, whereas the MCF-10A derivatives were photographed after 21 days.

SNP/chip analysis

Cells were harvested and genomic DNA was extracted using the QIAmp DNA Blood Mini Kit (Qiagen). SNP genotyping was performed on a 250 K NspI Affymetrix SNP array, according to the manufacturer's protocol (Affymetrix, Santa Clara, CA, USA). All `SNP Chip' images (`CEL files'), were extracted using the Affymetrix Genotype software, and analyzed using the dChip software package (Lin et al., 2004; Greshock et al., 2007).

Statistical analysis

Statistical analysis was performed using a two-tailed Student's t-test and analysis of variance with two factor designs between groups, calculated with Excel (Microsoft). A P-value <0.05 was considered statistically significant.

Supplementary Material

part1
part2
part3

Acknowledgements

We thank Dr Fred Bunz for providing TP53 knockout vectors and advice, and Joel Greschock for advice on copy number analysis. This work was supported by: NIH grant T32DK067872 (MBW), Susan G Komen Breast Cancer Foundation PDF104506 (MIV), DOD Breast Cancer Predoctoral Training Award BC083057 (MM), The Avon Foundation, R01CA109274, The Breast Cancer Research Foundation (BHP), R01CA107331 and R01GM58888 (DJW) and Maryland Cigarette Restitution Fund (KEB).

Footnotes

Conflict of interest Work by Drs Park and Weber has been funded by the NIH. Dr Park is a consultant for GlaxoSmithKline and has received research funding from GlaxoSmithKline though none of the work presented in this paper was funded by those funds. Dr Park is on the Scientific Advisory Board of Horizon Discovery LTD, and receives payment for these services. This is managed as per the Johns Hopkins School of Medicine Conflict of Interest Policy. Dr Denmeade is a Consultant and receives equity from GenSpera and Protox Therapeutics. Dr Bachman is currently employed by GlaxoSmithKline though all data presented in this paper were generated when Dr Bachman was a faculty member of the University of Maryland Greenebaum Cancer Center.

Supplementary Information accompanies the paper on the oncogene website (http://www.nature.com/onc)

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part1
NIHMS313786-supplement-part1.pdf (74KB, pdf)
part2
NIHMS313786-supplement-part2.pdf (33.4KB, pdf)
part3
NIHMS313786-supplement-part3.pdf (17.9KB, pdf)

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