The function of a heterozygous p53 mutation in a Li-Fraumeni syndrome patient.

p53 is one of the most extensively studied proteins in cancer research. Mutations in p53 generally abolish normal p53 function, and some mutants can gain new oncogenic functions. However, the mechanisms underlying p53 mutation-driven cancer remains to be elucidated. Our study investigated the function of a heterozygous p53 mutation (p.Asn268Glufs*4) in a Li-Fraumeni syndrome (LFS) patient. We used episomal technology to perform somatic reprogramming, and used molecular and cell biology methods to determine the p53 mutation levels in patient-originated induced pluripotent stem (iPS) cells at the RNA and protein levels. We found that p53 protein expression was not increased in this patient's somatic cells compared with those of a healthy control. p53 mutation facilitates the proliferation of tumor cells by inhibiting apoptosis and promoting cell division. It can inhibit the efficiency of somatic reprogramming by inhibiting OCT4 expression during reprogramming stage. Moreover, not all p53 mutant iPS cell lines have mutant p53 RNA sequences. A small percentage of mutant p53 mRNA is present in the somatic cells from the patient and his mother. In summary, this p53 mutation can promote tumor cell proliferation, inhibit somatic reprogramming, and exhibit random p53 allelic expression of heterozygous mutations in the patient and iPS cells which may be one of the reasons why the people with p53 mutations develop cancer at random. This finding suggested that mutant p53 allelic expression should be added to the risk forecasting of cancer.

Introduction

Somatic cell reprogramming is a valuable tool for understanding the mechanism of pluripotency recovery, because it enables the possibility of producing patient-specific pluripotent stem cells [1-3]. What’s more, researchers can get infinite patient samples and set up experimental platforms to study the pathogenesis of diseases in vitro [4].

As a tumor suppressor gene, p53 plays a significant role in promoting apoptosis and cell cycles arrest. Missense mutations of p53 can be a key factor of cell carcinogenesis and reduce the induction efficiency of induced pluripotent stem cells (iPS) [5-12]. Moreover, the p53 mutation might not only loss its anti-cancer functions, but also obtain oncogenic traits called gain of function (GOF), including malignant progression and invasion, metastasis and even chemotherapy resistance [13-16]. In cell reprogramming, oncogenes, such as Notch, can inhibit the generation of iPS cells [17], but no one knows how specific p53 mutations affect the iPS cell derivation process. Additionally, p53 does not fully follow the classic Knudson’s two-hit theory during carcinogenesis or cancer progression [18].Therefore, so many healthy people with the same p53 mutation can go their entire lives without developing cancer [9].

In the present study, we generated iPS cells from the peripheral blood of a male infant with LFS; the patient has a p53 heterozygous mutation inherited from his mother (22 years old) [19]. The p53 mutation facilitates the proliferation of tumor cells by inhibiting apoptosis and promoting cell division. Additionally, it reduced the reprogramming efficiency by inhibiting Oct4 expression. In three mutant p53 iPS cell lines, we found that the expression levels of WT p53 protein in one iPS line was different from that in the other two iPS cell lines. We speculated that the differential expression of WT p53 was related to allelic expression imbalance. Using p53 RNA sequencing, we confirmed this conclusion.

Materials and methods

Cell culture

Primary murine embryonic fibroblasts (MEFs) with p53 knockout were obtained from 13.5-day CD-1 IGS mouse embryos. HEK293T and MEF cells were cultured in standard DMEM containing 10% FBS (HyClone, Logan) and passaged routinely with trypsin-EDTA solution. Human iPSCs were maintained in a feeder-free culture system. Briefly, the wells of plates were precoated with Matrigel (BD Biosciences), and then we seeded the iPSCs and cultured them in PSCeasy medium (Cellapy).

Isolation and preparation of MNCs from peripheral blood

Blood samples were obtained from the Hematology and Oncology Department of Shanghai Children's Medical Center, and patient’s mother provided informed consent. MNCs were isolated from PB samples using standard Ficoll procedures; 8 ml of diluted blood (blood: PBS = 1:2) was loaded onto a 3 ml layer of Ficoll-Paque PREMIUM (p = 1.077 g/ml; Sigma) in a 15-ml conical tube.

Culture and expansion of MNCs from peripheral blood

We expanded PB MNCs for 4–10 days in a serum-free medium supplemented with a mixture of cytokines. We used erythroid culture medium (ECM). ECM included IMDM (50%; Invitrogen) and Ham’s F12 (50%; Invitrogen) with ITS-X (100×; Invitrogen), chemically defined lipid concentrate (100×; Invitrogen), L-glutamine (100×; Invitrogen), BSA (5 mg/ml; Sigma), ascorbic acid (0.05 mg/ml; Sigma), L-thioglycerol (200 μM; Sigma), IL-3 (10 ng/ml; PeproTech), SCF (100 ng/ml; PeproTech), erythropoietin (2 U/ml; PeproTech), dexamethasone (1 μM; Sigma), IGF-1 (40 ng/ml; PeproTech), and holotransferrin (100 μg/ml; R&D).

Nucleofection and generation of iPSCs

The following episomal vectors were used: pEV SFFV-OCT4-E2A-SOX2 (OS), pEV SFFV-MYC-E2A-KLF4 (MK), and pEV SFFV-BCL-XL (Bcl-XL). We added plasmids (4 μg OS (EF1-OS), 4 μg MK (EF1-MK) and 2 μg B (BCL-XL)) to a sterile Eppendorf tube and mixed them with 100 μl nucleofection buffer (Nucleofector™ Kits for Human CD34+ Cells, Lonza) and then transferred the mix to the cell pellet (1 × 106 cells). Using the plastic pipette provided by the kit, we transferred the mixture of plasmids and cells into the provided cuvette to run the program (U008) for nucleofection (2B; Lonza). After nucleofection, we directly transferred the mixture to a culture plate, which was already preseeded with feeder cells. The cells were then cultured in reprogramming medium, which was composed of knockout DMEM/F12 medium (Invitrogen) supplemented with 1% L-glutamine (Invitrogen), 2 mM nonessential amino acids (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 50 ng/ml FGF2 (Invitrogen), 1% ITS (BD Biosciences), and 50 μg/ml ascorbic acid (Sigma) for 7 days. The cells were then cultured in E8 medium (Invitrogen) until iPSCs were generated.

Generation of mouse iPS cells

Retroviral constructs pMXs-Klf4 (#13370), pMXs-Sox2 (#13367), pMXs-Oct4 (#13366), pMXs-c-Myc (#13375) [1], were obtained from Addgene. Reprogramming of primary (passage 2) MEFs was performed as previously described [12]. In brief, primary MEFs of the indicated genotypes were seeded in 100-mm-diameter dishes (5 × 105 cells per dish) that had been precoated with 0.1% gelatin (Sigma). They were transduced twice in the next two days at 24 h intervals by virus supernatant collected from Plat-E cells transfected with the previously mentioned retroviral plasmids. At the end of transduction, we changed the medium to mouse ES culture medium. After culturing for 10–12 days, colonies with ES-cell-like morphology became visible. They were then chosen after counting or picking for further expansion on feeder fibroblasts using standard ES culture methods.

Counting and picking of iPSC colonies

When the colonies became visible to the naked eye, we stained the human iPS cells with a Tra-1-60 antibody, counted them under a fluorescence microscope and picked them by hand. To pick them, we gently scratched a colony with a 10 μl pipette tip and transferred the single colony to a 12-well plate coated with Matrigel and filled with E8 medium. We usually selected 10 to 20 colonies from each donor. Mouse iPS cell colonies were counted using a published method [12].

Cell line construction

WT p53, mutant p53 p.Asn268Glufs*4 and p53 R175H coding DNA sequences (CDS) were cloned into a pLL CMV puro mammalian lentiviral expression vector.

To produce the lentivirus, each expression vector was transfected into 293T cells with second-generation lentiviral packaging plasmids pMD2.G and psPAX2 using the PolyExpress transfection reagent (Excellgen, Rockville). Forty-eight and 72 h after transfection, we harvested the culture medium, incubated it with Lenti-X concentrator (Clontech Laboratories, Mountain View), and centrifuged it to obtain concentrated lentivirus. p53-/- MEF cells were infected with the lentiviruses in the presence of 6 μg/mL polybrene (Sigma-Aldrich) for 24 h. Overexpression was confirmed by Western blotting.

Immunohistochemistry

IHC was performed in 3-μm formalin-fixed paraffin embedded tissue sections mounted on adhesive microscope slides. Sections were deparaffinized, rehydrated in graded alcohols and underwent antigen retrieval performed by microwave treatment in 0.01 M-citrate buffer at pH 6.0, during 9 min. The sections were then incubated overnight at 4°C with the primary antibody against p53 (1:100, monoclonal antibody; Cat. MAB-0674; MXB). The detection of the immune reaction was performed using the avidin-biotin-peroxidase method (1:100; Vector Laboratories, Peterborough, UK). DAB (3, 3′-diaminobenzidine) was used as chromogen and hematoxylin as nuclear counterstaining.

Immunofluorescence

To detect targeted antigens and p53 in pluripotent stem cells, we immobilized cells with PBS containing 4% polyformaldehyde at room temperature for 10 minutes. After washing with PBS, the cells were incubated in PBS containing 0.1% Triton X-100 for 20 minutes at room temperature. Then, we stained fixed cells with SSEA-4 (1:100; monoclonal antibody; MAB8490; Stemgent), TRA-1-60 (1/200; monoclonal antibody; 09–0010; Stemgent), OCT4 (1/200; monoclonal antibody; MAB4419A4; Millipore), Nanog (1/600; monoclonal antibody; sc-293121; Santa Cruz) and p53 (1/500; monoclonal antibody; ab1101; abcam). These primary antibodies were visualized by with goat anti-rabbit IgG bound to Alexa 488 and goat anti-rabbit IgG bound to Alexa 594 or goat anti-mouse IgG bound to Alexa Fluor 488. Nuclear staining was performed with DAPI, and fluorescence images were obtained using Zeiss inverted LSM confocal microscopy (Carl Zeiss).

Teratoma formation assay and histological analysis

We suspended the human iPSCs in PBS at 1 x 108 cells/ml and then injected 100 ml of cell suspension (1 x 107 cells) subcutaneously into the dorsal side of SCID mice. One month after the injection, we dissected the tumors from the mice. Teratomas were weighed and fixed in PBS containing 4% formaldehyde and embedded in paraffin wax. We then produced sections from the fixed teratomas and stained them with hematoxylin and eosin.

Gene expression analysis of LFS iPS cell lines

We used three LFS iPS cell lines, as well as H1 ESCs and H9 ESCs, and we extracted total RNA from each using the RNeasy plus kit (Qiagen) to assess their self-renewal abilities and p53 transcription levels. Real-time PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems) on a 7500 Fast Real-Time PCR System (Applied Biosystems). The primer sets were as follows: Oct4, 5′-ATTCAGCCAAACGACCATCT-3′ and 5′-GCTTCCTCCACCCACTTCT-3′; SOX2, 5′-CACACTGCCCCTCTCACAC A-3′ and 5′-CCCTCCCATTTCCCTCGTTT-3′; NANOG, 5′-GCCGAAGAATAGCAATGGTGTG-3′ and 5′-GGAAGATAGAGGCTG GGGTAG-3′. p53, 5′-CTGAGGCATAACTGCACCCT-3′ and 5′-GACAA GGGTGGTTGGGAGTAG-3′.

To determine the average copy numbers of residual or integrated episomal vectors in iPSC clones, real-time PCR analysis was performed. We extracted total DNA (genomic and episomal) from iPSCs at passage 10. Two sets of primers were used to detect vector DNA (in either the episomal or integrated form): EBNA1, 5′-TTTAATACGATTGAGGGCGTCT-3′ and 5′-GGTTTTGAAGGATGCGATTAAG-3′; and OSW, 5′-GGATTACAAGGATGACGACGA-3′ and 5′-AAGCCATACGGGAAGCAATA-3′.

Gene expression analysis of MEF and iPS cell lines

To detect the expression of pluripotent genes in MEF cells at different time points of reprogramming, we collected SSEA1-positive cells and extracted total RNA from the groups of p53 mutant, WT, or an empty vector control for 2, 4, 8 and 12 days using a RNeasy plus kit (Qiagen). Real-time PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems) on a 7500 Fast Real-Time PCR System (Applied Biosystems). The primer sets were as follows: OCT4, 5′- TCTTTCCACCAGGCCCCCGGCTC-3′ and 5′- TGCGGGCGGACATGGGGAGATCC-3′; SOX2, 5′-TTGCCTTAAACAAGACCACGAAA-3′ and 5′- TAGAGCTAGACTCCGGGCGATGA-3′; NANOG, 5′- CAGGTGTTTGAGGGT AGCTC-3′ and 5′- CGGTTCATCATGGTACAGTC-3′; GAPDH, 5′- TGTGTCCGTCGTGGATCTGA-3′ and 5′ TTGCTGTTGAAGTCGCAGGAG-3′.

Growth curve

Cell growth curves were compared among the cells of p53 mutant, WT, or an empty vector control according to the method [20]. Briefly, 1.5 E5 cells were seeded in a 12-well plate, and the growth curves were plotted by counting cells every 24 hours over three-day with excel software.

Karyotyping and G-banding

G-banding chromosome analysis of the iPSC lines was performed following the protocol published by Li et al [12]. A certified cytogenetic technologist interpreted the data.

Western blotting

Cell extracts were prepared, resolved on gels, transferred to nitrocellulose and incubated with antibodies against the N terminus of p53, which can recognize mutant and wild type of p53 (1:1,000; monoclonal antibody; ab1101; abcam), β-actin (1:500; monoclonal antibody; M1210-2; Huaan), BCL-2 (1:1,000; monoclonal antibody; sc-7382; Santa Cruz), and PUMA (1:500; monoclonal antibody; sc-374223; Santa Cruz). γH2AX-139 (1:1,000; monoclonal antibody; sc-517348; Santa Cruz).

Apoptosis

Apoptosis was measured by staining with annexin V–APC and Propidium Iodide (PI)-phycoerythrin (PE) (Annexin V-APC Apoptosis Detection kit, BD Pharmingen) followed by flow cytometry on a FACS flow cytometer (BD, Canto II). All experiments were performed in triplicate, and results were calculated as the mean ± S.D.

Statistical analysis

All experiments were repeated three times. Data are presented as the mean ± S.D. Two-tailed Student’s t tests were performed, and p < 0.05 was considered statistically significant.

Animals and ethics statement

SCID mice were bought from Shanghai SLAC Laboratory Animal CO. All mice used in this study were authorized by the Animal Care Use and Review committee of Shanghai Jiao Tong University. The study was conducted according to the Ethical Principles of Measures for Ethical Review of Biomedical Research Involving Human Beings and the Declaration of Helsinki. The ethics committee of the Children's Medical Center affiliated with Shanghai Jiao tong University approved the induction experiment for iPS cells (SCMCIRB-K2014050).

Results

p53 Asn268Glufs*4 mutation was found in a LFS patient

The tumor suppressor gene p53 encodes a tetrameric DNA-binding protein that regulates cell cycle and apoptosis [21-23]. A 6-month-old male infant was first diagnosed with composite ACC (adrenocortical carcinoma) and neuroblastoma in May 2017. In March 2018, the relapse of ACC was identified by abdominal computed tomography (CT) scanning and confirmed by resection (Fig 1A and 1B). We found that p53 protein expression was negative in this patient’s adrenocortical carcinoma tumor tissues by immunohistochemistry (IHC) (Fig 1C). Since gain-of-function mutations of p53 were reported to be stable for IHC [24-26], our data suggest that this new p53 mutation is not a gain-of-function mutation. Given that p53 gene mutation has a strong correlation with the diagnosis of infant ACC [19, 27], genetic testing for p53 status was performed on the patient and his parents with their agreements. We found a heterozygous insertion of c.801dupG that caused a p.Asn268Glufs*4 in the p53 gene in this patient and his mother, suggesting that this patient inherited the mutation from his healthy mother (Fig 1D). The active p53 is a homo-tetramer formed by four identical chains of 393 residues each, and the N-terminal region of p53 consists of an intrinsically disordered transactivation domain (TAD) and a proline-rich region. It is followed by the central, folded DNA-binding core domain that is responsible for sequence-specific DNA binding. Via a flexible linker, this domain is connected to a short tetramerization domain that regulates the oligomerization state of p53 (Fig 1E). Asn268Glufs*4 mutation is a nonsense mutation which is located in specific DNA binding domain, which caused early termination of this specific protein synthesis and may affect the function of p53.

Fig 1

Identification of a Asn268Glufs*4 mutation of p53 in a LFS patient.

a-b. The patient was first diagnosed with composite ACC and neuroblastoma at the age of 6 months. Relapse of ACC was diagnosed when he was 16 months old. CT of the mass arising from the left adrenal gland at initial presentation (red arrow) and in the right adrenal gland at relapse (blue arrow). Histologic appearance (H&E staining) of the adrenocortical carcinoma at diagnosis and relapse stage. c. No expression of p53 in the left adrenocortical carcinoma cells from the patient. d. Sanger sequencing of the patient and his mother. The mutation site of p53 is indicated by the red arrow. The p53 sequence is C.801 dup G on chromosome 17 in the patient and his mother. e The domain structure of full-length p53 consisting of an N-terminal transactivation domain (TAD), followed by a proline-rich region (PRR), a central DNA-binding domain (p53C), a tetramerization domain (TET), and an extreme C-terminus (CT)The p53 mutant position of the patient is indicated by the red arrow.

p.Asn268Glufs*4 mutation of p53 loses some functions of wild type p53

To explore the function of the mutant p53, we separately infected lentivirus-mediated p53 mutant, wild type (WT), or an empty vector (EV) control into p53-/- MEF. We identified full-length WT and the mutant (truncated) of p53 protein in the transformed cells (Fig 2A). As p53 known functional mutant R175H, the expression of BCL-2 in mutant cells was similar to that in control cells, and higher than that in p53 WT cells. The expression of PUMA in mutant cells was the same as that in the control cells, but PUMA levels relatively increased in WT cells (Fig 2A, S1C Fig). The analysis of apoptosis revealed that compared with unregulated control cells, overexpression of WT p53 enhanced apoptosis in p53-/- MEF cells (Fig 2B, S1E Fig). Only p53 WT induced DNA damage compared with the control (Fig 2D and 2E, S1C Fig). Unlike p53 R175H, p53 p.Asn268Glufs*4 mutant as well as its WT dramatically inhibited cell proliferation (Fig 2C, S1D Fig). These data suggest that p53 mutant lost WT p53 ability to induce apoptosis and DNA damage and thereby reduced the inhibition of cell division.

Fig 2

p.Asn268Glufs*4 mutation of p53 loses some functions of wild type p53.

a. Western blotting (WB) of expression of p53, BCL-2, and PUMA in p53-/- MEF transfected with lentiviruses-mediated p53 WT (WT), mutant (Mut), or an empty vector (EV) control. Arrow, WT p53; arrow head, p53 mutant. b. FACS analysis of apoptosis at Day 3 in the cells from a. * p < 0.05. c. Cell proliferation analysis. d. WB of γH2AX-139 expression. e. Quantitative analysis of γH2X-S139 protein expression in d.

The p53 p.Asn268Glufs*4 mutation inhibits iPS cell generation

Since p53 is critical for iPSC reprogramming [6, 10, 12], we explored the role of this p53 mutant in iPSC reprogramming. We first tested the expression of p53 protein in mononuclear cells from the patient and his mother. As shown in Fig 3A, the patient showed lower p53 WT protein levels compared with his mother and the healthy control and a truncated p53 protein was found only in the patient’s MNC, suggesting that the p53 mutant protein does not express in all cells with this gene mutation.

Fig 3

The p53 p.Asn268Glufs*4 mutation inhibits iPS cell generation.

a. WB of p53 protein levels. Mononuclear cells in healthy people with the same age as the patient were used as a control. Arrow, WT p53; arrow head, p53 mutant. b. iPS colony numbers per 1 × 106 monocyte cells used to generate iPSCs at Day 14 after transduction. ***, p < 0.001. c. The percentage of iPS cell lines established on Day 16 after transduction. **, p < 0.01. d. Chromosome numbers in three patient iPS cell lines. e. iPS colony numbers following introduction of WT p53, mutant p53 and vector into p53+/+ and p53-/- MEFs were counted on reprogramming Day 14 after transduction. f. Real-time PCR (RT-PCR) of expression of OCT4 in cells following introduction of WT p53 and mutant p53 compared with vector control at the indicated reprogramming time points.

Next, we generated iPSCs from the mononuclear cells of this patient, his mother and three healthy individuals, respectively. On the 14th day of reprogramming, we counted the number of iPS colonies. The number of iPS colonies from the patient was significantly less than his mother and the mean number of three healthy control (Fig 3B). The induction efficiency of the patient’s iPS was also declined (Fig 3C). This data shows that this p53 mutation gets a new function, which inhibits somatic cell reprogramming.

Since p53 is the best known ‘guardian’ of the genome and the loss of p53 function can induce the abnormal karyotype [28, 29], we randomly picked up three iPS cell lines and performed karyotype analysis. As shown in Fig 3D, all of the chromosome numbers of iPSCs were hypodiploidy. To confirm that p53 mutant inhibited somatic reprogramming, we separately introduced p53 mutant, WT, or an EV control into p53-/- and p53+/+ mouse embryonic fibroblast (MEF) cells and then MEFs were reprogrammed to iPS cells. The numbers of iPS colonies in the mutant and WT groups on the 14th day of reprogramming were significantly lower than that in the control group (Fig 3E). However, p53 R175H did not affect the reprogramming rate (S1F Fig). Compared the expression of pluripotent genes on Day 2nd, 4th, 8th, and 12th during reprogramming, Oct4 expression in p53 WT and mutant cells had been significantly less than that in the control (Fig 3F), whereas the expression of SOX2 and NANOG had no difference (S1A and S1B Fig). This data indicates that the mutant of p53 likes as its WT and inhibits Oct4 expression and reduces the reprogramming efficiency.

To investigate whether p53 p.Asn268Glufs*4 mutation influenced cell pluripotency, three p53 p.Asn268Glufs*4 iPS cell lines were picked up. Using RT-PCR, we found that the expression of pluripotency genes, including OCT4, SOX-2, NANOG, and Rex-1 in these three p53 mutant iPS cell lines was coincident with the H1 ESC at RNA level (S2A Fig). At protein levels, we also confirmed that all three iPSCs retained ES marker expression (e.g., Oct4, Sox2, NANOG and TRA-1-60, S2B Fig) by immunostaining. What’s more, the iPSCs with the p53 p.Asn268Glufs*4 mutation as normal iPS could differentiate into three primary germ layers and form teratomas in immunodeficient mice (S2C Fig). All of these data indicate that iPSCs with the p53 p.Asn268Glufs*4 mutation can maintain pluripotency. Ultimately, similar to previous reports [30-32], we could not detect the vector sequence (EBNA1 and OSW) in iPSCs by PCR after 10 times of passages (S2D Fig).

The heterozygous p53 mutant cells have random allelic expression of p53

To demonstrate whether the iPS was originated from this patient, we performed Sanger sequence analysis. The results showed that all iPS cell lines contained the same p53 mutation with patient’s somatic cells. (Fig 4A), which confirmed that the p53 p.Asn268Glufs*4 mutant is a germline mutation.

Fig 4

The p53 mutation causes random allelic expression in heterozygous iPS cell lines.

a. Sanger DNA sequencing of three patient iPS cell lines. **, p< 0.01. *, p< 0.05. b. RT-PCR of expression of p53 in iPSCs derived from an LFS patient compared with H1 cells. c. WB of p53 protein expression in iPSCs derived from an LFS patient compared with H1 cells. Arrow, WT p53; arrow head, p53 mutant. d. p53 cDNA sequence from three LFS patient-derived iPS cell lines. e. p53 cDNA sequence from the somatic cells of the patient and his mother.

Clinically, it is common for LFS patients to carry the p53 mutations. However, not all p53 mutations carriers will develop into LFS patients p53 does not fully follow the classic Knudson’s two-hit theory during carcinogenesis or cancer progression [33, 34]. Similar to the previous condition, the patient here inherited the disease-causing mutation, p53 p.Asn268Glufs*4 from his mother, but his mother (22 years old) had not yet developed the disease. Compared with the expression level of p53 between different iPS cell lines from the patient, we found that there was no difference at their mRNA levels (Fig 4B) whereas their protein levels were significantly different (Fig 4C). p53 protein levels in one of the iPS cell lines were same as in H1 ESCs, whereas the other two iPS cell lines expressed lower levels of p53 WT and mutant proteins (Fig 4C). To clarify this phenomenon, we performed Sanger sequencing of the p53 cDNAs from the three patient iPS cell lines and found that the cell line with normal amount of p53 protein only contained the p53 WT, while the other two with lower expression of p53 protein contained almost equivalent amounts of the WT and the mutated p53 sequences (Fig 4D) Then, we checked p53 mRNA and protein levels in other three randomly selected iPS cell lines, but we did not find any difference compared to the H1 ES control (S3A and S3B Fig). What’s more, all of the three iPS cell lines contained p53 WT RNA sequence (S3C Fig). These data indicated that there may be random allelic gene expression in p53 heterozygous mutations.

To confirm our hypothesis, we sequenced p53 cDNA from the patient’s and his mother’s mononuclear cells. We found that their mononuclear cells mainly contained the WT p53 sequence and low expression of the mutated p53 RNA (Fig 4E). It suggested that the p53 mutant allele was expressed in iPS cell lines and somatic cells. This finding indicates that checking the protein level of mutant p53 may be more important than sequencing p53 DNA and mutant p53 allelic expression is a potential predictor of cancer risk.

Discussion

In summary, the goal of the present study was to gain a better understanding of the specific roles of p53 mutations during iPSC reprogramming and p53 related tumorigenesis. Mutations in p53 usually not only abolish its normal function, but also gain additional oncogenic functions [13-16]. In a word, this specific p53 mutation loses the ability to induce apoptosis and inhibit proliferation, which facilitates tumorigenesis. Besides, this mutation reduced the efficiency of somatic cell reprogramming by inhibiting OCT4 expression. Random allelic expression of p53 in heterozygous p53 mutations caused variable WT p53 protein expression, which might be one of the reasons why people with the same p53 mutation had different states of health.

In the present study, we found that losing part of p53 function caused by a heterozygous mutation did not promote cell reprogramming, instead, it did significantly decrease induction frequency of iPS generation by inhibiting OCT4 expression during reprogramming. This result was contrary to the phenomenon related with p53 deletions and mutations (p53 isoform Δ133p53). For example, the high expression of the p53 isoform Δ133 improved the induction efficiency of iPSCs and ensured genomic integrity during reprogramming [35-37]. Consistent with our results, the OCT4 expression dramatically increased in p53 knockout MEF cells compared with WT p53 MEF cells [38]. Using chromosome counting, we found that three iPS cell lines were hypodiploidy, which was the same as that in the p53 knockout iPS cell lines [12].

Family history could not predict the presence of an underlying predisposition syndrome in most patients [39]. In mammals, monoallelic gene expression can result from X-chromosome inactivation, genomic imprinting and random monoallelic expression (RMAE) [40, 41]. Recently, many studies have found allelic imbalance in the chromatin state of autosomal genes [42-44]. Biallelic inactivation of p53 has a significant impact on clinical outcome in multiple myeloma [41].

In our paper, we found that the patient and his mother had the same p53 mutation, but his mother was a healthy carrier without any clinical symptoms. Using Sanger sequencing to analyze the p53 cDNA of the six patient-derived iPS cell lines, we found that four iPS cell lines only contained the p53 WT cDNA sequence, while the other two with low p53 expression contained both WT and mutant p53 cDNA sequences, indicating that p53 random allelic expression occurred in heterozygous mutations. When testing the cDNA sequence of the patient and his mother’s somatic cells, we also found very little mutant p53 RNA. This result confirmed that the random allelic expression of p53 in heterozygous p53 mutations varied the WT p53 expression. Random allelic expression of heterozygous p53 mutations may be a reason why the people with p53 mutations develop cancer at random. This finding suggested that mutated p53 allelic expression should be added to the risk forecasting of cancer.

Conclusion

Our data demonstrate that the mutation of p53 p.Asn268Glufs*4 maintains partial p53 function, which decreases the efficiency of somatic reprogramming by inhibiting OCT4 expression during the reprogramming stage and exhibites random p53 allelic expression in heterozygous p53 mutant cells. Random allelic expression of p53 in heterozygous mutation scenarios may be a reason why the people who carry p53 mutations develop cancer at random. Our finding also suggests that the mutant p53 allelic expression may be a risk forecasting of cancers.

Expression of pluripotent genes in p53 mutation cells.

a-b. RT-PCR of expression of SOX2 and NANOG in cells with p53 WT or mutant compared with an empty vector (EV) control. c. Western blot analysis of p53, BCL-2, and PUMA, γH2AX-139 expression after transfecting with lentiviruses carrying the p53 R175H, WT p53 and vector control plasmids into p53 KO MEF cells. d. Growth curve of p53 KO MEF cells with p53 WT or R175H. ** p<0.01. e. FACS analysis of apoptosis at day 3 after p53 KO MEF cells infection of p53 WT or R175H. ** p<0.01. f. iPS colony numbers following introduction of WT p53, R175H and vector into p53 KO MEFs were counted on reprogramming day 14 after transduction.

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Qualification of iPSCs from LFS patient.

a. RT-PCR of expression of pluripotency genes in iPSCs compared with H1 ESCs. b. Representative images of pluripotency markers OCT4, SOX-2, NANOG, and TRA-1-60 in iPSCs. c. Teratoma analysis of iPSCs with p53 mutation. H&E staining of representative teratoma with derivatives of three embryonic germ layers: blood vessel with blood (mesoderm), glands (endoderm), and epithelium (ectoderm). d. Vector sequence (OSW and EBNA1) was tested by PCR-based detection in iPSCs expanded for 10 passages.

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Analysis of random allelic expression of p53 in another three iPS cell lines.

a. RT-PCR of expression of p53 in another three iPS cell lines compared with H1 cells. b. WB of p53 protein levels in another three iPS cell lines compared with H1 cells. c. p53 cDNA sequence from another three iPS cell lines.

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6 Mar 2020

PONE-D-20-04840

The function of a heterozygous p53 mutation (p.Asn268Glufs*4) in a Li-Fraumeni syndrome (LFS) patient

PLOS ONE

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Reviewer #1: No

Reviewer #2: Partly

**********

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Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: In this manuscript the authors have described a heterozygous p53 mutation in a LFS patient. The authors claim that p.Asn268Glufs*4 mutation promotes tumorigenecity and inhibit the efficiency of somatic reprogramming by inhibiting OCT4 expression. The manuscript has some major issues that need to be resolved.

1. Authors need to thoroughly proofread the manuscript as it had grammatical mistakes.

2. Authors need to be consistent with gene and protein nomenclature of p53 protein throughout the manuscript.

3. The sentence “P53 has four identical chains of 393 residues” in the result section is faulty syntax. Please revise.

4. What did the authors mean by “Asn268Glufs*4 mutation was located in specific DNA binding domain to stop this specific protein synthesis”. Does that mean that the mutation was a nonsense mutation and hence no protein was synthesized?

5. Authors need to clarify why they chose 293T cells that has endogenous WT p53 to do lentiviral infection experiments. The authors need to choose a cleaner system to do these studies with a p53 null background and then compare WT p53 vs mutant p53 effects.

6. In figure 2a why are there two different lanes for mutant vs WT p53? Also, the p53 blot is a little confusing.

7. Authors wrote that “The expression of PUMA in mutant 15 cells was the same as that in the control cells, but PUMA levels were relatively decreased in WT cells (Fig. 2a).” but that is contrary to what is shown in the figure. PUMA levels were increased.

8. Authors need to clarify between which groups was statistical comparison performed. Was it between control vs WT and control vs mutant or WT vs mutant? Was there any difference between control and mutant?

9. The result “TP53 p.Asn268Glufs*4 mutation promotes the tumorigenesis” cannot be concluded using just apoptosis and DNA damage experiments. Especially since most of the difference between mutant and control are minimal and may not be biologically significant.

10. How are the authors concluding that mutant p53 protein is present in the patient’s MNC? The levels that they are seeing in 3a-3b could be just the endogenous WT p53 allele since patient is heterozygous.

The mutation studied by the authors appears to be a loss of function mutation leading to the patient having only one functional WT allele. And most of the functional data shown could be attributed to having lower p53 levels due to only one allele present in the patient rather than the mutation specifically.

Reviewer #2: The manuscript titled “The function of a heterozygous p53 mutation (p.Asn268Glufs*4) in a Li-Fraumeni

syndrome (LFS) patient” by Li, et al. presents a follow-up analysis of a TP53 mutation they found in a Li Fraumeni Syndrome patient. They utilize cell culture models and sequencing to determine the functional aspect of the mutation. While the study of specific mutations found in LFS patients may be interesting, provided they yield some generalizable knowledge, there are some serious flaws throughout the manuscript that must be addressed which are detailed below.

1. The authors claim (pg. 14) that the mutation studied, Asn268Glufs*4, was located within the DNA binding domain of p53 which stopped protein synthesis. It isn’t mentioned anywhere in the rest of the paper that this mutation created a truncated protein, neither is it shown that a smaller p53 protein is expressed. Instead, the authors show that the mutant expresses at the same molecular weight as both wildtype p53 and control samples. (Figures 2A, 3B, 4C, and S3B)

2. The entirety of Figure 2 uses the 293T cell line, which has endogenous wildtype p53, to show the consequence of overexpressing the Asn268Glufs*4 mutant as well as wildtype p53 on wildtype p53 target proteins, apoptosis, cell proliferation, and DNA damage. In order to avoid the dominant negative effect of mutant p53 on wildtype p53, and to compare the mutant with wildtype p53, the authors should use a cell line without p53 for these studies.

3. Figure 2A-the “p53 mutation” panel-how is expression of the specific mutation probed for? Is this from a lower point in the gel? The antibody listed under Materials and Methods recognizes p53 at the N-terminus and thus would recognize both mutant and wildtype p53.

4. Figure 2A-the authors state (pg.15) that PUMA levels were decreased in WT cells, when in fact PUMA was significantly increased.

5. Figure 2C-the authors state that “this TP53 mutation can promote tumor cell proliferation”. The graph shown depicts less proliferation than control. In order to make this claim, the mutant should show greater proliferation than the control cells.

6. Figure 2D-densitometry should be shown to be able to make the claim that the mutant induced DNA damage.

7. The authors state at the end of the first paragraph on pg. 15 that the “mutant could promote tumorigenesis”. An actual tumorigenicity assay would need to be performed to make this claim.

8. Overall Figure 3-A comparison between two completely different individuals cannot be made because there is no control over any other genetic difference. An isogenic system would need to be used for comparison.

9. Figure 3A-what is “control”?

10. Figure 3B-if the patient is being compared to the mother, p53 expression needs to be shown in the same Western blot.

11. Figure 3F-what is the p53 status of the MEFs used? If the MEFs contain wildtype p53, then the same problem exists as in the 293T system for Figure 2.

12. Figure 4B-the text states that there was no difference between the patient’s and the patient’s mother’s p53 RNA level, but this is not shown.

13. Figure S2C-the authors state that the images come from tumors formed using immunodeficient mice. These need to be labeled-what are the authors trying to show? Also, do H1 ES cells form tumors themselves?

14. Another point-since the authors state that the mutation being studied here may have lost its function, it would be helpful to show a known functional mutant found in LFS patients as a positive control.

**********

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Reviewer #2: No

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6 May 2020

The due date for submitting the revised version of your article is 20 Apr 2020.

Point-by-point response to the reviewers’ comments:

Reviewer 1:

Q1. Authors need to thoroughly proofread the manuscript as it had grammatical mistakes.

Response: We thank the reviewer to point out this. We have carefully checked and corrected the full manuscript again.

Q2. Authors need to be consistent with gene and protein nomenclature of p53 protein throughout the manuscript.

Response: We agree with this comment. We have modified as p53 gene and p53 protein.

Q3. The sentence “P53 has four identical chains of 393 residues” in the result section is faulty syntax. Please revise.

Response: We appreciate the reviewer to point out this mistake. We have corrected this as “The active p53 is a homo-tetramer formed by four identical chains of 393 residues each”.

Q4. What did the authors mean by “Asn268Glufs*4 mutation was located in specific DNA binding domain to stop this specific protein synthesis”. Does that mean that the mutation was a nonsense mutation and hence no protein was synthesized?

Response: I apologize for this confusion. We have modified it to “Asn268Glufs*4 mutation is a nonsense mutation which is located in specific DNA binding domain, which caused early termination of this specific protein synthesis and may affect the function of p53.”

Q5. Authors need to clarify why they chose 293T cells that has endogenous WT p53 to do lentiviral infection experiments. The authors need to choose a cleaner system to do these studies with a p53 null background and then compare WT p53 vs mutant p53 effects.

Response: We appreciate this important comment. We have carried out the experiment and replaced the data with p53-/- MEF (new Fig.2).

Q6. In figure 2a why are there two different lanes for mutant vs WT p53? Also, the p53 blot is a little confusing.

Response: We apologize for this confusion. We replaced this with new data showing p53 WT and mutant in the same film (new Fig. 2a).

Q7. Authors wrote that “The expression of PUMA in mutant 15 cells was the same as that in the control cells, but PUMA levels were relatively decreased in WT cells (Fig. 2a).” but that is contrary to what is shown in the figure. PUMA levels were increased.

Response: We apologize for these errors. We have corrected as: “PUMA levels relatively increased in WT cells”.

Q8. Authors need to clarify between which groups was statistical comparison performed. Was it between control vs WT and control vs mutant or WT vs mutant? Was there any difference between control and mutant?

Response: We apologize for this confusion. The statistical comparison was performed between control with p53 WT and p53 mutation. We have modified our result part to：“The analysis of apoptosis revealed that compared with unregulated control cells, overexpression of WT p53 enhanced apoptosis in p53-/- MEF cells (Fig. 2b, Fig.S1e). Only p53 WT induced DNA damage compared with the control (Fig. 2d-e, Fig. S1c). Unlike p53 R175H, p53 p.Asn268Glufs*4 mutant as well as its WT dramatically inhibited cell proliferation (Fig. 2c, Fig. S1d).”

Q9. The result “TP53 p.Asn268Glufs*4 mutation promotes the tumorigenesis” cannot be concluded using just apoptosis and DNA damage experiments. Especially since most of the difference between mutant and control are minimal and may not be biologically significant.

Response: We apologize for these errors. We have corrected as: “p.Asn268Glufs*4 mutation of p53 loses some functions of wild type p53”

Q10. How are the authors concluding that mutant p53 protein is present in the patient’s MNC? The levels that they are seeing in 3a-3b could be just the endogenous WT p53 allele since patient is heterozygous.

Response: We apologize for these errors. We replaced this with p53 WT and mutant band in the same film (new Fig. 3a).

Reviewer 2:

Q1. The authors claim (pg. 14) that the mutation studied, Asn268Glufs*4, was located within the DNA binding domain of p53 which stopped protein synthesis. It isn’t mentioned anywhere in the rest of the paper that this mutation created a truncated protein, neither is it shown that a smaller p53 protein is expressed. Instead, the authors show that the mutant expresses at the same molecular weight as both wildtype p53 and control samples. (Figures 2A, 3B, 4C, and S3B)

Response: We apologize for this confusion. We replaced this with p53 WT and mutant bands in the same film to new Figures 2A, 3A, 4C, and S3B

Q2. The entirety of Figure 2 uses the 293T cell line, which has endogenous wildtype p53, to show the consequence of overexpressing the Asn268Glufs*4 mutant as well as wildtype p53 on wildtype p53 target proteins, apoptosis, cell proliferation, and DNA damage. In order to avoid the dominant negative effect of mutant p53 on wildtype p53, and to compare the mutant with wildtype p53, the authors should use a cell line without p53 for these studies.

Response: We agree with this comment. We selected p53-/- MEF to repeat again. The results added to new Fig.2

Q3. Figure 2A-the “p53 mutation” panel-how is expression of the specific mutation probed for? Is this from a lower point in the gel? The antibody listed under Materials and Methods recognizes p53 at the N-terminus and thus would recognize both mutant and wildtype p53.

Response: I apologize for this confusion. We added the antibody to p53 information to material part: “Cell extracts were prepared, resolved on gels, transferred to nitrocellulose and incubated with antibodies against the N terminus of p53, which can recognize mutant and wild type of p53”.

Q4. Figure 2A-the authors state (pg.15) that PUMA levels were decreased in WT cells, when in fact PUMA was significantly increased.

Response：We apologize for these errors. We have modified as: “PUMA levels relatively increased in WT cells”.

Q5. Figure 2C-the authors state that “this TP53 mutation can promote tumor cell proliferation”. The graph shown depicts less proliferation than control. In order to make this claim, the mutant should show greater proliferation than the control cells.

Response: We apologize for these errors. And we modified to: “Only p53 WT induced DNA damage compared with the control (Fig. 2d-e, Fig. S1c). Unlike p53 R175H, p53 p.Asn268Glufs*4 mutant as well as its WT dramatically inhibited cell proliferation (Fig. 2c, Fig. S1d)”.

Q6. Figure 2D-densitometry should be shown to be able to make the claim that the mutant induced DNA damage.

Response: We agree with this comment. We have added the densitometry data in new Fig.2.

Q7. The authors state at the end of the first paragraph on pg. 15 that the “mutant could promote tumorigenesis”. An actual tumorigenicity assay would need to be performed to make this claim.

Response: We agree with this comment. We have corrected as: “p.Asn268Glufs*4 mutation of p53 loses some functions of wild type p53”

Q8. Overall Figure 3-A comparison between two completely different individuals cannot be made because there is no control over any other genetic difference. An isogenic system would need to be used for comparison.

Response: We agree with this comment. We cannot get the isogenic system control, so we deleted this panel from Fig.3.

Q9. Figure 3A-what is “control”?

Response: We apologize for this confusion. The Control was normal MNC. Now we have deleted this panel from Fig. 3.

Q10. Figure 3B-if the patient is being compared to the mother, p53 expression needs to be shown in the same Western blot.

Response: We agree with this comment. We have repeated this and showed p53 WT and mutant bands in the same film in new Fig.3a.

Q11. Figure 3F-what is the p53 status of the MEFs used? If the MEFs contain wildtype p53, then the same problem exists as in the 293T system for Figure 2.

Response: We agree with this comment. We added p53-/- MEF to do the experiments (Fig.3e-f, and Fig.S1a-S1b, S1f) again. The similar results were gained.

Q12. Figure 4B-the text states that there was no difference between the patient’s and the patient’s mother’s p53 RNA level, but this is not shown.

Response: We apologize for these errors. In Fig. 4B, iPS cells all come from patients. So, we modified the text states to “Compared with the expression level of p53 between different iPS cell lines from the patient, we found that there was no difference at their mRNA levels (Fig. 4b) whereas their protein levels were significantly different (Fig.4c).” in result part.

Q13. Figure S2C-the authors state that the images come from tumors formed using immunodeficient mice. These need to be labeled-what are the authors trying to show? Also, do H1 ES cells form tumors themselves?

Response: We apologize for this confusion. All iPS or ES which have the pluripotent ability can differentiate to three germ line tissues. So we modified to: “the iPSCs with the p53 p.Asn268Glufs*4 mutation as normal iPSCs could differentiate into three primary germ layers and form teratomas in immunodeficient mice (Fig. S2c). All of these data indicate that iPSCs with the p53 p.Asn268Glufs*4 mutation can maintain pluripotency”

Q14. Another point-since the authors state that the mutation being studied here may have lost its function, it would be helpful to show a known functional mutant found in LFS patients as a positive control.

Response: It’s a good suggestion. We have picked up p53 R175H as a positive control and performed new experiments. The result was shown in Fig.S1c-f.

Submitted filename: Rebuttal letter.docx

Click here for additional data file.

22 May 2020

The function of a heterozygous p53 mutation in a Li-Fraumeni syndrome patient

PONE-D-20-04840R1

Dear Dr. Li,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Sumitra Deb, PhD

Academic Editor

PLOS ONE

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: There were still many grammatical issues during sentence construction. Please proof read thoroughly once more before it can be ready for publishing.

Reviewer #2: (No Response)

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

27 May 2020

PONE-D-20-04840R1

The function of a heterozygous p53 mutation in a Li-Fraumeni syndrome patient

Dear Dr. Li:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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