Understanding cell cycle and cell death regulation provides novel weapons against human diseases.

Cell division, cell differentiation and cell death are the three principal physiological processes that regulate tissue homoeostasis in multicellular organisms. The growth and survival of cells as well as the integrity of the genome are regulated by a complex network of pathways, in which cell cycle checkpoints, DNA repair and programmed cell death have critical roles. Disruption of genomic integrity and impaired regulation of cell death may both lead to uncontrolled cell growth. Compromised cell death can also favour genomic instability. It is becoming increasingly clear that dysregulation of cell cycle and cell death processes plays an important role in the development of major disorders such as cancer, cardiovascular disease, infection, inflammation and neurodegenerative diseases. Research achievements in these fields have led to the development of novel approaches for treatment of various conditions associated with abnormalities in the regulation of cell cycle progression or cell death. A better understanding of how cellular life-and-death processes are regulated is essential for this development. To highlight these important advances, the Third Nobel Conference entitled 'The Cell Cycle and Cell Death in Disease' was organized at Karolinska Institutet in 2016. In this review we will summarize current understanding of cell cycle progression and cell death and discuss some of the recent advances in therapeutic applications in pathological conditions such as cancer, neurological disorders and inflammation.

What is the restriction point?

The restriction point (R) separates two functionally different parts of G1 in continuously cycling cells. G1-pm represents the postmitotic interval of G1 that lasts from mitosis to R. G1-ps represents the pre S phase interval of G1 that lasts from R to S. G1-pm is remarkably constant in length (its duration is about three hours) in the different cell types studied so far. G1-ps, however, varies considerably, indicating that entry into S is not directly followed after passage through R. Progression through G1-pm requires continuous stimulation by mitogenic signals (e.g. growth factors) and a high rate of protein synthesis. Interruption of the mitogenic signals or moderate inhibition of protein synthesis leads to a rapid exit from the cell cycle to G0 in normal (untransformed) cells. Upon restimulation with mitogenic signals, the cell returns to the same point in G1-pm from which it left the cell cycle. Thus the cell seems to have a memory for how far it has advanced through G1-pm, suggesting that a continuous structural alteration, for example chromatin decondensation, takes place in G1. The molecular background to transition from growth factor dependence in G1-pm to growth factor independence in G1-ps (a switch which represents commitment to a new cell cycle and passage through R) is still not fully understood. Cyclin-dependent kinase (cdk)-mediated hyperphosphorylation of the retinoblastoma protein (Rb), and concomitant liberation (and activation) of members of the E2F family of transcription factors, are probably important aspects of R control in normal cells. A key component here could be cdk2 activity which is controlled by cyclin E. When cdk2 activity starts to increase rapidly in G1, due to activation of a positive feedback loop, it reaches a critical level above which cdk inhibitors (CKIs) such as p21 and p27 are outweighed; the cell has then become independent of mitogenic and inhibitory signals and is committed to a new cell cycle. However, other components are probably also involved in R control. For instance, a 'cryptic' R (a G1-pm-like state) can be induced even in tumour cells that do not respond to growth factor starvation or protein synthesis inhibitors, and are therefore probably defective in the cdk-Rb-E2F pathway. Possibly, a certain degree of chromatin decondensation has to take place after mitosis in order to allow transcription of, for example, the cyclin E gene or other critical E2F targets. Although the molecular basis for restriction point control still remains unclear, we can expect rapid progress in this important field over the next few years.

Restoration of the growth suppression function of mutant p53 by a synthetic peptide derived from the p53 C-terminal domain.

We demonstrate here that synthetic 22-mer peptide 46, corresponding to the carboxy-terminal amino acid residues 361-382 of p53, can activate specific DNA binding of wild-type p53 in vitro and can restore the transcriptional transactivating function of at least some mutant p53 proteins in living cells. Introduction of peptide 46 in Saos-2 cells carrying a Tet-regulatable His-273 mutant p53 construct caused growth inhibition and apoptosis in the presence of mutant p53 but not in its absence, confirming that the effect of the peptide is mediated by reactivation of mutant p53. Moreover, peptide 46 caused apoptosis in mutant as well as wild-type p53-carrying human tumor cell lines of different origin, whereas p53 null tumor cells were not affected. These findings raise possibilities for developing drugs that restore the tumor suppressor function of mutant p53 proteins, thus selectively eliminating tumor cells.

Reactivation of mutant p53: molecular mechanisms and therapeutic potential.

The p53 tumor suppressor gene is the most frequently mutated gene in cancer. Most p53 mutations are missense point mutations that cluster in the DNA-binding core domain. This results in distortion of core domain folding and disruption of DNA binding and transcriptional transactivation of p53 target genes. Structural studies have demonstrated that mutant p53 core domain unfolding is not irreversible. Mutant p53 is expressed at high levels in many tumors. Therefore, mutant p53 is a promising target for novel cancer therapy. Mutant p53 reactivation will restore p53-dependent apoptosis, resulting in efficient removal of tumor cells. A number of strategies for targeting mutant p53 have been designed, including peptides and small molecules that restore the active conformation and DNA binding to mutant p53 and induce p53-dependent suppression of tumor cell growth in vitro and in vivo. This opens possibilities for the clinical application of mutant p53 reactivation in the treatment of cancer.

PRIMA-1(MET) induces nucleolar accumulation of mutant p53 and PML nuclear body-associated proteins.

We have previously identified PRIMA-1, a low molecular weight compound that restores the transcriptional transactivation function to mutant p53 and induction of apoptosis. To explore the molecular mechanism for PRIMA-1-induced mutant p53-dependent apoptosis, we examined the intracellular distribution of mutant p53 upon treatment with PRIMA-1(MET) by immunofluorescence staining. We found that PRIMA-1(MET) induced nucleolar translocation of mutant p53 and the promyelocytic leukemia (PML) nuclear body-associated proteins PML, CBP and Hsp70. Levels of Hsp70 were significantly enhanced by PRIMA-1(MET) treatment. PRIMA-Dead, a compound structurally related to PRIMA-1 but unable to induce mutant p53-dependent apoptosis, failed to induce nucleolar translocation of mutant p53. Our results suggest that redistribution of mutant p53 to nucleoli plays a role in PRIMA-1-induced apoptosis.

Hypoxia induces p53-dependent transactivation and Fas/CD95-dependent apoptosis.

p53 triggers apoptosis in response to cellular stress. We analyzed p53-dependent gene and protein expression in response to hypoxia using wild-type p53-carrying or p53 null HCT116 colon carcinoma cells. Hypoxia induced p53 protein levels and p53-dependent apoptosis in these cells. cDNA microarray analysis revealed that only a limited number of genes were regulated by p53 upon hypoxia. Most classical p53 target genes were not upregulated. However, we found that Fas/CD95 was significantly induced in response to hypoxia in a p53-dependent manner, along with several novel p53 target genes including ANXA1, DDIT3/GADD153 (CHOP), SEL1L and SMURF1. Disruption of Fas/CD95 signalling using anti-Fas-blocking antibody or a caspase 8 inhibitor abrogated p53-induced apoptosis in response to hypoxia. We conclude that hypoxia triggers a p53-dependent gene expression pattern distinct from that induced by other stress agents and that Fas/CD95 is a critical regulator of p53-dependent apoptosis upon hypoxia.

Mutated and non-mutated TP53 as targets in the treatment of leukaemia.

TP53 is mutated in 10-20% of cases of chronic lymphocytic leukaemia (CLL) and 3-8% of cases of acute myeloid leukaemia (AML). Recently, two classes of compounds that restore the function of p53 in tumours have been described. PRIMA-1 (p53-dependent reactivation and induction of massive apoptosis) restores the wild-type conformation of mutant TP53, whereas RITA (reactivation of p53 and induction of tumour cell apoptosis) increases intracellular levels of p53. We evaluated the effects of RITA alone and in combination with PRIMA-1 or conventional cytostatics on leukaemic cells isolated from AML and CLL patients. AML samples with -17, which are more resistant to daunorubicin and cytarabine compared with samples without -17, were effectively killed by PRIMA-1. RITA, which stabilizes the function of wild-type p53, induced apoptosis in AML cells. In contrast to that seen with PRIMA-1, AML patient samples without -17 were significantly more sensitive to RITA. Similarly, RITA exerted dose-dependent apoptosis and cytotoxicity in CLL cells, which was significantly more pronounced in samples without hemizygous TP53 deletion. Notably, a synergistic effect was observed in all CLL samples with RITA and fludarabine in combination. In both AML and CLL cells exposure to RITA resulted in induction of intracellular p53. We conclude that small molecules targeting p53 might be of clinical importance in the future for treating drug-resistant leukaemia.

Reactivation of mutant p53 through interaction of a C-terminal peptide with the core domain.

A synthetic 22-mer peptide (peptide 46) derived from the p53 C-terminal domain can restore the growth suppressor function of mutant p53 proteins in human tumor cells (G. Selivanova et al., Nat. Med. 3:632-638, 1997). Here we demonstrate that peptide 46 binds mutant p53. Peptide 46 binding sites were found within both the core and C-terminal domains of p53. Lys residues within the peptide were critical for both p53 activation and core domain binding. The sequence-specific DNA binding of isolated tumor-derived mutant p53 core domains was restored by a C-terminal polypeptide. Our results indicate that C-terminal peptide binding to the core domain activates p53 through displacement of the negative regulatory C-terminal domain. Furthermore, stabilization of the core domain structure and/or establishment of novel DNA contacts may contribute to the reactivation of mutant p53. These findings should facilitate the design of p53-reactivating drugs for cancer therapy.

PRIMA-1MET induces mitochondrial apoptosis through activation of caspase-2.

This corrects the article DOI: 10.1038/onc.2016.210.

Increased expression of insulin-like growth factor I receptor in malignant cells expressing aberrant p53: functional impact.

We investigated the functional impact of p53 on insulin-like growth factor I receptor (IGF-IR) expression in malignant cells. Using the BL-41tsp53-2 cell line, a transfectant carrying temperature-sensitive (ts) p53 and endogenous mutant p53 (codon 248), we demonstrated a drastic down-regulation of plasma membrane-bound IGF-IRs on induction of wild-type p53. However, a similar response was obtained by treatment of BL-41tsp53-2 cells expressing mutant ts p53 with a p53 antisense oligonucleotide. Thus, even if the negative effect of wild-type p53 predominates under a competitive condition, these data indicate that mutant p53 may be important for up-regulation of IGF-IR. To further elucidate this issue, three melanoma cell lines (BE, SK-MEL-5, and SK-MEL-28) that overexpressed p53 were investigated. The BE cell line has a "hot spot" mutation (codon 248) and expresses only codon 248-mutant p53. SK-MEL-28 has a point mutation at codon 145. SK-MEL-5 cells did not exhibit any p53 mutations, but the absence of p21Waf1 expression suggested functionally aberrant p53. Our data suggest that interaction with Mdm-2 may underlie p53 inactivation in these cells. Using p53 antisense oligonucleotides, we demonstrated a substantial down-regulation of cell surface expression of IGF-IR proteins in all melanoma cell lines after 24 h. This was paralleled by decreased tyrosine phosphorylation of IGF-IR and growth arrest, and, subsequently, massive cell death was observed (this was also seen in BL-41tsp53-2 cells with mutant conformation of ts p53). Taken together, our results suggest that up-regulation of IGF-IR as a result of expression of aberrant p53 may be important for the growth and survival of malignant cells.

p14ARF homozygous deletion or MDM2 overexpression in Burkitt lymphoma lines carrying wild type p53.

The hallmark of Burkitt lymphoma (BL) is a constitutively activated c-myc gene that drives tumor cell growth. A majority of BL-derived cell lines also carry mutant p53. In addition, the p16INK4a promoter is hypermethylated in most BL biopsies and BL cell lines, leading to silencing of this gene. Activation of c-myc and/or cell cycle dysregulation can induce ARF expression and p53-dependent apoptosis. We therefore investigated the p14ARF-MDM2-p53 pathway in BL cell lines. p14ARF was expressed and localized to nucleoli in all BL carrying mutant p53. Three out of seven BL carrying wt p53 had a homozygous deletion of the CDKN2A locus that encodes both p14ARF and p16INK4a. Three BL carrying wild type p53 retained the CDKN2A locus and overexpressed MDM2. DNA sequencing revealed a point mutation in CDKN2A exon 2 in one of these BL, Seraphine. However, this point mutation did not affect p14ARF's nucleolar localization or ability to induce p53. The Bmi-1 protein that negatively regulates the p14ARF promoter and co-operates with c-myc in tumorigenesis was expressed at low to moderate levels in all BL analysed. Our results indicate that inactivation of the ARF-MDM2-p53 pathway is an essential step during the development of Burkitt lymphoma, presumably as a mechanism to escape c-myc induced apoptosis.

Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts.

The expression of the retinoblastoma susceptibility (RB-1) gene was investigated in highly proliferating mouse embryonic stem (ES) cells and in slowly proliferating mouse embryonic fibroblasts. The RB protein was expressed at the same level in these two cell types. Mainly hyperphosphorylated RB was detected in exponentially-growing ES cells. Embryonic fibroblasts and embryonic stem cells were synchronized by colcemid block followed by mitotic shake-off. In embryonic fibroblasts, DNA replication started 10-15 h after exit from mitosis and RB was transiently dephosphorylated during the G1 phase as previously described. In ES cells, DNA replication started 2 h after release from the colcemid block but virtually no hypophosphorylated RB was observed after the release. Instead, there was a dramatic decrease in the total RB protein level between exit from mitosis and entry into S phase. These observations were made by using two different monoclonal antibodies, both in immunoblotting and immunoprecipitation experiments. Absence of hypophosphorylated RB and cell cycle-dependent change in total RB protein level may be relevant to the high proliferation rate and to the tumorigenic nature of mouse embryonic stem cells.

Mutant p53 detected in a majority of Burkitt lymphoma cell lines by monoclonal antibody PAb240.

The status of the p53 gene in lymphoblastoid cell lines (LCLs) and Burkitt lymphoma cell lines (BLs) was investigated. Southern blot analysis demonstrated that no major deletions or rearrangements had occurred in the p53 gene in any of the cell lines. The p53 protein was examined by immunoprecipitation using two monoclonal anti-p53 antibodies. PAb1801 recognizes both wild-type and mutant p53. PAb240 reacts exclusively with mutant p53. Fourteen LCLs reacted with PAb1801, but not with PAb240, suggesting that none of them expressed mutant p53. However, one LCL had mutant p53. This LCL differs from other LCLs in that it grows to higher cell densities and has a higher agarose clonability. All BLs expressed p53. Out of 15 BLs, nine (60%) carried mutant p53, as indicated by their reactivity with PAb240. Among the nine BLs with mutant p53, eight Epstein-Barr virus (EBV)-positive. Three out of the six BLs with wild-type p53 were EBV-positive. Multiple EBV-converted sublines all exhibited the same p53 status as the parental line. Our results indicate that the p53 gene is mutated in a majority of Burkitt lymphoma cell lines (BLs), and suggest that p53 mutation contributes to the malignant phenotype of these cell lines.

Wild-type p53-triggered apoptosis is inhibited by bcl-2 in a v-myc-induced T-cell lymphoma line.

Using a temperature sensitive p53 construct (ts p53), we have earlier shown that expression of wild-type (wt) p53 triggers apoptosis in a v-myc-induced T-cell lymphoma line that lacks endogenous p53, and in a Burkitt lymphoma line that carries mutant p53. We have suggested that apoptosis is elicited by the contradictory signals emanating from the constitutively activated myc gene and the growth arresting signal of wt p53 (Ramqvist et al., 1993; Wang et al., 1993). Work in other laboratories has shown that constitutive c-myc expression can induce apoptosis when cell proliferation is inhibited due to the lack of growth stimulating factors. Expression of bcl-2 could inhibit apoptosis. In order to test whether p53-induced apoptosis can be prevented by bcl-2, we have introduced a retrovirally driven bcl-2 construct into our v-myc-induced murine T-cell lymphoma line, previously transfected with ts p53. About 90% of the parental ts p53 transfected cells died of apoptosis within 3 days after induction of wt p53 expression at 32 degrees C. Two clones of ts p53/bcl-2 double transfectants that expressed high levels of bcl-2 from the introduced construct were completely protected from apoptosis, following transfer of the cells to 32 degrees C. One clone that expressed the exogenous bcl-2 only at a low level was partially protected from wt p53-induced apoptosis. Clones of the parental ts p53 carrying cells transfected with the puromycin resistance gene vector, without the bcl-2 gene underwent 90% apoptosis. These results suggest that bcl-2 may prevent apoptosis in cells simultaneously exposed to the proliferation-stimulating effect of activated myc and the growth arresting signal of wt p53.

Is conversion of solid into more anoxic ascites tumors associated with p53 inactivation?

Most solid tumors are unable to grow in the ascites form, unless selected by prolonged serial transfer of peritoneal fluid (Klein, 1955). Established ascites tumor cells grow under highly crowded, virtually anoxic conditions (Warburg and Hiepler, 1953). Hypoxia was recently identified as a powerful inducer of p53 dependent apoptosis (Graeber et al., 1996). We wished to examine whether the conversion of relatively well-vascularized solid mouse tumors into freely growing ascitic cell variants favors cell with mutated or deleted p53. We have sequenced exons 4-9 of p53 cDNA from two serially transplanted methylcholanthrene induced sarcomas (MCIM and MSWBS) that were available in the original solid and the gradually converted ascites form. We have also examined five additional solid tumors, four carcinomas and one sarcoma and six additional ascites tumors, five carcinomas and one sarcoma. Sequence analysis showed that all solid tumors carried exclusively wild type p53. Among the eight ascites tumors, five carried mutant p53 and three had only the wild type gene. In one of the two isogenic pairs, the original solid tumor line had only wild type, whereas the derived ascites line had only mutant p53. In the second pair, the solid tumor was wild type whereas the ascitic variant was heterozygous. The naturally occurring alternatively spliced p53 (p53as) mRNA was detected in all solid tumors, but not in five of the eight ascites tumors. Our findings indicate that conversion of solid into ascites tumors favors the selection of cell variants with mutated p53 and of cells that lack the alternatively spliced form of p53.

Abrogation of p53-induced G1 arrest by the HPV 16 E7 protein does not inhibit p53-induced apoptosis.

Wild type (wt) p53 expressed from a temperature-sensitive construct (ts p53) can induce both G1 cell cycle arrest and apoptosis in the p53-negative J3D mouse T lymphoma line (Wang et al, 1995). The human papillomavirus (HPV) 16 E7 protein has been shown to prevent p53-induced G1 cell cycle arrest following DNA damage. We asked whether inhibition of p53-induced G1 arrest by overexpression of the HPV16 E7 protein in the ts p53-transfected J3D cells would interfere with p53-induced apoptosis. Whereas a majority of the ts p53-expressing J3D cells were arrested in the G1 phase 22 h after induction of wt p53 by temperature shift to 32 degrees C, the E7/ts p53-expressing cells showed only a minor increase in the number of cells in G1 at this time point. In addition, the E7/ts p53-expressing cells showed a much less dramatic reduction in number of cells in S phase than the ts p53-expressing cells. This demonstrates that E7 at least partially rescues the cells from p53-induced G1 arrest. In contrast, overexpression of HPV16 E7 did not have any effect on the kinetics nor the frequency of p53-triggered apoptotic death, as shown by FACS analysis, trypan blue exclusion, and DNA fragmentation analysis. These findings support the notion that p53-induced G1 arrest and p53-induced apoptosis are two separate independent pathways.

Wild-type p53 induces apoptosis in a Burkitt lymphoma (BL) line that carries mutant p53.

All Burkitt lymphoma (BL) biopsies and cell lines carry a c-myc/Ig translocation. The resulting constitutive activation of c-myc is regarded as an essential factor for the progressive growth of the tumor cells. At least 60% of BL cell lines carry a mutated p53 gene as well. It has been shown that the growth of mutant p53 carrying tumor cells could be inhibited by the introduction of wild-type p53. In order to examine whether this also applies to the presumably 'myc-driven' BL cell, we have transfected the Epstein-Barr virus (EBV) negative BL41 cell line with a temperature sensitive p53 mutant (p53-Val135) that expresses p53 with a largely mutant conformation at 37.5 degrees C and mostly wild-type conformation at 32 degrees C. At 37.5 degrees C, the p53-Val135 transfected cells behaved like the parental or neo transfected control cells. However, expression of exogenous wild-type p53 at 32 degrees C resulted in a rapid reduction of the number of viable cells while the parental and neo control cells remained unaffected. Cell death was due to apoptosis as shown by chromatin and nuclear condensation and specific DNA fragmentation. The first signs of apoptosis were evident after 10 h at 32 degrees C and after 3 days 90-100% of the cells had undergone apoptosis. These findings indicate an incompatibility between expression of wild-type p53 and progressive growth of BL cells if their neoplastic development has included a p53 mutation. The question whether apoptosis was induced in by the wild-type protein per se or by the contradictory signals of a constitutively activated c-myc and wild-type p53 needs further investigation.