Mismatch repair (MMR) efficiency and MSH2 gene mutation in human colorectal carcinoma cell line COLO320HSR

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLHL. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT 116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.     

Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.     

Mismatch repair (MMR) efficiency and <Emphasis Type="Italic">MSH2</Emphasis> gene mutation in human colorectal carcinoma cell line COLO320HSR

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLH1. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.     

Comparison of geno- and cytotoxicity of methylnitrosourea on MMR-proficient and MMR-deficient human tumor cell lines

Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells.     

Ferroptosis-inducing agents enhance TRAIL-induced apoptosis through upregulation of death receptor 5

Ferroptosis is considered genetically and biochemically distinct from other forms of cell death. In this study, we examined whether ferroptosis shares cell death pathways with other types of cell death. When human colon cancer HCT116, CX-1, and LS174T cells were treated with ferroptotic agents such as sorafenib (SRF), erastin, and artesunate, data from immunoblot assay showed that ferroptotic agents induced endoplasmic reticulum (ER) stress and the ER stress response-mediated expression of death receptor 5 (DR5), but not death receptor 4. An increase in the level of DR5, which is activated by binding to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and initiates apoptosis, was probably responsible for synergistic apoptosis when cells were treated with ferroptotic agent in combination with TRAIL. This collateral effect was suppressed in C/EBP (CCAAT-enhancer-binding protein)-homologous protein (CHOP)-deficient mouse embryonic fibroblasts or DR5 knockdown HCT116 cells, but not in p53-deficient HCT116 cells. The results from in vitro studies suggest the involvement of the p53-independent CHOP/DR5 axis in the synergistic apoptosis during the combinatorial treatment of ferroptotic agent and TRAIL. The synergistic apoptosis and regression of tumor growth were also observed in xenograft tumors when SRF and TRAIL were administered to tumor-bearing mice.     

NSMF promotes the replication stress-induced DNA damage response for genome maintenance

Proper activation of DNA repair pathways in response to DNA replication stress is critical for maintaining genomic integrity. Due to the complex nature of the replication fork (RF), problems at the RF require multiple proteins, some of which remain unidentified, for resolution. In this study, we identified the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF) as a key replication stress response factor that is important for ataxia telangiectasia and Rad3-related protein (ATR) activation. NSMF localizes rapidly to stalled RFs and acts as a scaffold to modulate replication protein A (RPA) complex formation with cell division cycle 5-like (CDC5L) and ATR/ATR-interacting protein (ATRIP). Depletion of NSMF compromised phosphorylation and ubiquitination of RPA2 and the ATR signaling cascade, resulting in genomic instability at RFs under DNA replication stress. Consistently, NSMF knockout mice exhibited increased genomic instability and hypersensitivity to genotoxic stress. NSMF deficiency in human and mouse cells also caused increased chromosomal instability. Collectively, these findings demonstrate that NSMF regulates the ATR pathway and the replication stress response network for genome maintenance and cell survival.     

Reduced ATR or Chk1 Expression Leads to Chromosome Instability and Chemosensitization of Mismatch Repair–deficient Colorectal Cancer Cells

Genomic instability in colorectal cancer is categorized into two distinct classes: chromosome instability (CIN) and microsatellite instability (MSI). MSI is the result of mutations in the mismatch repair (MMR) machinery, whereas CIN is often thought to be associated with a disruption in the APC gene. Clinical data has recently shown the presence of heterozygous mutations in ATR and Chk1 in human cancers that exhibit MSI, suggesting that those mutations may contribute to tumorigenesis. To determine whether reduced activity in the DNA damage checkpoint pathway would cooperate with MMR deficiency to induce CIN, we used siRNA strategies to partially decrease the expression of ATR or Chk1 in MMR-deficient colorectal cancer cells. The resultant cancer cells display a typical CIN phenotype, as characterized by an increase in the number of chromosomal abnormalities. Importantly, restoration of MMR proficiency completely inhibited induction of the CIN phenotype, indicating that the combination of partial checkpoint blockage and MMR deficiency is necessary to trigger CIN. Moreover, disruption of ATR and Chk1 in MMR-deficient cells enhanced the sensitivity to treatment with the commonly used colorectal chemotherapeutic compound, 5-fluorouracil. These results provide a basis for the development of a combination therapy for those cancer patients.     

Role of the mismatch repair system and p53 in the clastogenicity and cytotoxicity induced by bleomycin

The mismatch repair (MMR) system and p53 protein play a pivotal role in maintaining genomic stability and modulate cell chemosensitivity. Aim of this study was to examine the effects of either MMR-deficiency or p53 inactivation, or both, on cellular responses to bleomycin. The MMR-deficient colon carcinoma cell line HCT116 and its MMR-proficient subline HCT116/3-6, both expressing wild-type p53, were transfected with an expression vector encoding a dominant-negative p53 mutant, or with the empty vector. Four transfected clones, having the following phenotypes, MMR-proficient/p53 wild-type, MMR-proficient/p53 mutant, MMR-deficient/p53 wild-type, MMR-deficient/p53 mutant, were subjected to treatment with bleomycin. Loss of MMR function alone was associated with increased resistance to apoptosis, chromosomal damage and inhibition of colony formation caused by bleomycin. Loss of p53 alone resulted in abrogation of G1 arrest and increased sensitivity to apoptosis and chromosomal damage induced by the drug, but did not affect clonogenic survival after bleomycin treatment. Disabling both p53 and MMR function led to abrogation of G1 arrest and to a moderate impairment of drug-induced apoptosis. Chromosomal damage was reduced in the MMR-deficient/p53 mutant clone with respect to the MMR-proficient/p53 wild-type one, when evaluated 48 h after bleomycin treatment, but was comparable in both clones 96 h after drug exposure. Clonogenic survival of the MMR-deficient/p53 mutant clone was similar to that of the MMR-deficient/p53 wild-type one. The effects of MMR-deficiency on cellular responses to bleomycin were confirmed using the MMR-proficient lymphoblastoid cell line TK6 and its MMR-deficient subline MT1, both expressing wild-type p53. In conclusion, our data show that loss of MMR and p53 function exerts opposite and independent effects on apoptosis and chromosomal damage induced by bleomycin. Moreover, inactivation of MMR confers resistance to the cytotoxic activity of the anticancer agent in cells expressing either wild-type or mutant p53.     

DNA mismatch repair-dependent response to fluoropyrimidine-generated damage

Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G(2) arrest in HCT116 human colon cancer cells, as well as Mlh1(-/-) murine embryonic fibroblasts, after treatment with 5-fluoro-2'-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2(+/+) (compared with isogenic Msh2(-/-)) murine embryonic stem cells. Because MMR status had little effect on cellular responses (i.e. G(2) arrest and lethality) to the thymidylate synthase inhibitor, Tomudex, and a greater level of [(3)H]FdUrd incorporation into DNA was found in MMR-deficient cells, we concluded that the differential FdUrd cytotoxicity between MMR-competent and MMR-deficient cells was mediated at the level of DNA incorporation. Analyses of ATPase activation suggested that the hMSH2-hMSH6 heterodimer only recognized FdUrd moieties (as the base 5-fluorouracil (FU) in DNA) when mispaired with guanine, but not paired with adenine. Furthermore, analyses of incorporated FdUrd using methyl-CpG-binding domain 4 glycosylase indicated that there was more misincorporated FU:Gua in the DNA of MMR-deficient HCT116 cells. Our data provide the first demonstration that MMR specifically detects FU:Gua (in the first round of DNA replication), signaling a sustained G(2) arrest and lethality.     

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.     

Chromosomal instability at common fragile sites in Seckel syndrome

Seckel syndrome (SCKL) is a rare, genetically heterogeneous disorder, with dysmorphic facial appearance, growth retardation, microcephaly, mental retardation, variable chromosomal instability, and hematological disorders. To date, three loci have been linked to this syndrome, and recently, the gene encoding ataxia-telangiectasia and Rad3-related protein (ATR) was identified as the gene mutated at the SCKL1 locus. The ATR mutation affects splicing efficiency, resulting in low levels of ATR in affected individuals. Elsewhere, we reported increased instability at common chromosomal fragile sites in cells lacking the replication checkpoint gene ATR. Here, we tested whether cells from patients carrying the SCKL1 mutation would show increased chromosome breakage following replication stress. We found that, compared with controls, there is greater chromosomal instability, particularly at fragile sites, in SCKL1-affected patient cells after treatment with aphidicolin, an inhibitor of DNA polymerase alpha and other polymerases. The difference in chromosomal instability between control and patient cells increases at higher levels of aphidicolin treatment, suggesting that the low level of ATR present in these patients is not sufficient to respond appropriately to replication stress. This is the first human genetic syndrome associated with increased chromosome instability at fragile sites following replication stress, and these findings may be related to the phenotypic findings in patients with SCKL1.     

Requirement of BAX for efficient adenovirus-induced apoptosis

Infection of human epithelial cells with adenoviruses induces an apoptosis paradigm that is efficiently suppressed by the expression of viral E1B-19K protein, which is a functional homolog of the cellular antiapoptosis protein BCL-2. The mechanisms of adenovirus (Ad)-induced apoptosis appear to involve the cellular BCL-2 family proapoptotic proteins. Recent genetic studies with fibroblasts derived from mutant mouse embryos indicate that a class of the BCL-2 family proapoptotic proteins (designated BH-123 or multidomain proteins) such as BAX and BAK constitutes an essential component of the core apoptosis machinery in animal cells. We have examined the role of BAX in Ad-induced apoptosis in human epithelial cells using two colon cancer cell lines, HCT116Bax (Bax(+/-)) and HCT116BaxKO (Bax(-/-)) (L. Zhang, J. Yu, B. H. Park, K. W. Kinzler, and B. Vogelstein, Science 290:989-992, 2000). Infection of Bax(+/-) cells with an Ad type 2 mutant (dl250) defective in expression of the E1B-19K protein resulted in enhanced cytopathic effect, large plaques on cell monolayers, fragmentation of cellular DNA, and enhanced cell death. These mutant phenotypes were not efficiently expressed in Bax(-/-) cells, suggesting that BAX is essential for Ad-induced apoptosis. Infection of Bax(+/-) cells with dl250 induced increased levels of an N-terminally processed form of BAX. Cells infected with the 19K mutant also contained enhanced levels of truncated BAX in membrane-inserted form. Our results suggest that at least a part of the mechanism utilized by E1B-19K to suppress apoptosis during Ad infection may involve modulation of the activities of BAX.     

Mismatch Repair Status and the Response of Human Cells to Cisplatin

The emergence of resistance to cisplatin is a serious drawback of cancer therapy. To help elucidate the molecular basis of this resistance, we examined matched ovarian cancer cell lines that differ in their DNA mismatch repair (MMR) status and the response to cisplatin. Checkpoint activation by cisplatin was identical in both lines. However, sensitive cells delayed S-phase transition, arrested at G2/M and died by apoptosis. The G2/M block was characterized by selective disappearance of homologous recombination (HR) proteins, which likely resulted in incomplete repair of the cisplatin adducts. In contrast, resistant cells transiently arrested at G2/M, maintained constant levels of HR proteins and ultimately resumed cell cycle progression. The net contribution of MMR to the cisplatin response was examined using matched semi-isogenic (HCT116±chr3) or strictly isogenic (293T-Lα-/+) cell lines. Delayed transition through S-phase in response to cisplatin was also observed in the MMR-proficient HCT116+chr3 cells. Unlike in the ovarian cell lines, however, both HCT116+chr3 and HCT116 permanently arrested at G2/M with an intact complement of HR proteins and died by apoptosis. A similar G2/M arrest was observed in the strictly isogenic 293T-Lα-/+ cells. This confirmed that although MMR undoubtedly contributes towards the cytotoxicity of cisplatin, it is only one of several pathways that modulate the cellular response to this drug. However, our data highlighted the importance of HR to cisplatin cytotoxicity and suggested that HR status might represent a novel prognostic marker and possibly also a therapeutic target, the inhibition of which would substantially sensitize cells to cisplatin chemotherapy.     

Checkpoint signaling, base excision repair, and PARP promote survival of colon cancer cells treated with 5-fluorodeoxyuridine but not 5-fluorouracil

The fluoropyrimidines 5-fluorouracil (5-FU) and FdUrd (5-fluorodeoxyuridine; floxuridine) are the backbone of chemotherapy regimens for colon cancer and other tumors. Despite their widespread use, it remains unclear how these agents kill tumor cells. Here, we have analyzed the checkpoint and DNA repair pathways that affect colon tumor responses to 5-FU and FdUrd. These studies demonstrate that both FdUrd and 5-FU activate the ATR and ATM checkpoint signaling pathways, indicating that they cause genotoxic damage. Notably, however, depletion of ATM or ATR does not sensitize colon cancer cells to 5-FU, whereas these checkpoint pathways promote the survival of cells treated with FdUrd, suggesting that FdUrd exerts cytotoxicity by disrupting DNA replication and/or inducing DNA damage, whereas 5-FU does not. We also found that disabling the base excision (BER) repair pathway by depleting XRCC1 or APE1 sensitized colon cancer cells to FdUrd but not 5-FU. Consistent with a role for the BER pathway, we show that small molecule poly(ADP-ribose) polymerase 1/2 (PARP) inhibitors, AZD2281 and ABT-888, remarkably sensitized both mismatch repair (MMR)-proficient and -deficient colon cancer cell lines to FdUrd but not to 5-FU. Taken together, these studies demonstrate that the roles of genotoxin-induced checkpoint signaling and DNA repair differ significantly for these agents and also suggest a novel approach to colon cancer therapy in which FdUrd is combined with a small molecule PARP inhibitor.     

Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53^+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53^−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53^−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.