DNA-PKcs Inhibition Sensitizes Cancer Cells to Carbon-Ion Irradiation via Telomere Capping Disruption

Heavy-ion irradiation induces a higher frequency of DNA double strand breaks (DSBs) which must be properly repaired. Critical shortening of telomeres can trigger DNA damage responses such as DSBs. Telomeres are very sensitive to oxidative stress such as ionizing radiation. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the central component in the non-homologous end joining (NHEJ) repair complex and participates in telomere maintenance. Therefore, it is expected to enhance the cell killing effect of heavy-ion irradiation via DNA-PKcs inhibition. To test this hypothesis, cellular radiosensitivity was measured by the clonal genetic assay. DNA damage repair was relatively quantified by long PCR. Apoptosis was quantified by flow-cytometric analysis of annexin V/PI double staining, and senescence was analyzed by galactosidase activity. Telomere length was semi-quantified by real-time PCR. P53 and p21 expression was determined by western blotting. Our data demonstrated that MCF-7 and HeLa cells with DNA-PKcs inhibition were more susceptible to carbon-ion irradiation than Those without DNA-PKcs inhibition. Even though NHEJ was inhibited by the DNA-PKcs specific inhibitor, NU7026, most DNA damage induced by carbon-ion irradiation was repaired within 24 hours after irradiation in both cell lines. However, potential lethal damage repair (PLDR) could not restore cellular inactivation in DNA-PKcs inhibited cells. MCF-7 cells showed extensive senescence and accelerated telomere length reduction, while HeLa cells underwent significant apoptosis after irradiation with NU7026 incubation. In addition, both cell lines with shorter telomere were more susceptible to carbon-ion radiation. Our current data suggested that DNA-PKcs inhibition could enhance cellular sensitivity to carbon-ion radiation via disturbing its functional role in telomere end protection. The combination of DNA-PKcs inhibition and carbon-ion irradiation may be an efficient method of heavy-ion therapy.     

Tubacin Kills Epstein-Barr Virus (EBV)-Burkitt Lymphoma Cells by Inducing Reactive Oxygen Species and EBV Lymphoblastoid Cells by Inducing Apoptosis

Tubacin is a small molecule inhibitor of histone deacetylase 6 and blocks aggresome activity. We found that Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) cells were generally killed by lower doses of tubacin than EBV-transformed lymphoblastoid cells (LCLs) or EBV-negative BL cells. Tubacin induced apoptosis of LCLs, which was inhibited by pretreatment with a pancaspase inhibitor but not by butylated hydroxyanisole, which inhibits reactive oxygen species. In contrast, tubacin killed EBV-positive BL cells in a caspase-3-independent pathway that involved reactive oxygen species and was blocked by butylated hydroxyanisole. Previously, we showed that bortezomib, a proteasome inhibitor, induces apoptosis of EBV LCLs and that LCLs are killed by lower doses of bortezomib than EBV-positive BL cells. Here we found that the combination of bortezomib and tubacin acted in synergy to kill EBV-positive BL cells and LCLs. Tubacin or the combination of bortezomib and tubacin did not induce EBV lytic replication. These findings suggest that the combination of a proteasome inhibitor and an HDAC6 inhibitor may represent a useful strategy for the treatment of certain EBV-associated B cell lymphomas.Epstein-Barr virus (EBV)⁴ is associated with several human lymphoid malignancies, including Hodgkin disease, Burkitt lymphoma (BL), T cell lymphomas, and post-transplant lymphoproliferative disease (1, 2). Tissues from patients with EBV post-transplant lymphoproliferative disease typically have a type 3 latency pattern in which each of the EBV latency-associated proteins, including EBV nuclear antigens (EBNA-1, -2, -3A, -3B, and -3C) and latent membrane proteins (LMP1 and LMP2) are expressed. A type 3 latency pattern is also seen in lymphoblastoid cell lines (LCLs), derived from primary B cells transformed with EBV in vitro. Tissues from patients with EBV-positive BL usually have a type 1 latency pattern with expression of EBNA-1 but not the other latency-associated proteins. When grown in cell culture, BL cell lines can have a type 1 or a type 3 pattern of latency.The treatment of EBV-associated lymphoid malignancies often requires cytotoxic chemotherapy, which is not always successful. Inhibition of proteasomes and aggresomes represents new therapeutic targets for malignancies (3–5). Degradation of proteins is required for vital cell functions and is carried out both in proteasomes and aggresomes. Misfolded or unfolded proteins are polyubiquitinated by a complex of proteins and subsequently degraded by proteasomes. However, if ubiquitinated proteins escape degradation by proteasomes and aggregate, they accumulate into aggresomes (6). Aggresome formation can be abrogated by disrupting the microtubule cytoskeleton or by overexpression of the p50 subunit of dynactin (7). HDAC6 (histone deacetylase 6) is a microtubule-associated deacetylase that can induce microtubule disassembly and promote chemotactic cell motility (8–10). HDAC6 contains a dynein motor binding domain, two catalytic domains with histone deacetylase activity, and a carboxyl-terminal domain that binds polyubiquitinated misfolded proteins (11). The carboxyl catalytic domain of HDAC6 possesses α-tubulin deacetylase activity (12). HDAC6 is required for transport of misfolded proteins for aggresome formation and to prevent apoptosis in response to misfolded protein stress (11). HDAC6 inhibitors disrupt aggresomes (5). Tubacin inhibits the carboxyl catalytic domain of HDAC6, increases the level of acetylated α-tubulin, and blocks aggresome activity (4, 12, 13).Bortezomib is an inhibitor of the 26 S proteasome (3). Previously, we showed that bortezomib induces apoptosis of EBV-transformed B cells and prolongs survival of mice inoculated with EBV-transformed B cells (14). In contrast, EBV-negative Burkitt lymphoma cells were much less sensitive to killing by bortezomib. Since bortezomib has been shown to interact synergistically with tubacin to induce apoptosis in multiple myeloma cells (4), we studied the effect of tubacin on EBV-transformed B cells and Burkitt lymphoma cells both in the absence and presence of bortezomib. We show that tubacin kills LCLs by apoptosis and induction of caspase-3, whereas tubacin kills EBV-positive BL cells by induction of reactive oxygen species. Bortezomib and tubacin acted in synergy to kill EBV-positive BL cells and LCLs. These findings suggest that the combination of tubacin and bortezomib may have potential as a model for the treatment of certain EBV-associated lymphomas.     

p53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.     

p53 Suppresses Metabolic Stress-Induced Ferroptosis in Cancer Cells

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Distinctive Uptake of Neutral Red by Mitotic Cancer Cells

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.     

Gambogenic Acid Kills Lung Cancer Cells through Aberrant Autophagy

Lung cancer is one of the most common types of cancer and causes 1.38 million deaths annually, as of 2008 worldwide. Identifying natural anti-lung cancer agents has become very important. Gambogenic acid (GNA) is one of the active compounds of Gamboge, a traditional medicine that was used as a drastic purgative, emetic, or vermifuge for treating tapeworm. Recently, increasing evidence has indicated that GNA exerts promising anti-tumor effects; however, the underlying mechanism remains unclear. In the present paper, we found that GNA could induce the formation of vacuoles, which was linked with autophagy in A549 and HeLa cells. Further studies revealed that GNA triggers the initiation of autophagy based on the results of MDC staining, AO staining, accumulation of LC3 II, activation of Beclin 1 and phosphorylation of P70S6K. However, degradation of p62 was disrupted and free GFP could not be released in GNA treated cells, which indicated a block in the autophagy flux. Further studies demonstrated that GNA blocks the fusion between autophagosomes and lysosomes by inhibiting acidification in lysosomes. This dysfunctional autophagy plays a pro-death role in GNA-treated cells by activating p53, Bax and cleaved caspase-3 while decreasing Bcl-2. Beclin 1 knockdown greatly decreased GNA-induced cell death and the effects on p53, Bax, cleaved caspase-3 and Bcl-2. Similar results were obtained using a xenograft model. Our findings show, for the first time, that GNA can cause aberrant autophagy to induce cell death and may suggest the potential application of GNA as a tool or viable drug in anticancer therapies.     

mRNA Expression Profiles for Prostate Cancer following Fractionated Irradiation Are Influenced by p53 Status

We assessed changes in cell lines of varying p53 status after various fractionation regimens to determine if p53 influences gene expression and if multifractionated (MF) irradiation can induce molecular pathway changes. LNCaP (p53 wild-type), PC3 (p53 null), and DU145 (p53 mutant) prostate carcinoma cells received 5 and 10 Gy as single-dose (SD) or MF (0.5 Gy × 10, 1 Gy × 10, and 2 Gy × 5) irradiation to simulate hypofractionated and conventionally fractionated prostate radiotherapies, respectively. mRNA analysis revealed 978 LNCaP genes differentially expressed (greater than two-fold change, P < .05) after irradiation. Most were altered with SD (69%) and downregulated (75%). Fewer PC3 (343) and DU145 (116) genes were induced, with most upregulated (87%, 89%) and altered with MF irradiation. Gene ontology revealed immune response and interferon genes most prominently expressed after irradiation in PC3 and DU145. Cell cycle regulatory (P = 9.23 × 10^-73, 14.2% of altered genes, nearly universally downregulated) and DNA replication/repair (P = 6.86 × 10^-30) genes were most prominent in LNCaP. Stress response and proliferation genes were altered in all cell lines. p53-activated genes were only induced in LNCaP. Differences in gene expression exist between cell lines and after varying irradiation regimens that are p53 dependent. As the duration of changes is ≥24 hours, it may be possible to use radiation-inducible targeted therapy to enhance the efficacy of molecular targeted agents.     

Pro-Oxidant Activity of Amine-Pyridine-Based Iron Complexes Efficiently Kills Cancer and Cancer Stem-Like Cells

Differential redox homeostasis in normal and malignant cells suggests that pro-oxidant-induced upregulation of cellular reactive oxygen species (ROS) should selectively target cancer cells without compromising the viability of untransformed cells. Consequently, a pro-oxidant deviation well-tolerated by nonmalignant cells might rapidly reach a cell-death threshold in malignant cells already at a high setpoint of constitutive oxidative stress. To test this hypothesis, we took advantage of a selected number of amine-pyridine-based Fe(II) complexes that operate as efficient and robust oxidation catalysts of organic substrates upon reaction with peroxides. Five of these Fe(II)-complexes and the corresponding aminopyridine ligands were selected to evaluate their anticancer properties. We found that the iron complexes failed to display any relevant activity, while the corresponding ligands exhibited significant antiproliferative activity. Among the ligands, none of which were hemolytic, compounds 1, 2 and 5 were cytotoxic in the low micromolar range against a panel of molecularly diverse human cancer cell lines. Importantly, the cytotoxic activity profile of some compounds remained unaltered in epithelial-to-mesenchymal (EMT)-induced stable populations of cancer stem-like cells, which acquired resistance to the well-known ROS inducer doxorubicin. Compounds 1, 2 and 5 inhibited the clonogenicity of cancer cells and induced apoptotic cell death accompanied by caspase 3/7 activation. Flow cytometry analyses indicated that ligands were strong inducers of oxidative stress, leading to a 7-fold increase in intracellular ROS levels. ROS induction was associated with their ability to bind intracellular iron and generate active coordination complexes inside of cells. In contrast, extracellular complexation of iron inhibited the activity of the ligands. Iron complexes showed a high proficiency to cleave DNA through oxidative-dependent mechanisms, suggesting a likely mechanism of cytotoxicity. In summary, we report that, upon chelation of intracellular iron, the pro-oxidant activity of amine-pyrimidine-based iron complexes efficiently kills cancer and cancer stem-like cells, thus providing functional evidence for an efficient family of redox-directed anti-cancer metallodrugs.     

6-溴异香兰素BVAN08诱发肝癌细胞HepG2纺锤体损伤和有丝分裂灾变死亡

研究香兰素衍生物中的6.溴异香兰素(BVAN08)对细胞纺锤体结构的影响及诱发灾变死亡的相关机制,为开发该化合物为新的抗癌药物提供理论依据.通过光学显微镜观察BvAN08作用后细胞形态学变化,流式细胞术检测细胞周期,纺锤体功能检测点实验和原位免疫荧光杂交实验分析细胞有丝分裂进程和纺锤体结构.western印记检测BVAN08作用后相关蛋白质的变化.结果表明20～60 μmol/L BVAN08作用后,HepG2细胞变圆不再贴壁生长、随后脱落死亡,具有浓度依赖性量效关系;明显诱导细胞G2/M期阻滞、导致细胞有丝分裂指数升高,并出现大量的非二倍体和多倍体细胞;破坏细胞纺锤体的结构,多中心体细胞显著增加;该化合物促使细胞周期转录调节因子FoxM1及其下游靶分子细胞周期蛋白B1和CdK1的降解、阻止有丝分裂过程而导致有丝分裂灾变死亡.研究揭示BVAN08通过破坏纺锤体结构、诱发M期阻滞,导致细胞有丝分裂灾变死亡,FoxM1失活可能参与其作用机制.     

Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53

Galectin-7 was initially described as a marker of epithelial differentiation expressed in the stratified epithelium of various tissues. Like other members of the galectin family, its expression level is often significantly altered in cancer cells. In breast cancer, its expression is significantly augmented in aggressive molecular subtypes, most notably in estrogen receptor-negative tumors and in cell lines with a basal-like phenotype. Studies using experimental mouse models have further shown high expression of galectin-7 was sufficient to increase the metastatic behavior of poorly metastatic breast cancer cells, rendering them more resistant to apoptosis. This expression pattern in breast cancer cells is unexpected because galectin-7 was originally identified as a p53-induced gene. To address this paradox, we have examined the molecular mechanisms regulating galectin-7 in breast cancer cells. Our results showed that transfection of breast cancer cells with expression vectors encoding mutant p53 was sufficient to induce galectin-7 at both mRNA and protein levels. Doxorubicin treatment of breast cancer cells harboring a mutant p53 also induced galectin-7. This induction was specific since knockdown of endogenous mutant p53 inhibited doxorubicin-induced galectin-7 expression. The p53-induced galectin-7 expression in breast cancer cells correlated with increased NF-κB activity and was inhibited by NF-κB inhibitors, indicating that the ability of mutant p53 to induce galectin-7 was dependent on NF-κB activity. The implication of NF-κB was further supported by data showing that NF-κB bound to the endogenous galectin-7 promoter and that TNFα-induced galectin-7 expression was abolished by NF-κB inhibitors. Taken together, our data provide an explanation to the observed high galectin-7 expression levels in cancer cells and suggest that galectin-7 could be part of a common pathway used by mutant p53 to promote cancer progression.     

Hematoporphyrin Monomethyl Ether-Mediated Photodynamic Therapy Selectively Kills Sarcomas by Inducing Apoptosis

We investigated the antitumor effect and mechanism of hematoporphyrin monomethyl ether-mediated photodynamic therapy (HMME-PDT) in sarcomas. Intracellular uptake of HMME by osteosarcoma cells (LM8 and K7) was time- and dose-dependent, while this was not observed for myoblast cells (C2C12) and fibroblast cells (NIH/3T3). HMME-PDT markedly inhibited the proliferation of sarcoma cell lines (LM8, MG63, Saos-2, SW1353, TC71, and RD) (P<0.05), and the killing effect was improved with increased HMME concentration and energy intensity. Flow cytometry analysis revealed that LM8, MG63, and Saos-2 cells underwent apoptosis after treatment with HMME-PDT. Additionally, apoptosis was induced after HMME-PDT in a three-dimensional culture of osteosarcoma cells. Hoechst 33342 staining confirmed apoptosis. Cell death caused by PDT was rescued by an irreversible inhibitor (Z-VAD-FMK) of caspase. However, cell viability was not markedly decreased compared with the HMME-PDT group. Expression levels of caspase-1, caspase-3, caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP) proteins were markedly up-regulated in the treatment groups and increased with HMME concentration as determined by western blot analysis. In vivo, tumor volume markedly decreased at 7–16 days post-PDT. Hematoxylin and eosin staining revealed widespread necrotic and infiltrative inflammatory cells in the HMME-PDT group. Immunohistochemistry analysis also showed that caspase-1, caspase-3, caspase-6, caspase-9, and PARP proteins were significantly increased in the HMME-PDT group. These results indicate that HMME-PDT has a potent killing effect on osteosarcoma cells in vitro and significantly inhibits tumor growth in vivo, which is associated with the caspase-dependent pathway.     

Mitotic Catastrophe Results in Cell Death by Caspase-Dependentand Caspase-Independent Mechanisms

Exposure of MDA-MB-231 and MCF-7/VP human breast carcinoma cells to theanthracyclines doxorubicin and WP631 induced polyploidy, formation of multinucleated cellsand cell death by mitotic catastrophe through caspase-dependent and caspase-independentmechanisms. In both cell lines, the antiproliferative effect of WP631 was higher than that ofdoxorubicin and a transient halt in G2/M was observed without cell senescence, while p53-dependent apoptosis did not occur in these cells. Mitotic catastrophe was linked to necrosis, butalso to apoptosis-like death, estimated by differential cell staining with Annexin-V-fluoresceinand propidium iodide. Drug-induced changes in the expression of c-myc and p21WAF1, and in theirrespective protein levels, were observed. They depended on the cell line, the anthracycline usedand its concentration, and they were consistent with the cell cycle progression through G2 tomitosis. Significant activation of caspase-2 and caspase-3 was only observed in MDA-MB-231cells treated with doxorubicin but not with WP631, indicating that caspases may be notmandatory for the occurrence of cell death through mitotic catastrophe. In MCF-7/VP cells,which do not express functional caspase-3, mitotic catastrophe was also induced.     

An Antimitotic and Antivascular Agent BPR0L075 Overcomes Multidrug Resistance and Induces Mitotic Catastrophe in Paclitaxel-Resistant Ovarian Cancer Cells

Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC₅₀ = 2–7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.     

Geminin and p53: Deterrents to Re-replication in Human Cancer Cells

No abstract available

Key words

rereplication, p53, checkpoint pathway, gene amplification, Cdc6, Cdt1, cancer     

Therapeutic Exploitation of Checkpoint Defects in Cancer Cells Lacking p53 Function

Cytotoxic agents form the basis of most cancer therapies. These agents primarily affect rapidly proliferating cells, so their use incurs morbidity associated with damage to tissues such as bone marrow and gastrointestinal mucosa. Clinical outcome would be improved if it were possible to develop therapeutics with more specific activity against p53-deficient cancers, which account for over 50% of all cases. p53 deficiency alters the cellular response to DNA damage in that it leaves cells with attenuated DNA damage checkpoint controls and a reduced propensity for apoptotic cell death. Thus, the DNA repair capacity of these cells is reduced but survival is increased. This promotes genomic instability and contributes to the resistance of p53-deficient cells to cytotoxic agents. Disabling the residual G2 checkpoint function of p53-deficient cells may favour cell death following DNA damage. Several potential strategies for G2 checkpoint abrogation show promise for the specific sensitization of cancer cells. Here we detail how the G2 DNA damage checkpoint is influenced by p53 status and how the loss of p53 function in cancer cells can be exploited to enhance the cytotoxicity of anti-cancer agents.     

p53-based Cancer Therapy

Inactivation of p53 functions is an almost universal feature of human cancer cells. This has spurred a tremendous effort to develop p53 based cancer therapies. Gene therapy using wild-type p53, delivered by adenovirus vectors, is now in widespread use in China. Other biologic approaches include the development of oncolytic viruses designed to replicate and kill only p53 defective cells and also the development of siRNA and antisense RNA''s that activate p53 by inhibiting the function of the negative regulators Mdm2, MdmX, and HPV E6. The altered processing of p53 that occurs in tumor cells can elicit T-cell and B-cell responses to p53 that could be effective in eliminating cancer cells and p53 based vaccines are now in clinical trial. A number of small molecules that directly or indirectly activate the p53 response have also reached the clinic, of which the most advanced are the p53 mdm2 interaction inhibitors. Increased understanding of the p53 response is also allowing the development of powerful drug combinations that may increase the selectivity and safety of chemotherapy, by selective protection of normal cells and tissues.Thirty years of research on p53 have produced a detailed understanding of its structure and function. The almost universal loss of p53 activity in tumors has spurred an enormous effort to develop new cancer treatments based on this fact. Sophisticated animal models have shown that activation of the p53 response in even advanced tumors can be curative (Martins et al. 2006; Ventura et al. 2007; Xue et al. 2007). The p53 gene therapy, Gendicine, is approved in China and its US counterpart, Advexin, has shown activity in number of clinical trials. The p53 protein level is raised in many tumors by virtue of an increase in the protein''s half life and this tumor specific alteration in p53 processing has attracted tumor immunologists, who are now testing a number of p53 based vaccines in cancer patients (Speetjens et al. 2009).In more conventional approaches a range of small druglike molecules targeting the p53 system have been developed and several are now in clinical trials. Of critical importance has been the development of small-molecule inhibitors of the p53–Mdm2 protein interaction such as the Nutlins (Vassilev et al. 2004), which have shown activity against human xenografts in preclinical models. Advanced structural approaches have provided compelling support for the idea that some mutant p53 proteins can be targets for small molecules that would cause them to regain wild-type function (Joerger et al. 2006). Cell based screening methods have identified small molecules that can activate both mutant and wild-type p53 proteins in tumor cells to induce apoptosis. These screens, and RNAi based approaches, have revealed many new targets for therapy in the p53 pathway. In an exciting new approach, that has been validated in other tumor suppressor pathways, the search is on for targets in pathways that will show synthetic lethal interactions with loss of p53 function. Finally drug combinations have been developed that can selectively kill cancer cells that lack p53 function while protecting normal cells (Sur et al. 2009). The next few years hold out the prospect of new p53 based therapies that will be of wide application in cancer and other diseases.