Identification of a Small Molecule That Overcomes HdmX-Mediated Suppression of p53.

Inactivation of the p53 tumor suppressor by mutation or overexpression of negative regulators occurs frequently in cancer. As p53 plays a key role in regulating proliferation or apoptosis in response to DNA-damaging chemotherapies, strategies aimed at reactivating p53 are increasingly being sought. Strategies to reactivate wild-type p53 include the use of small molecules capable of releasing wild-type p53 from key, cellular negative regulators, such as Hdm2 and HdmX. Derivatives of the Hdm2 antagonist Nutlin-3 are in clinical trials. However, Nutlin-3 specifically disrupts Hdm2-p53, leaving tumors harboring high levels of HdmX resistant to Nutlin-3 treatment. Here, we identify CTX1, a novel small molecule that overcomes HdmX-mediated p53 repression. CTX1 binds directly to HdmX to prevent p53-HdmX complex formation, resulting in the rapid induction of p53 in a DNA damage-independent manner. Treatment of a panel of cancer cells with CTX1 induced apoptosis or suppressed proliferation and, importantly, CTX1 demonstrates promising activity as a single agent in a mouse model of circulating primary human leukemia. CTX1 is a small molecule HdmX inhibitor that demonstrates promise as a cancer therapeutic candidate. Mol Cancer Ther; 15(4); 574-82. ©2016 AACR.

The role of TLR8 signaling in acute myeloid leukemia differentiation.

Acute myeloid leukemia (AML) is an aggressive disease with a poor 5-year survival of 21% that is characterized by the differentiation arrest of immature myeloid cells. For a rare subtype of AML (acute promyeloctyic leukemia, 5-10% of cases), all-trans retinoic acid therapy removes the differentiation block, yielding over a 90% cure rate. However, this treatment is not effective for the other 90-95% of AML patients, suggesting that new differentiation strategies are needed. Interestingly, differentiation is induced in normal hematopoietic cells through Toll-like receptor (TLR) stimulation and TLRs are expressed on AML cells. We present evidence that the TLR8 activation promotes AML differentiation and growth inhibition in a TLR8/MyD88/p38-dependent manner. We also show that that TLR7/TLR8 agonist, R848, considerably impairs the growth of human AML cells in immunodeficient mice. Our data suggests TLR8 activation has direct anti-leukemic effects independent of its immunomodulating properties that are currently under investigation for cancer therapy. Taken together, our results suggest that treatment with TLR8 agonists may be a promising new therapeutic strategy for AML.

ATP depletion triggers acute myeloid leukemia differentiation through an ATR/Chk1 protein-dependent and p53 protein-independent pathway.

Despite advances in oncology drug development, most commonly used cancer therapeutics exhibit serious adverse effects. Often the toxicities of chemotherapeutics are due to the induction of significant DNA damage that is necessary for their ability to kill cancer cells. In some clinical situations, the direct induction of significant cytotoxicity is not a requirement to meet clinical goals. For example, differentiation, growth arrest, and/or senescence is a valuable outcome in some cases. In fact, in the case of acute myeloid leukemia (AML), the use of the differentiation agent all-trans-retinoic acid (ATRA) has revolutionized the therapy for a subset of leukemia patients and led to a dramatic survival improvement. Remarkably, this therapeutic approach is possible even in many elderly patients, who would not be able to tolerate therapy with traditional cytotoxic chemotherapy. Because of the success of ATRA, there is widespread interest in identifying differentiation strategies that may be effective for the 90-95% of AML patients who do not clinically respond to ATRA. Utilizing an AML differentiation agent that is in development, we found that AML differentiation can be induced through ATP depletion and the subsequent activation of DNA damage signaling through an ATR/Chk1-dependent and p53-independent pathway. This study not only reveals mechanisms of AML differentiation but also suggests that further investigation is warranted to investigate the potential clinical use of low dose chemotherapeutics to induce differentiation instead of cytotoxicity. This therapeutic approach may be of particular benefit to patients, such as elderly AML patients, who often cannot tolerate traditional AML chemotherapy.

Securinine, a myeloid differentiation agent with therapeutic potential for AML.

As the defining feature of Acute Myeloid Leukemia (AML) is a maturation arrest, a highly desirable therapeutic strategy is to induce leukemic cell maturation. This therapeutic strategy has the potential of avoiding the significant side effects that occur with the traditional AML therapeutics. We identified a natural compound securinine, as a leukemia differentiation-inducing agent. Securinine is a plant-derived alkaloid that has previously been used clinically as a therapeutic for primarily neurological related diseases. Securinine induces monocytic differentiation of a wide range of myeloid leukemia cell lines as well as primary leukemic patient samples. Securinine's clinical potential for AML can be seen from its ability to induce significant growth arrest in cell lines and patient samples as well as its activity in significantly impairing the growth of AML tumors in nude mice. In addition, securinine can synergize with currently employed agents such as ATRA and decitabine to induce differentiation. This study has revealed securinine induces differentiation through the activation of DNA damage signaling. Securinine is a promising new monocytic differentiation inducing agent for AML that has seen previous clinical use for non-related disorders.

p53-Dependent p21-mediated growth arrest pre-empts and protects HCT116 cells from PUMA-mediated apoptosis induced by EGCG.

The tumor suppressor protein p53 plays a key role in regulation of negative cellular growth in response to EGCG. To further explore the role of p53 signaling and elucidate the molecular mechanism, we employed colon cancer HCT116 cell line and its derivatives in which a specific transcriptional target of p53 is knocked down by homologous recombination. Cells expressing p53 and p21 accumulate in G1 upon treatment with EGCG. In contrast, same cells lacking p21 traverse through the cell cycle and eventually undergo apoptosis as revealed by TUNEL staining. Treatment with EGCG leads to induction of p53, p21 and PUMA in p21 wild-type, and p53 and PUMA in p21(-/-) cells. Ablation of p53 by RNAi protects p21(-/-) cells, thus indicating a p53-dependent apoptosis by EGCG. Furthermore, analysis of cells lacking PUMA or Bax with or without p21 but with p53 reveals that all the cells expressing p53 and p21 survived after EGCG treatment. More interestingly, cells lacking both PUMA and p21 survived ECGC treatment whereas those lacking p21 and Bax did not. Taken together, our results present a novel concept wherein p21-dependent growth arrest pre-empts and protects cells from otherwise, in its absence, apoptosis which is mediated by activation of pro-apoptotic protein PUMA. Furthermore, we find that p53-dependent activation of PUMA in response to EGCG directly leads to apoptosis with out requiring Bax as is the case in response to agents that induce DNA damage. p21, thus can be used as a molecular switch for therapeutic intervention of colon cancer.

Securinine induces p73-dependent apoptosis preferentially in p53-deficient colon cancer cells.

The identification of agents that preferentially kill cancer cells while protecting normal cells offers the potential to overcome toxicities found in many existing chemotherapeutic agents. Because p53 is frequently inactivated in cancer, agents that preferentially kill p53-null cells and protect wild-type p53-expressing cells are highly desirable chemotherapeutic agents. By using pairs of isogenic colon cancer cell lines that differ only in p53 expression (RKO and HCT116), securinine was found to exhibit these properties. Securinine (30 microM) induces apoptosis in 73% of p53-null HCT116 cells (LD(50) 17.5 microM) as opposed to 17.6% of HCT116 parental cells (LD(50) 50 microM) at 72 h after treatment. The mechanism of securinine-mediated death in p53-deficient cells involves the induction of the p53 family member, p73. Interestingly, the proapoptotic protein p73 is down-regulated in colon cancer cells expressing p53. This differential regulation of p73 in a p53-dependent fashion reveals a novel pathway for preferentially targeting cancer cells. In contrast to p53-deficient cells, cells expressing p53 are protected from cell death through the p53-mediated up-regulation of p21. These studies reveal a novel approach to specifically target colon cancer cells lacking p53 as well as identify a novel clinically relevant pathway to selectively induce p73 in p53-null cells.

Restoration of p53 functions protects cells from concanavalin A-induced apoptosis.

A great majority of human cancers encounter disruption of the p53 network. Identification and characterization of molecular components important in both p53-dependent and p53-independent apoptosis might be useful in developing novel therapies. Previously, we reported that concanavalin A (Con A) induced p73-dependent apoptosis of cells lacking functional p53. In the present study, we investigated the mechanism and role of p53 in protection from apoptosis induced by Con A. Treatment with Con A resulted in apoptosis of p53-null ovarian cancer, SKOV3, or Li-Fraumeni syndrome, MDAH041 (041), cells. However, their isogenic pairs, SKP53 and TR9-7, expressing wild-type p53 were much less sensitive and were protected by G(1) arrest. Inhibition of p53 function rendered these cells sensitive to Con A. Con A-induced apoptosis was accompanied by upregulation of forkhead box O1a (FOXO1a) and Bcl-2-interacting mediator (Bim), which were strongly inhibited after p53 expression and rescued after p53 ablation. Moreover, ablation of Bim by short hairpin RNA protected cells from apoptosis. Taken together, our study suggests that Con A induces apoptosis of cells lacking p53 by activating FOXO1a-Bim signaling and that expression of p53 protects these cells by inducing G(1) arrest and by downregulating the expression of both FOXO1a and Bim, identifying a novel cross-talk between FOXO1a and p53 transcription factors.

Sumoylation of vimentin354 is associated with PIAS3 inhibition of glioma cell migration.

The invasive phenotype of glioblastoma multiforme (GBM) is a hallmark of malignant process, yet the molecular mechanisms that dictate this locally invasive behavior remain poorly understood. Over-expression of PIAS3 effectively changes cell shape and inhibits GBM cell migration. We focused on the molecular target(s) of PIAS3 stimulated sumoylation, which play an important role in the inhibition of GBM cell motility. Here we report, through the immunoprecipitation with SUMO1 antibody, followed by proteomic analysis, the identification of vimentin (vimentin354), a nuclear component in GBM cells, as the main target of sumoylation promoted by PIAS3.

Transposon-based mutagenesis identifies short RIP1 as an activator of NFkappaB.

We used a vector based on the Sleeping Beauty transposon to search for constitutive activators of NFkappaB in cultured cells. Dominant mutations were produced by random insertion of a tetracycline-regulated promoter, which provided robust and exceptionally well-regulated expression of downstream genes. The ability to regulate the mutant phenotype was used to attribute the latter to the insertional event. In one such mutant, the promoter was inserted in the middle of the gene encoding receptor-interacting protein kinase 1 (RIP1). The protein encoded by the hybrid transcript lacks the putative kinase domain of RIP1, but potently stimulates NFkappaB, AP-1 and Ets-1 activity. Similarly to TNFalpha treatment, expression of the short RIP1 was toxic to cells that failed to upregulate NFkappaB. The effects of short RIP1 did not require endogenous RIP1 or cytokine treatment and coincided with reduced responsiveness to TNFalpha. Additional evidence indicates that a similar short RIP1 could be produced naturally from the ripk1 locus. Interestingly, elevated expression of short RIP1 resulted in the loss of full length RIP1 from cells, pointing to a novel mechanism through which the abundance of RIP1 and the status of the related signaling cascades may be regulated.

Functional assays for classification of BRCA2 variants of uncertain significance.

The assessment of the influence of many rare BRCA2 missense mutations on cancer risk has proved difficult. A multifactorial likelihood model that predicts the odds of cancer causality for missense variants is effective, but is limited by the availability of family data. As an alternative, we developed functional assays that measure the influence of missense mutations on the ability of BRCA2 to repair DNA damage by homologous recombination and to control centriole amplification. We evaluated 22 missense mutations from the BRCA2 DNA binding domain (DBD) that were identified in multiple breast cancer families using these assays and compared the results with those from the likelihood model. Thirteen variants inactivated BRCA2 function in at least one assay; two others truncated BRCA2 by aberrant splicing; and seven had no effect on BRCA2 function. Of 10 variants with odds in favor of causality in the likelihood model of 50:1 or more and a posterior probability of pathogenicity of 0.99, eight inactivated BRCA2 function and the other two caused splicing defects. Four variants and four controls displaying odds in favor of neutrality of 50:1 and posterior probabilities of pathogenicity of at least 1 x 10(-3) had no effect on function in either assay. The strong correlation between the functional assays and likelihood model data suggests that these functional assays are an excellent method for identifying inactivating missense mutations in the BRCA2 DBD and that the assays may be a useful addition to models that predict the likelihood of cancer in carriers of missense mutations.

DNA synthesis from unbalanced nucleotide pools causes limited DNA damage that triggers ATR-CHK1-dependent p53 activation.

p53-dependent G(1) and G(2) cell cycle checkpoints are activated in response DNA damage that help to maintain genomic stability. p53 also helps to protect cells from damage that occurs during S phase, for example, when the cells are starved for DNA precursors or irradiated with a low dose of UV. p53 is activated in normal cells starved for pyrimidine nucleotides by treatment with N-(phosphonacetyl)-l-aspartate (PALA). The treated cells progress through a first S phase with kinetics similar to those of untreated cells. However, the DNA of the treated cells begins to become damaged rapidly, within 12 h, as revealed by a comet assay, which detects broken DNA, and by staining for phosphorylated histone H2AX, which accumulates at sites of DNA damage. Because the cells survive, the damage must be reversible, suggesting single-strand breaks or gaps as the most likely possibility. The transiently damaged DNA stimulates activation of ATR and CHK1, which in turn catalyze the phosphorylation and accumulation of p53. Although PALA-induced DNA damage occurs only in dividing cells, the p53 that is activated is only competent to transcribe genes such as p21 and macrophage inhibitory cytokine 1 (whose products regulate G(2) and G(1) or S phase checkpoints, respectively) after the cells have exited the S phase during which damage occurs. We propose that p53 is activated by stimulation of mismatch repair in response to the misincorporation of deoxynucleotides into newly synthesized DNA, long before the lack of pyrimidine nucleoside triphosphates causes the rate of DNA synthesis to slow appreciably.

Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers.

Ovarian carcinomas with mutations in the tumour suppressor BRCA2 are particularly sensitive to platinum compounds. However, such carcinomas ultimately develop cisplatin resistance. The mechanism of that resistance is largely unknown. Here we show that acquired resistance to cisplatin can be mediated by secondary intragenic mutations in BRCA2 that restore the wild-type BRCA2 reading frame. First, in a cisplatin-resistant BRCA2-mutated breast-cancer cell line, HCC1428, a secondary genetic change in BRCA2 rescued BRCA2 function. Second, cisplatin selection of a BRCA2-mutated pancreatic cancer cell line, Capan-1 (refs 3, 4), led to five different secondary mutations that restored the wild-type BRCA2 reading frame. All clones with secondary mutations were resistant both to cisplatin and to a poly(ADP-ribose) polymerase (PARP) inhibitor (AG14361). Finally, we evaluated recurrent cancers from patients whose primary BRCA2-mutated ovarian carcinomas were treated with cisplatin. The recurrent tumour that acquired cisplatin resistance had undergone reversion of its BRCA2 mutation. Our results suggest that secondary mutations that restore the wild-type BRCA2 reading frame may be a major clinical mediator of acquired resistance to platinum-based chemotherapy.

Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFkappaB.

gp130-linked cytokines such as interleukin-6 (IL-6) stimulate the formation of tyrosine-phosphorylated signal transducer and activator of transcription 3 (P-STAT3), which activates many genes, including the STAT3 gene itself. The resulting increase in the concentration of unphosphorylated STAT3 (U-STAT3) drives a second wave of expression of genes such as RANTES, IL6, IL8, MET, and MRAS that do not respond directly to P-STAT3. Thus, U-STAT3 sustains cytokine-dependent signaling at late times through a mechanism completely distinct from that used by P-STAT3. Many U-STAT3-responsive genes have kappaB elements that are activated by a novel transcription factor complex formed when U-STAT3 binds to unphosphorylated NFkappaB (U-NFkappaB), in competition with IkappaB. The U-STAT3/U-NFkappaB complex accumulates in the nucleus with help from the nuclear localization signal of STAT3, activating a subset of kappaB-dependent genes. Additional genes respond to U-STAT3 through an NFkappaB-independent mechanism. The role of signal-dependent increases in U-STAT3 expression in regulating gene expression is likely to be important in physiological responses to gp130-linked cytokines and growth factors that activate STAT3, and in cancers that have constitutively active P-STAT3.

DNA replication licensing factor minichromosome maintenance deficient 5 rescues p53-mediated growth arrest.

Inactivation of p53 signaling by mutation of p53 itself or abrogation of its normal function by other transfactors, such as MDM2, is a key event in the development of most human cancers. To identify novel regulators of p53, we have used a phenotype-based selection in which a total cDNA library in a retroviral vector has been introduced into TR9-7ER cells, which arrest when p53 is expressed from a tetracycline-regulated promoter. We have isolated several clones derived from cells that are not growth-arrested when p53 is overexpressed. In one clone, the levels of p53, p21, and MDM2 are comparable with those in TR9-7ER cells and, therefore, the abrogation of growth arrest by an exogenous cDNA is likely to be distal to p21. Using reverse transcription-PCR, we were able to isolate a cDNA of approximately 2.2 kb, which was found to have 99% identity to the nucleotides between about 80 and 2,288 of the open reading frame of a gene encoding DNA replication licensing factor. It encodes complete peptide of 734 residues of this protein also called minichromosome maintenance deficient 5 (MCM5) or cell division cycle 46 (Saccharomyces cerevisiae). Northern and Western blot analyses revealed that the expression of MCM5 and its transcriptional regulator, E2F1, is negatively regulated by p53. When MCM5 cDNA was reintroduced into fresh TR9-7ER cells, numerous colonies that grow in the absence of tetracycline were formed. This novel observation establishes a role for MCM5 in negating the growth arrest function of p53.

Macrophage inhibitory cytokine 1 mediates a p53-dependent protective arrest in S phase in response to starvation for DNA precursors.

p53 is essential for the cellular responses to DNA damage that help to maintain genomic stability. Protective p53-dependent cell-cycle checkpoints are activated in response to a wide variety of stresses, including not only DNA damage but also arrest of DNA synthesis and of mitosis. In addition to its role in activating the G(1) and G(2) checkpoints, p53 also helps to protect cells in S phase when they are starved for DNA precursors by treatment with the specific aspartate transcarbamylase inhibitor N-phosphonacetyl-l-aspartate (PALA), which blocks the synthesis of pyrimidine nucleotides. Even though p53 is activated, PALA-treated cells expressing low levels of p53 or lacking expression of p21 do not arrest in G(1) or G(2) but are blocked in S phase instead. In the complete absence of p53, PALA-treated cells continue to synthesize DNA slowly and eventually progress through S phase, suffering severe DNA damage that in turn triggers apoptosis. Expression of the secreted protein macrophage inhibitory cytokine 1 (MIC-1), a member of the TGF-beta superfamily, increases substantially after PALA treatment, and application of exogenous MIC-1 or its constitutive expression from a cDNA provides remarkable protection of p53-null cells from PALA-mediated apoptosis, arguing that the p53-dependent secretion of MIC-1 provides a major part of such protection. Stimulation of MIC-1-dependent S phase arrest in normal gut epithelial cells might help to revitalize the clinical use of PALA, which has been limited by gut toxicity.

Alternating current electrical stimulation enhanced chemotherapy: a novel strategy to bypass multidrug resistance in tumor cells.

BACKGROUND: Tumor burden can be pharmacologically controlled by inhibiting cell division and by direct, specific toxicity to the cancerous tissue. Unfortunately, tumors often develop intrinsic pharmacoresistance mediated by specialized drug extrusion mechanisms such as P-glycoprotein. As a consequence, malignant cells may become insensitive to various anti-cancer drugs. Recent studies have shown that low intensity very low frequency electrical stimulation by alternating current (AC) reduces the proliferation of different tumor cell lines by a mechanism affecting potassium channels while at intermediate frequencies interfere with cytoskeletal mechanisms of cell division. The aim of the present study is to test the hypothesis that permeability of several MDR1 over-expressing tumor cell lines to the chemotherapic agent doxorubicin is enhanced by low frequency, low intensity AC stimulation. METHODS: We grew human and rodent cells (C6, HT-1080, H-1299, SKOV-3 and PC-3) which over-expressed MDR1 in 24-well Petri dishes equipped with an array of stainless steel electrodes connected to a computer via a programmable I/O board. We used a dedicated program to generate and monitor the electrical stimulation protocol. Parallel cultures were exposed for 3 hours to increasing concentrations (1, 2, 4, and 8 microM) of doxorubicin following stimulation to 50 Hz AC (7.5 microA) or MDR1 inhibitor XR9576. Cell viability was assessed by determination of adenylate kinase (AK) release. The relationship between MDR1 expression and the intracellular accumulation of doxorubicin as well as the cellular distribution of MDR1 was investigated by computerized image analysis immunohistochemistry and Western blot techniques. RESULTS: By the use of a variety of tumor cell lines, we show that low frequency, low intensity AC stimulation enhances chemotherapeutic efficacy. This effect was due to an altered expression of intrinsic cellular drug resistance mechanisms. Immunohistochemical, Western blot and fluorescence analysis revealed that AC not only decreases MDR1 expression but also changes its cellular distribution from the plasma membrane to the cytosol. These effects synergistically contributed to the loss of drug extrusion ability and increased chemo-sensitivity. CONCLUSION: In the present study, we demonstrate that low frequency, low intensity alternating current electrical stimulation drastically enhances chemotherapeutic efficacy in MDR1 drug resistant malignant tumors. This effect is due to an altered expression of intrinsic cellular drug resistance mechanisms. Our data strongly support a potential clinical application of electrical stimulation to enhance the efficacy of currently available chemotherapeutic protocols.

p130/p107/p105Rb-dependent transcriptional repression during DNA-damage-induced cell-cycle exit at G2.

The progression of normal cells from G2 into mitosis is stably blocked when their DNA is damaged. Tumor cells lacking p53 arrest only transiently in G2, but eventually enter mitosis. We show that an important component of the stable G2 arrest in normal cells is the transcriptional repression of more than 20 genes encoding proteins needed to enter into and progress through mitosis. Studies from a number of labs including our own have shown that, by inducing p53 and p21/WAF1, DNA damage can trigger RB-family-dependent transcriptional repression. Our studies reported here show that p130 and p107 play a key role in transcriptional repression of genes required for G2 and M in response to DNA damage. For plk1, repression is partially abrogated by loss of p130 and p107, and is completely abrogated by loss of all three RB-family proteins. Mouse cells lacking RB-family proteins do not accumulate with a 4N content of DNA when exposed to adriamycin, suggesting that all three RB-family proteins contribute to G2 arrest in response to DNA damage. Stable arrest in the presence of functional p53-to-RB signaling is probably due to the ability of cells to exit the cell cycle from G2, a conclusion supported by our observation that KI67, a marker of cell-cycle entry, is downregulated in both G1 and G2 in a p53-dependent manner.

Mutagenesis by reversible promoter insertion to study the activation of NF-kappaB.

Genetic dissection of signaling pathways in mammalian cells involves screening or selecting phenotypic mutants obtained by a variety of techniques. Limitations in current methods include inadequate genome coverage and difficulty in validating the link between mutation and phenotype. We describe an improved method for insertional mutagenesis with retroviral vectors and show that the ability to induce mutations increases greatly if a randomly inserted promoter directs transcription into the host DNA. The mutant phenotype is due to the expression of a hybrid transcript derived from the vector and the insertion site. Because other alleles of the affected gene remain intact, the phenotype is dominant, but is reversible by inactivating the promoter, for example, by site-specific recombination. Importantly, in mutant clones with multiple inserts, limited excision yields progeny with different patterns of inserts remaining. Characterizing these progeny allows the mutant phenotype to be associated with a specific target gene. Relative simplicity and robust target validation make the method suitable for a broad range of applications. We have used this technique to search for proteins that regulate NF-kappaB-dependent signaling in human cells. Two validated targets are the relA gene, which codes for the NF-kappaB p65 subunit, and the NF-kappaB regulator act1. Overexpression of the corresponding proteins, caused by insertion of a promoter into the first intron of each gene, leads to NF-kappaB-dependent secretion of factors that activate NF-kappaB through cell-surface receptors, establishing an autocrine loop.

Very low intensity alternating current decreases cell proliferation.

Electric fields impact cellular functions by activation of ion channels or by interfering with cell membrane integrity. Ion channels can regulate cell cycle and play a role in tumorigenesis. While the cell cycle may be directly altered by ion fluxes, exposure to direct electric current of sufficient intensity may decrease tumor burden by generating chemical products, including cytotoxic molecules or heat. We report that in the absence of thermal influences, low-frequency, low-intensity, alternating current (AC) directly affects cell proliferation without a significant deleterious contribution to cell survival. These effects were observed in normal human cells and in brain and prostate neoplasms, but not in lung cancer. The effects of AC stimulation required a permissive role for GIRK2 (or K(IR)3.2) potassium channels and were mimicked by raising extracellular potassium concentrations. Cell death could be achieved at higher AC frequencies (>75 Hz) or intensities (>8.5 microA); at lower frequencies/intensities, AC stimulation did not cause apoptotic cellular changes. Our findings implicate a role for transmembrane potassium fluxes via inward rectifier channels in the regulation of cell cycle. Brain stimulators currently used for the treatment of neurological disorders may thus also be used for the treatment of brain (or other) tumors.

Ablation of either p21 or Bax prevents p53-dependent apoptosis induced by green tea polyphenol epigallocatechin-3-gallate.

Treatment with epigallocatechin-3-gallate (EGCG), a polyphenolic compound of green tea, results in activation of p53 and induction of apoptosis in prostate cancer LnCaP cells. However, no direct evidence has delineated the role of p53 and p53-dependent pathways in EGCG-mediated apoptosis. To understand the mechanism of negative growth regulation of prostate cancer cells by EGCG we undertook a genetic approach and generated an isogenic pair of prostate carcinoma cells PC3 (p53-/-) by stably introducing a cDNA encoding wild-type p53. Treatment of the resultant cells, PC3-p53, with EGCG led to, as reported earlier in LnCaP cells, an increase in p53 protein, which exacerbated both G1 arrest and apoptosis. This response was accompanied by an increase in the levels of p21 and Bax. The cells lacking p53 continued to cycle and did not undergo apoptosis upon treatment with similar concentrations of EGCG, thus establishing the action of EGCG in a p53-dependent manner. This observation was revalidated in another prostate cancer LNCaP cells harboring wild-type p53. Inactivation of p53 using small interfering RNA (siRNA) rendered these cells resistant to EGCG-mediated apoptosis. Because p53 activation led to increase in p21 and Bax, we investigated whether these two proteins are important in this process. Ablation of p21 protein by siRNA prevented G1 arrest and apoptosis in PC3-p53 cells. The p53-dependent increase in Bax expression altered the Bax/Bcl-2 ratio and paralleled the activation of caspase 9 and 3 and cleavage of PARP. Transfection of cells with Bax siRNA abolished these effects and inhibited apoptosis but did not affect the accumulation of the cells in G1. In summary, using isogenic cell lines and siRNA, we have clearly demonstrated that EGCG activates growth arrest and apoptosis primarily via p53-dependent pathway that involves the function of both p21 and Bax such that down-regulation of either molecule confers a growth advantage to the cells.