P53 Status Of Cells Research Articles

CAF hierarchy driven by pancreatic cancer cell p53-status creates a pro-metastatic and chemoresistant environment via perlecan

Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.

Acriflavine enhances the antitumor activity of the chemotherapeutic drug 5-fluorouracil in colorectal cancer cells.

5-Fluorouracil (5-FU)-based chemotherapy improves the overall survival rates of patients with colorectal cancer (CRC). However, only a small proportion of patients respond to 5-FU when used as a single agent. The aim of the present study was to investigate whether the anticancer property of 5-FU is potentiated by combination treatment with acriflavine (ACF) in CRC cells. Additionally, the potential underlying molecular mechanisms of the cytotoxic effect of ACF were determined. The cytotoxic effects of ACF, 5-FU and irinotecan on different CRC cell lines with different p53 status were investigated using an MTT assay. SW480 cells that express a mutated form of p53 and two other CRC cell lines were used, HCT116 and LS174T, with wild-type p53. To determine the effect of ACF on the sensitivity of cells to 5-FU, cells were co-treated with the 30% maximal inhibitory concentration (IC30) of ACF and various concentrations of 5-FU, or pretreated with the IC30 of ACF and various concentrations of 5-FU. To assess the mechanism of action of ACF, cells were treated with IC30 values of the compound and then the reverse transcription-quantitative polymerase chain reaction was used to evaluate mRNA levels of hypoxia-inducible factor-1α (HIF-1α) and topoisomerase 2. Results indicate that pretreatment with ACF markedly sensitized CRC cells to the cytotoxic effects of 5-FU, whereas simultaneous treatment with ACF and 5-FU were not able to alter the resistance of CRC cells to 5-FU. In comparison with irinotecan, ACF was a more potent agent for enhancing the antitumor activity of 5-FU. ACF did not alter the mRNA levels of either HIF-1α or topoisomerase 2. The results of the present study reveal for the first time that pretreatment of CRC cells with ACF markedly increases the cytotoxic effects of 5-FU, regardless of the p53 status of cells.

Abstract 1319: Vulnerability and resistance in glioblastoma to G2-specific drugs

Abstract Glioblastoma multiforme (GBM) is the most common and most lethal primary malignant brain tumor, affecting 25,000 patients per year. Approximately 87% of GBMs have abnormalities in the p53 pathway that affects G2 checkpoint. In the absence of p53, Wee1 kinase has been shown to regulate G2 checkpoint upon chemotherapeutic insult. Wee1 inhibits CDC2 function, thereby promoting G2 phase cell cycle arrest, thus preventing damaged cells from entering into premature mitosis without repairing the DNA. We hypothesize that in p53-deficient tumor cells, inhibitors of Wee1 would be effective to heighten DNA damage due to genetic and pharmacologic loss of both the G1 and G2 checkpoints, respectively. MK-1775 (Merck) is a potent and selective small molecule inhibitor of the Wee1 kinase that has demonstrated enhanced cytotoxic effect in combination with several chemotherapeutic agents in preclinical studies in vitro and in vivo. Here, we selected an array of short term cultures of glioma patient derived xenograft (PDX) models (GBM12, GBM43, GBM79, GBM84) and long term established glioma cell lines (U87, SF767, T98G) with aberrant and wild type G1 checkpoint pathways and investigated cytotoxic effects of MK-1775 as a single agent and in combination with Temozolomide (TMZ). Our results demonstrated differential sensitivity to MK-1775 and TMZ as single agents for different glioma cell lines in vitro. However, no correlation between MK-1775, TMZ sensitivity and p53 status of the cell lines was observed. Combination therapy with MK-1775 and TMZ showed a synergistic effect for one PDX model, GBM43, with combination index (CI) of 0.4. Combination therapy of all other glioma cell lines either showed an additive effect or antagonistic effect with CI of >0.75 irrespective of their p53 status. Our results demonstrated that synergistic effects of MK-1775 in combination with chemotherapy (TMZ) are not solely dependent on p53 status of glioma cells and require a more comprehensivce understanding of the molecular mechanism that dictates GBM's dependence on Wee1 for escaping DNA damage therapy. Supported by the Ben & Catherine Ivy Foundation Citation Format: Harshil D. Dhruv, Andrew D. Nelson, Brock Armstrong, Julie L. Boerner, Jann N. Sarkaria, Nhan L. Tran, Michael Berens. Vulnerability and resistance in glioblastoma to G2-specific drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1319. doi:10.1158/1538-7445.AM2014-1319

Identification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53-independent pathways.

Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer's glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD+) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly, however, we uncovered a novel role for p53 in the regulation of cancer cell NAD+ and its reduced form NADH. Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratio of NADH:NAD+. This effect was specific for p53+/+ cancer cells and correlated with (i) reduced activity of NAD+-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53+/+ cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-induced DNA damage in p53+/+ cancer cells, but importantly had no additive effect in non-cancer cells. Our results identify a unique strategy by which the NADH/NAD+ cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism.

Temporally distinct roles of ATM and ROS in genotoxic-stress-dependent induction and maintenance of cellular senescence.

Cells exposed to genotoxic stress induce cellular senescence through a DNA damage response (DDR) pathway regulated by ATM kinase and reactive oxygen species (ROS). Here, we show that the regulatory roles for ATM kinase and ROS differ during induction and maintenance of cellular senescence. Cells treated with different genotoxic agents were analyzed using specific pathway markers and inhibitors to determine that ATM kinase activation is directly proportional to the dose of the genotoxic stress and that senescence initiation is not dependent on ROS or the p53 status of cells. Cells in which ROS was quenched still activated ATM and initiated the DDR when insulted, and progressed normally to senescence. By contrast, maintenance of a viable senescent state required the presence of ROS as well as activated ATM. Inhibition or removal of either of the components caused cell death in senescent cells, through a deregulated ATM-ROS axis. Overall, our work demonstrates existence of an intricate temporal hierarchy between genotoxic stress, DDR and ROS in cellular senescence. Our model reports the existence of different stages of cellular senescence with distinct regulatory networks.

Identification of a heteromeric complex that promotes DNA replication origin firing in human cells.

Treslin/TICRR (TopBP1-interacting, replication stimulating protein/TopBP1-interacting, checkpoint, and replication regulator), the human ortholog of the yeast Sld3 protein, is an essential DNA replication factor that is regulated by cyclin-dependent kinases and the DNA damage checkpoint. We identified MDM two binding protein (MTBP) as a factor that interacts with Treslin/TICRR throughout the cell cycle. We show that MTBP depletion by means of small interfering RNA inhibits DNA replication by preventing assembly of the CMG (Cdc45-MCM-GINS) holohelicase during origin firing. Although MTBP has been implicated in the function of the p53 tumor suppressor, we found MTBP is required for DNA replication irrespective of a cell's p53 status. We propose that MTBP acts with Treslin/TICRR to integrate signals from cell cycle and DNA damage response pathways to control the initiation of DNA replication in human cells.

Abstract 3684: Differential mechanisms of green tea polyphenols-induced apoptosis in human prostate cancer cells: Role of p53.

Abstract Tumor suppressor gene p53 is frequently inactivated in a variety of human cancers including prostate cancer and is associated with therapeutic resistance. We have previously demonstrated that green tea polyphenols (GTP) induces apoptosis in prostate cancer cells irrespective of p53 status (Carcinogenesis 33:377-84, 2012). However, the molecular mechanisms underlying these observations are not properly understood. In the present study we investigated the mechanisms of GTP-induced apoptosis in human prostate cancer cells with and without functional p53. Human prostate cancer LNCaP cells were stably transfected with short hairpin-53 (LNCaPshp53) and control vector (LNCaPshV), exposed to GTP in dose and time dependent manner. GTP exposure induced p53 stabilization and activation of downstream targets p21/waf1 and Bax in dose-dependent manner specifically in LNCaPshV cells. However, GTP-induced FAS upregulation through the activation of c-jun N-terminal kinase resulting in FADD phosphorylation, caspase-8 activation and truncation of BID leading to apoptosis was common in both LNCaPshV and LNCaPshp53 cells. In parallel, treatment of cells with GTP resulted in inhibition of survival pathway, mediated by Akt deactivation and loss of BAD phosphorylation more prominently in LNCaPshp53 cells. These distinct routes of cell death converged to a common pathway leading to loss of mitochondrial transmembrane potential, cytochrome c release and activation of terminal caspases resulting in PARP cleavage. GTP-induced apoptosis was attenuated with JNK inhibitor, SP600125 in both cell lines; whereas PI3K-Akt inhibitor, LY294002 resulted in increased cell death in LNCaPshp53 cells, establishing the role of two distinct pathways of GTP-mediated apoptosis. Furthermore, GTP exposure resulted in the inhibition of class I HDAC protein, accumulation of acetylated histone H3 in total cellular chromatin resulting in increased accessibility to bind with the promoter sequences of p21/waf1 and Bax irrespective of p53 status of cells, consistent with the effects elicited by HDAC inhibitor, trichostatin A. These results demonstrate that GTP induces prostate cancer cell death by two distinct mechanisms irrespective of p53 status, thus identifying for the first time through defined molecular targets for their chemopreventive and/or therapeutic actions. Citation Format: Karishma Gupta, Vijay S. Thakur, Natarajan Bhaskaran, Akbar Nawab, Melissa A. Babcook, Mark W. Jackson, Sanjay Gupta. Differential mechanisms of green tea polyphenols-induced apoptosis in human prostate cancer cells: Role of p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3684. doi:10.1158/1538-7445.AM2013-3684

Impairment of Glioma Stem Cell Survival and Growth by a Novel Inhibitor for Survivin–Ran Protein Complex

Glioblastoma multiforme (GBM) is a devastating disease. Recent studies suggest that the stem cell properties of GBM contribute to the development of therapy resistance. The expression of Survivin and Ran was evaluated by immunohistochemistry with GBM tissues, and quantitative reverse transcriptase (qRT)-PCR and immunocytochemistry with patient-derived GBM sphere cultures. With a computational structure-based drug design, 11 small-molecule compounds were designed, synthesized, and evaluated as inhibitor candidates for the molecular interaction of Survivin protein. The molecular mechanism of the lead compound, LLP-3, was determined by Western blot, ELISA, in situ proximity ligation assay, and immunocytochemistry. The effects of LLP-3 treatment on GSCs were evaluated both in vitro and in vivo. Quantitative immunohistochemistry was carried out to compare Survivin expression in tissues from 44 newly diagnosed and 31 recurrent post-chemoradiation GBM patients. Lastly, the sensitivities of temozolomide-resistant GBM spheres to LLP-3 were evaluated in vitro. Survivin and Ran were strongly expressed in GBM tissues, particularly in the perivasculature, and also in patient-derived GSC cultures. LLP-3 treatment disrupted the Survivin-Ran protein complex in cancer cells and abolished the growth of patient-derived GBM spheres in vitro and in vivo. This inhibition was dependent on caspase activity and associated with p53 status of cells. Immunohistochemistry showed that Survivin expression is significantly increased in recurrent GBM compared with newly diagnosed tumors, and temozolomide-resistant GBM spheres exhibited high sensitivities to LLP-3 treatment. Disruption of the Survivin-Ran complex by LLP-3 abolishes survival and growth of GSCs both in vitro and in vivo, indicating an attractive novel therapeutic approach for GBM.

P53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status.

Green Tea Polyphenols Induce p53-Dependent and p53-Independent Apoptosis in Prostate Cancer Cells through Two Distinct Mechanisms

Inactivation of the tumor suppressor gene p53 is commonly observed in human prostate cancer and is associated with therapeutic resistance. We have previously demonstrated that green tea polyphenols (GTP) induce apoptosis in prostate cancer cells irrespective of p53 status. However, the molecular mechanisms underlying these observations remain elusive. Here we investigated the mechanisms of GTP-induced apoptosis in human prostate cancer LNCaP cells stably-transfected with short hairpin-RNA against p53 (LNCaPshp53) and control vector (LNCaPshV). GTP treatment induced p53 stabilization and activation of downstream targets p21/waf1 and Bax in a dose-dependent manner specifically in LNCaPshV cells. However, GTP-induced FAS upregulation through activation of c-jun-N-terminal kinase resulted in FADD phosphorylation, caspase-8 activation and truncation of BID, leading to apoptosis in both LNCaPshV and LNCaPshp53 cells. In parallel, treatment of cells with GTP resulted in inhibition of survival pathway, mediated by Akt deactivation and loss of BAD phosphorylation more prominently in LNCaPshp53 cells. These distinct routes of cell death converged to a common pathway, leading to loss of mitochondrial transmembrane potential, cytochrome c release and activation of terminal caspases, resulting in PARP-cleavage. GTP-induced apoptosis was attenuated with JNK inhibitor, SP600125 in both cell lines; whereas PI3K-Akt inhibitor, LY294002 resulted in increased cell death prominently in LNCaPshp53 cells, establishing the role of two distinct pathways of GTP-mediated apoptosis. Furthermore, GTP exposure resulted in inhibition of class I HDAC protein, accumulation of acetylated histone-H3 in total cellular chromatin, resulting in increased accessibility of transcription factors to bind with the promoter sequences of p21/waf1 and Bax, regardless of the p53 status of cells, consistent with effects elicited by an HDAC inhibitor, trichostatin A. These results demonstrate that GTP induces prostate cancer cell death by two distinct mechanisms regardless of p53 status, thus identifying specific well-defined molecular mechanisms that may be targeted by chemopreventive and/or therapeutic strategies.

Antitumor Activity of NVP-BKM120—A Selective Pan Class I PI3 Kinase Inhibitor Showed Differential Forms of Cell Death Based on p53 Status of Glioma Cells

The aim of this study was to show preclinical efficacy and clinical development potential of NVP-BKM120, a selective pan class I phosphatidylinositol-3 kinase (PI3K) inhibitor in human glioblastoma (GBM) cells in vitro and in vivo. The effect of NVP-BKM120 on cellular growth was assessed by CellTiter-Blue assay. Flow cytometric analyses were carried out to measure the cell-cycle, apoptosis, and mitotic index. Mitotic catastrophe was detected by immunofluorescence. The efficacy of NVP-BKM120 was tested using intracranial U87 glioma model. We tested the biologic effects of a selective PI3K inhibitor NVP-BKM120 in a set of glioma cell lines. NVP-BKM120 treatment for 72 hours resulted in a dose-dependent growth inhibition and effectively blocked the PI3K/Akt signaling cascade. Although we found no obvious relationship between the cell line's sensitivity to NVP-BKM120 and the phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) statuses, we did observe a differential sensitivity pattern with respect to p53 status, with glioma cells containing wild-type p53 more sensitive than cells with mutated or deleted p53. NVP-BKM120 showed differential forms of cell death on the basis of p53 status of the cells with p53 wild-type cells undergoing apoptotic cell death and p53 mutant/deleted cells having a mitotic catastrophe cell death. NVP-BKM120 mediates mitotic catastrophe mainly through Aurora B kinase. Knockdown of p53 in p53 wild-type U87 glioma cells displayed microtubule misalignment, multiple centrosomes, and mitotic catastrophe cell death. Parallel to the assessment of the compound in in vitro settings, in vivo efficacy studies using an intracranial U87 tumor model showed an increased median survival from 26 days (control cohort) to 38 and 48 days (treated cohorts). Our present findings establish that NVP-BKM120 inhibits the PI3K signaling pathways, leading to different forms of cell death on the basis of p53 statuses. Further studies are warranted to determine if NVP-BKM120 has potential as a glioma treatment.

Hexane Extract of Raphanus sativus L. Roots Inhibits Cell Proliferation and Induces Apoptosis in Human Cancer Cells by Modulating Genes Related to Apoptotic Pathway

Raphanus sativus, a common cruciferous vegetable has been attributed to possess a number of pharmacological and therapeutic properties. It has been used in indigenous system of medicine for the treatment of various human ailments in India. This present study evaluated the chemopreventive efficacy of different parts of R. sativus such as root, stem and leaves, extracted with solvents of varying polarity and investigated the molecular mechanism leading to growth arrest and apoptotic cell death in human cancer cell lines. Of the different parts, significant growth inhibitory effect was observed with hexane extract of R. sativus root. Analysis of hexane extract by GC-MS revealed the presence of several isothiocyanates (ITCs) such as 4-(methylthio)-3-butenyl isothiocyanate (MTBITC), 4-(methylthio)-3-butyl isothiocyanate (erucin), 4-methylpentyl isothiocyanate, 4-pentenyl isothiocyanate and sulforaphene. R. sativus root extract induced cell death both in p53 proficient and p53 deficient cell lines through induction of apoptotic signaling pathway regardless of the p53 status of cells. The molecular mechanisms underlying R. sativus-induced apoptosis may involve interactions among Bcl(2) family genes, as evidenced by up-regulation of pro-apoptotic genes and down-regulation of anti-apoptotic genes along with activation of Caspase-3. Our findings present the first evidence that hexane extract of R. sativus root exerts potential chemopreventive efficacy and induces apoptosis in cancer cell lines through modulation of genes involved in apoptotic signaling pathway.

Abstract B61: Regulation of mammary stem/progenitor cells by p53

Abstract Introduction: Breast cancer is the most frequent cancer among women in the United States. Understanding the biological behavior of mammary stem cells (MaSCs) and progenitor cells sheds light on the mechanism of mammary gland malignant transformation. The p53 tumor suppressor protein (TP53 in human, Trp53 in mouse) regulates cell growth and protects cells from malignant transformation and has been shown to also participate in regulation of tissue-specific stem cells. Methods: Total mammary cells were isolated from 8–10 weeks old mice of different p53 genotype (Trp53+/+, Trp53+/−, Trp53−/−). Mammosphere assay were used to compare the self-renewal and ionizing radiation (IR) response of mammosphere-initiating cells. Limiting dilution and transplantation was done to compare the frequency of long-term regenerative mammary stem cells of Trp53+/+ and Trp53−/− epithelium. 3 weeks old BALB/c Trp53+/+ and Trp53−/− mice were injected with BrdU for 7 consecutive days and chased for 9 weeks. BrdU staining was done to compare the frequency of label-retaining cells in their mammary glands. Results: Mammosphere assays showed that decreased p53 dosage in Trp53+/− and Trp53−/− mice led to increased numbers and size of mammospheres. Meanwhile, the number of secondary and tertiary mammospheres was not affected by (IR) regardless of their genotype. Serial dilution and transplantation experiments also showed that the Trp53−/− epithelium had significantly increased frequency of long-term regenerative MaSCs compared to Trp53+/+ epithelium. The BrdU labeling experiment showed that Trp53−/− mammary gland contains less label-retaining epithelial cells (LRECs) than Trp53+/+ epithelium. Conclusions: Our data suggest that p53 negatively regulates the self-renewal of mammary stem cells. The MaSCs pool expands with decreased p53 dosage, which may results in a higher transformation risk. Meanwhile, the p53-mediated apoptosis pathway is compromised in the mammosphere-initiating cells, suggesting different functional status of p53 in cells of different differentiation stage. Citation Information: Cancer Res 2009;69(23 Suppl):B61.

Nutlin-3 inhibits the NFκB Pathway in a p53 Dependent Manner: Implications in Lung Cancer Therapy

Nutlins were identified as the first potent and specific small molecule Mdm2 antagonists that inhibit the p53- Mdm2 interaction. We show in this study that Nutlin-3 can downregulate TNFα induced activation of the NF-κB reporter in lung cancer cells. Activation of p53 dependent transcription is not compromised when Nutlin-3 is combined with TNFα. Instead, this combination treatment decreases cell viability in a p53 dependent manner. We show that Nutlin-3 strikingly inhibits the protein expression of NF-κB target genes ICAM-1 and MCP-1 while other targets like Bcl-xL and FLIP are not affected, thereby suggesting that the inhibition is promoter specific. This inhibition of ICAM-1 and MCP-1 by Nutlin-3 is again dependent on the p53 status in cells. Furthermore, we show that Nutlin-3 strongly inhibits protein expression of ICAM-1 and MCP-1 induced by IL1, another NF-κB activating stimuli. Nutlin-3 does not inhibit Akt phosphorylation, IκBα phosphorylation, IκBα degradation, p65 modification or p65 DNA binding in the cell lines tested. This study suggests the potential of Nutlin-3 as a bitargeted anti-cancer drug by simultaneously causing p53 activation and NF-κB suppression. It also suggests that Nutlin-3 could be evaluated for treatment of lung cancer as a single agent or in combination therapy by targeting its effect on ICAM-1 and MCP-1 which are known to be critical for cancer cell invasion, thereby downregulating tumor formation and metastasis. This study also suggests biomarkers of response for evaluation of Nutlin-3 in the clinic.

A phase II study of ARQ 501 in patients with advanced squamous cell carcinoma of the head and neck

16509 Background: ARQ 501 is a DNA damage checkpoint pathway activator whose effect is to induce selective cell death in cancer cells, independent of the tumor cell's p53 status. Current evidence implicates a rapid and sustained increase of the pro-apoptotic protein E2F-1 by ARQ 501 as the mechanism of action. Cancer cells are selectively affected due to their pre-existing DNA damage. In initial clinical trials, there was evidence of activity in some patients with head and neck cancer and pre-clinical investigations also supported this application. Therefore, a phase 2 trial is warranted in this patient population. Methods: A phase 2 study in patients with locally advanced, recurrent or metastatic squamous cell carcinoma of the head and neck who had received up to 3 prior systemic therapies was initiated to assess overall response rate, progression free survival at six months, and to further characterize safety. Cycles consist of four weekly administrations of ARQ 501 at a dose of 450 mg/m2. Dose escalation is allowed if a patient successfully completed a full cycle of therapy and had no related grade 2 adverse events. Results: 59 patients have been enrolled to date and 47 have received at least one infusion of ARQ 501. Data is available for 34 patients (27M / 7F, median age, 57 years). Of the 47 patients treated, 11 patients did not reach a post baseline assessment (2 deaths, 8 PD prior to week 8 and 1 withdrew consent). Although at the current date, most patients have not reached their first tumor evaluation, 5 have been assessed for response per RECIST at eight weeks and one patient is demonstrating stable disease. The drug has been well tolerated with the most common adverse event being anemia (11% ) with corresponding edema, fatigue (both 5%), dyspnea (4%), and hyperbilirubinemia (2%). Conclusions: ARQ 501 is well tolerated in patients with advanced, recurrent or persistent squamous cell carcinoma of the head and neck. Enrollment is ongoing and further results will be presented. No significant financial relationships to disclose.

Methionine inhibits cellular growth dependent on the p53 status of cells

Background Methionine, an essential amino acid, is important for normal growth and development, as it is required for both protein and polyamine synthesis as well as in methylation reactions. It has been reported that high concentrations of methionine inhibit cellular growth and gene expression in the human breast tumor-derived MCF-7 cells. These effects are thought to be mediated by the modulation of p53. However, the generalizability of this observation and the precise role of p53 in methionine-induced growth suppression needs to be determined. Methods To determine if the inhibition of cell growth by methionine applies to other cell lines and to characterize further the role of p53 in methionine-induced growth suppression, we have assessed the effects of methionine on cellular growth and proliferation and p53 expression in cells expressing native p53, eg, breast cancer MCF-7 cells and prostate cancer LNCaP cells, and also in cells expressing a mutated (point) form of p53, eg, prostate cancer DU-145 cells. These cell lines were treated with varying concentrations of l-methionine. The effects of l-methionine on cell growth were assyed by using cell viability assays and immunostaining for Ki-67, a cell proliferation marker. The effects of methionine on p53 expression were assessed by using reverse transcriptase–polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. The role of p53 in l-methionine–mediated growth suppression was evaluated using short-interference RNA for p53 (siRNA-p53), immunoprecipitation, and direct DNA sequencing. Results We demonstrated that methionine at a concentration of 1 to 5 mg/mL inhibited the growth of both MCF-7 and LNCaP cells. In association with the inhibition of growth, methionine also inhibited native p53 expression at the mRNA and protein levels, respectively. Furthermore, transfection with siRNA-p53, to knock down p53 expression, increased cell growth and proliferation of the LNCaP cells even when they were exposed to methionine. In contrast, the same treatment did not diminish growth or proliferation of the DU-145 cells. Also, the expression of mutated p53 at the mRNA or protein levels was not altered. Conclusion Our results extend a prior observation to other cell lines and demonstrate that high concentrations of methionine suppress the expression of native but not mutated p53. These inhibitory effects on cellular growth are, in part, due to inhibition of cellular proliferation probably via a p53-dependent pathway.

P53 enhances the Δ-24 conditionally replicative adenovirus anti-glioma effect

Previous studies have demonstrated that the conditionally replicative adenovirus Ad5Delta24 is a powerful cytolytic agent against glioma selectively affecting cells with a defective p16/Rb/E2F pathway. The p53 protein is also known to be an apoptotic factor for glioma cells. In this study, we examined the simultaneous delivery of the combination of exogenous p53 and Ad5Delta24 adenovirus in glioma cells. Infecting cells with low doses of adenovirus p53 and Ad5Delta24 resulted in an additive effect on cell death. The cell death induced by both agents was independent of the p53 status of cells. Flow cytometry revealed that the potent anti-tumor effect induced by the mixture of Ad5CMV-p53 and Ad5Delta24 adenoviruses was due to a combination of apoptosis and cell lysis. Our results indicate that Ad5CMV-p53 enhances the oncolytic effect of the Ad5Delta24 adenovirus, and the combination of adenovirus Ad5Delta24 and Ad5CMV-p53 may thus be a potential therapeutic tool for gliomas.

Generation of loss of heterozygosity and its dependency on p53 status in human lymphoblastoid cells

Loss of heterozygosity (LOH) is a critical event in the development of human cancers. LOH is thought to result from either a large deletion or recombination between homologous alleles during repair of DNA double-strand breaks (DSBs). These types of genetic alterations produce mutations in the TK gene mutation assay, which detects a wide mutational spectrum, ranging from point mutations to LOH-type mutations. TK6, a human lymphoblastoid cell line, is heterozygous for the thymidine kinase (TK) gene and has a wild-type p53 gene. The related cell lines, TK6-E6 and WTK-1, which are p53-deficient and p53-mutant (Ile237), respectively, are also heterozygous for the TK gene and LOH-type mutation can be detected in these cells. Therefore, comparative studies of TK mutation frequency and spectrum with these cell lines are useful for elucidating the role of p53 in generating LOH and maintaining genomic stability in human cells. We demonstrate here that LOH and its associated genomic instability strongly depend on the p53 status in these cells. TK6-E6 and WTK-1 are defective in the G1/S checkpoint and in apoptosis. Unrepaired DSBs that escape from the checkpoint can potentially initiate genomic instability after DNA replication, resulting in LOH and a variety of chromosome changes. Moreover, genomic instability is enhanced in WTK-1 cells. It is likely that the mutant p53 protein in WTK-1 cells increases LOH in a dominant-negative manner due to its abnormal recombination capacity. We discuss the mutator phenotype and genomic instability associated with p53 inactivation with the goal of elucidating the mechanisms of mutation and DNA repair in untargeted mutagenesis.

Molecular mechanisms of TNF-alpha-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways.

The present study was designed to examine the roles of p53, reactive oxygen species (ROS), and ceramide, and to determine their mutual relationships during tumor necrosis factor (TNF)-alpha-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-alpha stimulated ceramide formation via the activation of both neutral and acid sphingomyelinases (SMases), accompanied by superoxide anion (O2-*) production, and induced mitochondrial depolarization and cytochrome c release, whereas p53-deficient cells were partially resistant to TNF-alpha and lacked O2-* generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-alpha by accumulation of O2-*. z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone), but not z-DEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone), blocked TNF-alpha-induced ceramide formation through both SMases as well as O2-* generation. Caspase-8 was processed by TNF-alpha regardless of p53 status of cells or the presence of antioxidants. Two separate signaling cascades, p53-mediated ROS-dependent and -independent pathways, both of which are initiated by caspase-8 activation, thus contribute to ceramide formation in TNF-alpha-induced apoptosis of human glioma cells.

Fundamental principals of tumor necrosis factor-alpha gene therapy approach and implications for patients with lung carcinoma

Apoptosis, known as programmed cell death, is defined as a cell’s preferred form of death under hectic conditions through genetically conserved and complex pathways. There is a decisive balance between stimulatory and inhibitory signaling pathways to maintain homeostasis in cells. In order to shift the balance towards apoptosis, the modulation of both apoptotic and anti-apoptotic pathways represents an attractive target for cancer therapeutics. Currently, chemotherapy and radiotherapy are among the most commonly used treatment modalities against lung cancer. Tumor suppressor gene, p53, is required in order for both of these treatment methods to work as anti-tumor agents. As a result, tumors lacking p53 display resistance to both chemotherapy and radiotherapy. However, death ligands induce apoptosis regardless of p53 status of cells. Thus, these methods constitute a complementary therapeutic approach to currently employed conventional treatment modalities. At present, death ligands are being evaluated as potential cancer therapeutic agents. Since resistance to tumor necrosis factor (TNF)-alpha-mediated apoptosis represented an obstacle for the treatment of patients with lung carcinoma in the earlier attempts, an extensive research was recently initiated to understand molecular mechanism of TNF-alpha signaling. NF-κB transcription factors have been demonstrated to modulate the apoptotic program, mostly as blockers of apoptosis in different cell types. In this review, we concentrate on the current progress in the understanding of TNF-alpha-mediated apoptosis for lung carcinoma. Representative models of NF-κB-inhibiting gene therapy strategies from various labs including ours are also provided as examples of up-to-date approaches to defeat TNF resistance. In order to give the reader better understanding and appreciation of such approaches, previously unpublished in vivo assays are also incorporated into this review. Current progress in clinical trials using adenovirus-mediated delivery of TNF-alpha is also discussed.