Predicting Positive p53 Cancer Rescue Regions Using Most Informative Positive (MIP) Active Learning

Many protein engineering problems involve finding mutations that produce proteins with a particular function. Computational active learning is an attractive approach to discover desired biological activities. Traditional active learning techniques have been optimized to iteratively improve classifier accuracy, not to quickly discover biologically significant results. We report here a novel active learning technique, Most Informative Positive (MIP), which is tailored to biological problems because it seeks novel and informative positive results. MIP active learning differs from traditional active learning methods in two ways: (1) it preferentially seeks Positive (functionally active) examples; and (2) it may be effectively extended to select gene regions suitable for high throughput combinatorial mutagenesis. We applied MIP to discover mutations in the tumor suppressor protein p53 that reactivate mutated p53 found in human cancers. This is an important biomedical goal because p53 mutants have been implicated in half of all human cancers, and restoring active p53 in tumors leads to tumor regression. MIP found Positive (cancer rescue) p53 mutants in silico using 33% fewer experiments than traditional non-MIP active learning, with only a minor decrease in classifier accuracy. Applying MIP to in vivo experimentation yielded immediate Positive results. Ten different p53 mutations found in human cancers were paired in silico with all possible single amino acid rescue mutations, from which MIP was used to select a Positive Region predicted to be enriched for p53 cancer rescue mutants. In vivo assays showed that the predicted Positive Region: (1) had significantly more (p<0.01) new strong cancer rescue mutants than control regions (Negative, and non-MIP active learning); (2) had slightly more new strong cancer rescue mutants than an Expert region selected for purely biological considerations; and (3) rescued for the first time the previously unrescuable p53 cancer mutant P152L

An Aqueous Extract of Fagonia cretica Induces DNA Damage, Cell Cycle Arrest and Apoptosis in Breast Cancer Cells via FOXO3a and p53 Expression

Background - Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings - Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as ?-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance - Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression

Mutation analysis of the p73 gene in nonastrocytic brain tumours

Loss of heterozygosity (LOH) involving the distal chromosome 1p36region occurs frequently in nonastrocytic brain tumours, but the tumour suppressor gene targeted by this deletion is unknown. p73is a novel gene that has high sequence homology and similar gene structure to thep53 gene; it has been mapped to 1p36, and may thus represent a candidate for this tumour suppressor gene. To determine whether p73is involved in nonastrocytic brain tumour development, we analysed 65 tumour samples including 26 oligodendrogliomas, 4 ependymomas, 5 medulloblastomas, 10 meningiomas, 2 meningeal haemangiopericytomas, 2 neurofibrosarcomas, 3 primary lymphomas, 8 schwannomas and 5 metastatic tumours to the brain, for p73 alterations. Characterization of allelic loss at 1p36–p35 showed LOH in about 50% of cases, primarily involving oligodendroglial tumours (22 of 26 cases analysed; 85%) and meningiomas (4 of 10; 40%). PCR-SSCP and direct DNA sequencing of exons 2 to 14 of p73 revealed a missense mutation in one primary lymphoma: a G-to-A transition, with Glu291Lys change. 8 additional cases displayed no tumour-specific alterations, as 3 distinct polymorphic changes were identified: a double polymorphic change of exon 5 was found in one ependymoma and both samples derived from an oligodendroglioma, as follows: a G-to-A transition with no change in Pro 146, and a C-to-T variation with no change in Asn 204: a delG at exon 3/+12 position was identified in 4 samples corresponding to 2 oligodendrogliomas, 1 ependymoma and 1 meningioma, and a C-to-T change at exon 2/+10 position was present in a metastatic tumour. Although both LOH at 1p36 and p73 sequence changes were evidenced in 4 cases, it is difficult to establish a causal role of the p73 variations and nonastrocytic brain tumours development. © 2001 Cancer Research Campaign http://www.bjcancer.co

Transcriptome Characterization by RNA-seq Unravels the Mechanisms of Butyrate-Induced Epigenomic Regulation in Bovine Cells

Short-chain fatty acids (SCFAs), especially butyrate, affect cell differentiation, proliferation, and motility. Butyrate also induces cell cycle arrest and apoptosis through its inhibition of histone deacetylases (HDACs). In addition, butyrate is a potent inducer of histone hyper-acetylation in cells. Therefore, this SCFA provides an excellent in vitro model for studying the epigenomic regulation of gene expression induced by histone acetylation. In this study, we analyzed the differential in vitro expression of genes induced by butyrate in bovine epithelial cells by using deep RNA-sequencing technology (RNA-seq). The number of sequences read, ranging from 57,303,693 to 78,933,744, were generated per sample. Approximately 11,408 genes were significantly impacted by butyrate, with a false discovery rate (FDR) <0.05. The predominant cellular processes affected by butyrate included cell morphological changes, cell cycle arrest, and apoptosis. Our results provided insight into the transcriptome alterations induced by butyrate, which will undoubtedly facilitate our understanding of the molecular mechanisms underlying butyrate-induced epigenomic regulation in bovine cells

p53 Target Gene SMAR1 Is Dysregulated in Breast Cancer: Its Role in Cancer Cell Migration and Invasion

Tumor suppressor SMAR1 interacts and stabilizes p53 through phosphorylation at its serine-15 residue. We show that SMAR1 transcription is regulated by p53 through its response element present in the SMAR1 promoter. Upon Doxorubicin induced DNA damage, acetylated p53 is recruited on SMAR1 promoter that allows activation of its transcription. Once SMAR1 is induced, cell cycle arrest is observed that is correlated to increased phospho-ser-15-p53 and decreased p53 acetylation. Further we demonstrate that SMAR1 expression is drastically reduced during advancement of human breast cancer. This was correlated with defective p53 expression in breast cancer where acetylated p53 is sequestered into the heterochromatin region and become inaccessible to activate SMAR1 promoter. In a recent report we have shown that SMAR1 represses Cyclin D1 transcription through recruitment of HDAC1 dependent repressor complex at the MAR site of Cyclin D1 promoter. Here we show that downmodulation of SMAR1 in high grade breast carcinoma is correlated with upregulated Cyclin D1 expression. We also established that SMAR1 inhibits tumor cell migration and metastases through inhibition of TGFβ signaling and its downstream target genes including cutl1 and various focal adhesion molecules. Thus, we report that SMAR1 plays a central role in coordinating p53 and TGFβ pathways in human breast cancer

Activation of the p53 pathway by the MDM2 inhibitor nutlin-3a overcomes BCL2 overexpression in a preclinical model of diffuse large B-cell lymphoma associated with t(14;18)(q32;q21)

p53 is frequently wild type (wt) in diffuse large B-cell lymphoma (DLBCL) associated with t(14;18)(q32;q21) that overexpresses BCL2. Nutlin-3a is a small molecule that activates the p53 pathway by disrupting p53–MDM2 interaction. We show that nutlin-3a activates p53 in DLBCL cells associated with t(14;18)(q32;q21), BCL2 overexpression and wt p53, resulting in cell cycle arrest and apoptosis. Nutlin-3a treatment had similar effects on DLBCL cells of activated B-cell phenotype with wt p53. Cell cycle arrest was associated with upregulation of p21. Nutlin-3a-induced apoptosis was accompanied by BAX and PUMA upregulation, BCL-XL downregulation, serine-70 dephosphorylation of BCL2, direct binding of BCL2 by p53, caspase-9 upregulation and caspase-3 cleavage. Cell death was reduced when p53-dependent transactivation activity was inhibited by pifithrin-α (PFT-α), or PFT-μ inhibited direct p53 targeting of mitochondria. Nutlin-3a sensitized activation of the intrinsic apoptotic pathway by BCL2 inhibitors in t(14;18)-positive DLBCL cells with wt p53, and enhanced doxorubicin cytotoxicity against t(14;18)-positive DLBCL cells with wt or mutant p53, the latter in part via p73 upregulation. Nutlin-3a treatment in a xenograft animal lymphoma model inhibited growth of t(14;18)-positive DLBCL tumors, associated with increased apoptosis and decreased proliferation. These data suggest that disruption of the p53–MDM2 interaction by nutlin-3a offers a novel therapeutic approach for DLBCL associated with t(14;18)(q32;q21)