Transcription factors that interact with p53 and Mdm2.

The tumor suppressor p53 is activated upon cellular stresses such as DNA damage, oncogene activation, hypoxia, which transactivates sets of genes that induce DNA repair, cell cycle arrest, apoptosis, or autophagy, playing crucial roles in the prevention of tumor formation. The central regulator of the p53 pathway is Mdm2 which inhibits transcriptional activity, nuclear localization and protein stability. More than 30 cellular p53-binding proteins have been isolated and characterized including Mdm2, Mdm4, p300, BRCA1/2, ATM, ABL and 53BP-1/2. Most of them are nuclear proteins; however, not much is known about p53-binding transcription factors. In this review, we focus on transcription factors that directly interact with p53/Mdm2 through direct binding including Dmp1, E2F1, YB-1 and YY1. Dmp1 and YB-1 bind only to p53 while E2F1 and YY1 bind to both p53 and Mdm2. Dmp1 has been shown to bind to p53 and block all the known functions for Mdm2 on p53 inhibition, providing a secondary mechanism for tumor suppression in Arf-null cells. Although E2F1-p53 binding provides a checkpoint mechanism to silence hyperactive E2F1, YB-1 or YY1 interaction with p53 subverts the activity of p53, contributing to cell cycle progression and tumorigenesis. Thus, the modes and consequences for each protein-protein interaction vary from the viewpoint of tumor development and suppression.

Positive and negative regulation of prostate stem cell antigen expression by Yin Yang 1 in prostate epithelial cell lines.

Prostate cancer is influenced by epigenetic modification of genes involved in cancer development and progression. Increased expression of Prostate Stem Cell Antigen (PSCA) is correlated with development of malignant human prostate cancer, while studies in mouse models suggest that decreased PSCA levels promote prostate cancer metastasis. These studies suggest that PSCA has context-dependent functions, and could be differentially regulated during tumor progression. In the present study, we identified the multi-functional transcription factor Yin Yang 1 (YY1) as a modulator of PSCA expression in prostate epithelial cell lines. Increased YY1 levels are observed in prostatic intraepithelial neoplasia (PIN) and advanced disease. We show that androgen-mediated up-regulation of PSCA in prostate epithelial cell lines is dependent on YY1. We identified two direct YY1 binding sites within the PSCA promoter, and showed that the upstream site inhibited, while the downstream site, proximal to the androgen-responsive element, stimulated PSCA promoter activity. Thus, changes in PSCA expression levels in prostate cancer may at least partly be affected by cellular levels of YY1. Our results also suggest multiple roles for YY1 in prostate cancer which may contribute to disease progression by modulation of genes such as PSCA.

Dmp1 physically interacts with p53 and positively regulates p53's stability, nuclear localization, and function.

The transcription factor Dmp1 is a Ras/HER2-activated haplo-insufficient tumor suppressor that activates the Arf/p53 pathway of cell-cycle arrest. Recent evidence suggests that Dmp1 may activate p53 independently of Arf in certain cell types. Here, we report findings supporting this concept with the definition of an Arf-independent function for Dmp1 in tumor suppression. We found that Dmp1 and p53 can interact directly in mammalian cells via the carboxyl-terminus of p53 and the DNA-binding domain of Dmp1. Expression of Dmp1 antagonized ubiquitination of p53 by Mdm2 and promoted nuclear localization of p53. Dmp1-p53 binding significantly increased the level of p53, independent of the DNA-binding activity of Dmp1. Mechanistically, p53 target genes were activated synergistically by the coexpression of Dmp1 and p53 in p53(-/-);Arf(-/-) cells, and genotoxic responses of these genes were hampered more dramatically in Dmp1(-/-) and p53(-/-) cells than in Arf(-/-) cells. Together, our findings identify a robust new mechanism of p53 activation mediated by direct physical interaction between Dmp1 and p53.

Critical roles of DMP1 in human epidermal growth factor receptor 2/neu-Arf-p53 signaling and breast cancer development.

Human epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3'-kinase-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpression. Both Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu mice, and mammary tumorigenesis was significantly accelerated in both Dmp1+/- and Dmp1-/- mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu carcinomas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/-, Dmp1-/-, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study shows the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis.

The Arf-inducing transcription factor Dmp1 encodes a transcriptional activator of amphiregulin, thrombospondin-1, JunB and Egr1.

Dmp1 (Dmtf1) encodes a Myb-like transcription factor implicated in tumor suppression through direct activation of the Arf-p53 pathway. The human DMP1 gene is frequently deleted in non-small cell lung cancers, especially those that retain wild-type INK4a/ARF and/or p53. To identify novel genes that are regulated by Dmp1, transcriptional profiles of lung tissue from Dmp1-null and wild-type mice were generated using the GeneChip Microarray. Comparative analysis of gene expression changes between the two groups resulted in identification of numerous genes that may be regulated by Dmp1. Notably, amphiregulin (Areg), thrombospondin-1 (Tsp-1), JunB, Egr1, adrenomedullin (Adm), Bcl-3 and methyl-CpG binding domain protein 1 (Mbd1) were downregulated in the lungs from Dmp1-null mice while Gas1 and Ect2 genes were upregulated. These target genes were chosen for further analyses since they are involved in cell proliferation, transcription, angiogenesis/metastasis, apoptosis, or DNA methylation, and thus could account for the tumor suppressor phenotype of Dmp1. Dmp1 directly bound to the genomic loci of Areg, Tsp-1, JunB and Egr1. Significant upregulation or downregulation of the novel Dmp1 target genes was observed upon transient expression of Dmp1 in alveolar epithelial cells, an effect which was nullified by the inhibition of de novo mRNA synthesis. Interestingly, these genes and their protein products were significantly downregulated or upregulated in the lungs from Dmp1-heterozygous mice as well. Identification of novel Dmp1 target genes not only provides insights into the effects of Dmp1 on global gene expression, but also sheds light on the mechanism of haploid insufficiency of Dmp1 in tumor suppression.

MMTV mouse models and the diagnostic values of MMTV-like sequences in human breast cancer.

Mouse mammary tumor virus (MMTV) long terminal repeat (LTR)-driven transgenic mice are excellent models for breast cancer as they allow for the targeted expression of various oncogenes and growth factors in neoplastic transformation of mammary glands. Numerous MMTV-LTR-driven transgenic mouse models of breast cancer have been created in the past three decades, including MMTV-neu/ErbB2, cyclin D1, cyclin E, Ras, Myc, int-1 and c-rel. These transgenic mice develop mammary tumors with different latency, histology and invasiveness, reflecting the oncogenic pathways activated by the transgene. Recently, homologous sequences of the env gene of MMTV have been identified in approximately 40% of human breast cancers, but not in normal breast or other types of cancers, suggesting possible involvement of mammary tumor virus in human breast carcinogenesis. Accumulating evidence demonstrates the association of MMTV provirus with progesterone receptor, p53 mutations and advanced-stage breast cancer. Thus, the detection of MMTV-like sequences may have diagnostic value to predict the clinical outcome of breast cancer patients.

Role of DMP1 and its future in lung cancer diagnostics.

Lung cancer is the most lethal carcinoma worldwide. Mutations of p53, inactivation of p16(INK4a), and overexpression of cyclins E, A and B are independently associated with poor prognoses of patients, while the prognostic value of cyclin D1 or RB expression is inconclusive. Cyclin D binding myb-like protein 1 (Dmp1) encodes a DNA binding protein that receives signals from oncogenic Ras and functions as a tumor suppressor by activating the Arf-p53 [corrected] pathway. Dmp1 has been shown to be haplo-insufficient for tumor suppression in mouse models including K-ras-mediated lung carcinogenesis. The human DMP1 gene is located on chromosome 7q21, and our recent results revealed that the hDMP1 gene is deleted, but not mutated or silenced, in approximately 40 % of human non-small-cell lung carcinomas. These cases typically retained wild-type ARF and p53 and expressed very low levels of the hDMP1 protein. Thus, hDMP1 loss could be a novel diagnostic marker for non-small-cell lung carcinomas.

Emerging roles of DMP1 in lung cancer.

The Ras-activated transcription factor DMP1 can stimulate Arf transcription to promote p53-dependent cell arrest. One recent study deepens the pathophysiologic significance of this pathway in cancer, first, by identifying DMP1 losses in human lung cancers that lack ARF/p53 mutations, and second, by demonstrating that Dmp1 deletions in the mouse are sufficient to promote K-ras-induced lung tumorigenesis via mechanisms consistent with a disruption of Arf/p53 suppressor function. These findings prompt further investigations of the prognostic value of DMP1 alterations in human cancers and the oncogenic events that can cooperate with DMP1 inactivation to drive tumorigenesis.

Signal transduction involving the dmp1 transcription factor and its alteration in human cancer.

Dmp1 (cyclin D-interacting myb-like protein 1; also called Dmtf1) is a transcription factor that has been isolated in a yeast two-hybrid screen through its binding property to cyclin D2. Dmp1 directly binds to and activates the Arf promoter and induces Arf-p53-dependent cell cycle arrest in primary cells. D-type cyclins usually inhibit Dmp1-mediated transcription in a Cdk-independent fashion; however, Dmp1 shows synergistic effects with D-cyclins on the Arf promoter. Ras or Myc oncogene-induced tumor formation is accelerated in both Dmp1(+/-) and Dmp1(-/-) mice with no significant differences between Dmp1(+/-) and Dmp1(-/-). Thus, Dmp1 is haplo-insufficient for tumor suppression. Tumors from Dmp1(-/-) or Dmp1(+/-) mice often retain wild-type Arf and p53, suggesting that Dmp1 is a physiological regulator of the Arf-p53 pathway. The Dmp1 promoter is activated by oncogenic Ras-Raf signaling, while it is repressed by physiological mitogenic stimuli, overexpression of E2F proteins, and genotoxic stimuli mediated by NF-κB. The human DMP1 gene (hDMP1) is located on chromosome 7q21 and is hemizygously deleted in approximately 40% of human lung cancers, especially those that retain normal INK4a/ARF and P53 loci. Thus, hDMP1 is clearly involved in human carcinogenesis, and tumors with hDMP1 deletion may constitute a discrete disease entity.

Mutually exclusive inactivation of DMP1 and ARF/p53 in lung cancer.

Dmp1 (Dmtf1) is activated by oncogenic Ras-Raf signaling and induces cell-cycle arrest in an Arf, p53-dependent fashion. The survival of K-ras(LA) mice was shortened by approximately 15 weeks in both Dmp1(+/-) and Dmp1(-/-) backgrounds, the lung tumors of which showed significantly decreased frequency of p53 mutations compared to Dmp1(+/+). Approximately 40% of K-ras(LA) lung tumors from Dmp1(+/+) mice lost one allele of the Dmp1 gene, suggesting the primary involvement of Dmp1 in K-ras-induced tumorigenesis. Loss of heterozygosity (LOH) of the hDMP1 gene was detectable in approximately 35% of human lung carcinomas, which was found in mutually exclusive fashion with LOH of INK4a/ARF or that of P53. Thus, DMP1 is a pivotal tumor suppressor for both human and murine lung cancers.

PKC-alpha and TAK-1 are intermediates in the activation of c-Jun NH2-terminal kinase by hypoxia-reoxygenation.

c-Jun NH(2)-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-alpha/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-alpha blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.

Acidosis regulates the stability, hydrophobicity, and activity of the BH3-only protein Bnip3.

Bnip3 is a prodeath member of the so-called BH3-only subfamily of Bcl-2 proteins. A major function of this class of proteins is to regulate the permeability state of the outer mitochondrial membrane by forming homoand hetero-oligomers inside the membrane. We reported previously that Bnip3 accumulates in cardiac myocytes during exposure to hypoxia, but coincident acidosis is required to activate the death program. Acidosis increased the rate of intracellular accumulation of Bnip3 and promoted a tighter association with mitochondria. Here we report that acidic pH mediates increased half-lives of Bnip3 dimers and monomers (>3-) as well as that of a faster-migrating fragment (>10-) and confers protection against degradation by protease. Hydrophobic partitioning experiments revealed that Bnip3 monomers and oligomers from hypoxia-acidic cell fractions associated significantly with the detergent layer, whereas protein from hypoxia-neutral myocytes did not. Acidosis promoted homodimerization of Bcl-xL but did not increase its association with detergent. Neutralization of the extracellular medium of cardiac myocyte cultures under hypoxia-acidosis resulted in rapid degradation of accumulated Bnip3 (half life, <2 h), coincident with cessation of the death program. Bnip3 monomers appear to be the active species because substitution of alanine for histidine at position 173 within the transmembrane (TM) domain prevented homodimerization but did not inhibit the death function. These results demonstrate a pH-sensitive shift in the stability and apparent hydrophobicity of Bnip3 monomers that correlates closely with membrane binding and function.

Redox stress and the contributions of BH3-only proteins to infarction.

Ischemia followed by reperfusion is the primary cause of tissue injury and infarction during heart attack and stroke. The initiating stimulus is believed to involve reactive oxygen species that are produced during reperfusion when electron transport resumes in the mitochondria after suppression by ischemia. Programmed death has been shown to be a significant component of infarction, and evidence indicates that multiple pathways are initiated during both ischemia and reperfusion phases. Major infarction is preceded by severe ischemia that includes hypoxia, intracellular acidosis, glucose depletion, loss of ATP, and elevation of cytoplasmic calcium. The superimposition of a reactive oxygen surge on the latter condition provides the impetus for maximal damage. Compelling evidence implicates mitochondria not only as the source of initiating ROS but also as the focal sensors that translate the redox stress signal into a cellular-death response. Pivotal to this response are the BH3-only proteins that are activated by death signals and regulate mitochondrial communication with executioner proteins in the cytoplasm. The BH3-only proteins do this by controlling the activity of pores and channels in the outer mitochondrial membrane. To date at least six BH3-only proteins have been shown to contribute to ischemia-reperfusion death pathways in heart and/or brain; these include Bnip3, PUMA, Bid, Bad, HGTD-P, and Noxa. Here we review the evidence for these cell-death pathways and discuss their relevance to ischemic disease and infarction.

A unique pathway of cardiac myocyte death caused by hypoxia-acidosis.

Chronic hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. The condition parallels myocardial ischemia in vivo, where ischemic tissue becomes rapidly hypoxic and acidotic. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the extracellular pH (pH[o]) drops below 6.5. A microarray analysis of 20 000 genes (cDNAs and expressed sequence tags) screened with cDNAs from aerobic and hypoxic cardiac myocytes identified >100 genes that were induced by >2-fold and approximately 20 genes that were induced by >5-fold. One of the most strongly induced transcripts was identified as the gene encoding the pro-apoptotic Bcl-2 family member BNIP3. Northern and western blot analyses confirmed that BNIP3 was induced by 12-fold (mRNA) and 6-fold (protein) during 24 h of hypoxia. BNIP3 protein, but not the mRNA, accumulated 3.5-fold more rapidly under hypoxia-acidosis. Cell fractionation experiments indicated that BNIP3 was loosely bound to mitochondria under conditions of neutral hypoxia but was translocated into the membrane when the myocytes were acidotic. Translocation of BNIP3 coincided with opening of the mitochondrial permeability pore (MPTP). Paradoxically, mitochondrial pore opening did not promote caspase activation, and broad-range caspase inhibitors do not block this cell death pathway. The pathway was blocked by antisense BNIP3 oligonucleotides and MPTP inhibitors. Therefore, cardiac myocyte death during hypoxia-acidosis involves two distinct steps: (1) hypoxia activates transcription of the death-promoting BNIP3 gene through a hypoxia-inducible factor-1 (HIF-1) site in the promoter and (2) acidosis activates BNIP3 by promoting membrane translocation. This is an atypical programmed death pathway involving a combination of the features of apoptosis and necrosis. In this article, we will review the evidence for this unique pathway of cell death and discuss its relevance to ischemic heart disease. The article also contains new evidence that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes.

Mitochondrial signals initiate the activation of c-Jun N-terminal kinase (JNK) by hypoxia-reoxygenation.

C-Jun N-terminal kinase (JNK) is part of the mitogen-activated protein kinase (MAPK) family of signaling pathways that are induced in response to extracellular stimuli. JNK is primarily a stress-response pathway and can be activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through non-receptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). The molecular initiators of MAPKs by non-receptor stimuli have not been described. Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We show here that the activation of JNK by this condition is initiated in the mitochondria and requires coupled electron transport, ROI generation, and calcium flux. These signals cause the selective, sequential activation of the calcium-dependent, proline-rich kinase Pyk2 and the small GTP binding factors Rac-1 and Cdc42. Interruption of these interactions with inactivated dominant negative mutant proteins, blocking calcium flux, or inhibiting electron transport through mitochondrial complexes II, III, or IV prevents JNK activation and results in a proapoptotic phenotype that is characteristic of JNK inhibition in this model of ischemia-reperfusion. The signaling pathway is unique for the reoxygenation stimulus and provides a framework for other non-receptor-mediated pathways of MAPK activation.