GC-selective DNA-binding antibiotic, Mithramycin A, reveals multiple points of control in the regulation of Hdm2 protein synthesis

The primary role of the Hdm2/Mdm2 oncoprotein is to regulate the levels and activity of the transcription factor p53. Hdm2 synthesis is itself tightly controlled and, as demonstrated by a recently described SNP (SNP309) in the hdm2-P2 promoter, minor variations in Hdm2 expression have phenotypic consequences on radiation sensitivity and cancer predisposition. To further define mechanisms regulating Hdm2 expression, we have investigated the effects of the GC-selective DNA-binding drug, Mithramycin A (MA) on hdm2 mRNA transcription, trafficking, and translation. Firstly we show that the constitutive hdm2-P1 promoter is inhibited by MA. We define, for the first time, the minimal sequence elements that are required for P1-promoter activity and identify those which confer MA sensitivity. Secondly, MA induces p53-dependent transcription from the hdm2-P2 promoter. Thirdly, and critically, MA also inhibits Hdm2 synthesis at the post-transcriptional level, with negative effects on hdm2 mRNA nuclear export and translation. This study highlights the complex interplay between the pathways that regulate Hdm2 protein synthesis in cancer cells, and furthermore emphasizes the export of hdm2 mRNA from the nucleus to the cytoplasm as a key point of control in this process.<br/><br/

CtBPs promote cell survival through the maintenance of mitotic fidelity

CtBPs (CtBP1 and CtBP2) act in the nucleus as transcriptional corepressors and in the cytoplasm as regulators of Golgi fission. Studies in which the expression or function of CtBPs has been inhibited have independently identified roles for CtBPs in both suppressing apoptosis and promoting cell cycle progression. Here we have analyzed the consequences of ablating CtBP expression in breast cancer-derived cell lines. We find loss of CtBP expression suppresses cell proliferation through a combination of apoptosis, reduction in cell-cycle progression, and aberrations in transit through mitosis. This third phenotype includes errors in mitotic chromosome segregation that are associated with decreased association of the chromosome passenger protein aurora B with mitotic chromatin, and which are likely to be a primary cause of the pro-apoptotic and anti-proliferative effects of CtBP loss. We also show that loss of CtBP expression results in the activation of the transcription factor p53, and that loss of p53 function renders cells more susceptible to CtBP siRNA-induced apoptosis.<br/

MNK1 and EIF4E are downstream effectors of MEKs in the regulation of the nuclear export of HDM2 mRNA

Regulation of the synthesis, function and degradation of HDM2 (Mdm2 in mouse) plays a key role in controlling the abundance and activity of the transcription factor p53, with consequent implications for the proliferation and survival of normal and cancer cells. We have previously identified the regulation of export of HDM2 mRNA from the nucleus as a novel point of control of HDM2 synthesis. This process is dependent on the activity of the growth factor-regulated MAP-kinase kinases ( MEKs). Here, we provide evidence that the eIF4E kinase MNK1 is a key downstream effector of MEKs in this regulatory pathway. We show that HDM2 mRNA export in breast cancer cells is promoted by overexpressed eIF4E in a MEK- and MNK1-dependent manner, and inhibition of MNK1 suppresses endogenous HDM2 mRNA export pathways. This MNK1- and eIF4E-dependent HDM2 regulation occurs through sequences in the 30 untranslated region of HDM2 mRNA, and consequently HDM2 mRNA transcripts from both the constitutive P1 and inducible P2 promoters are regulated by this pathway. eIF4E is a known oncogene that is overexpressed in human tumours, including the majority of breast cancers. This pathway, therefore, may play an important role in the dysregulation of HDM2 oncoprotein expression that occurs in many human tumours

GC-selective DNA-binding antibiotic, Mithramycin A, reveals multiple points of control in the regulation of Hdm2 protein synthesis

The primary role of the Hdm2/Mdm2 oncoprotein is to regulate the levels and activity of the transcription factor p53. Hdm2 synthesis is itself tightly controlled and, as demonstrated by a recently described SNP (SNP309) in the hdm2-P2 promoter, minor variations in Hdm2 expression have phenotypic consequences on radiation sensitivity and cancer predisposition. To further define mechanisms regulating Hdm2 expression, we have investigated the effects of the GC-selective DNA-binding drug, Mithramycin A (MA) on hdm2 mRNA transcription, trafficking, and translation. Firstly we show that the constitutive hdm2-P1 promoter is inhibited by MA. We define, for the first time, the minimal sequence elements that are required for P1-promoter activity and identify those which confer MA sensitivity. Secondly, MA induces p53-dependent transcription from the hdm2-P2 promoter. Thirdly, and critically, MA also inhibits Hdm2 synthesis at the post-transcriptional level, with negative effects on hdm2 mRNA nuclear export and translation. This study highlights the complex interplay between the pathways that regulate Hdm2 protein synthesis in cancer cells, and furthermore emphasizes the export of hdm2 mRNA from the nucleus to the cytoplasm as a key point of control in this process.<br/

DNA damage triggers DRB-resistant phosphorylation of human p53 at the CK2 site

The sequence-specific DNA binding activity of p53 is negatively regulated by a C-terminal domain whose phosphorylation in vitro can activate the latent DNA binding function of the protein. The DNA binding activity of p53 is a core component of its stress-activated transcription function, yet it is not yet clear whether phosphorylation within the C-terminal domain plays a role in the p53 damage response in vivo. As the casein kinase 2 (CK2) site at serine 392 is the C-terminal phosphorylation motif that exhibits the most pronounced conservation at the primary amino acid level, we have focused on determining whether the CK2 site is modified in vivo and whether radiation effects the extent of that phosphorylation. Using antibodies that can detect serine 392-phosphorylation of p53, we demonstrate that UV radiation can trigger extensive phosphorylation at the CK2 site. The CK2 inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), can partially inhibit the UV-induced phosphorylation at serine 392, suggesting that CK2 is one of the major serine 392-kinases. However, a striking increase in UV-induced serine 392 phosphorylation and p53 transactivation function at higher levels of DRB suggests that a DRB-resistant/stress-activated pathway may target serine 392 in vivo. These data demonstrate that radiation-induced phosphorylation of p53 can occur in vivo at serine 392 and implicate a CK2-independent signal cascade that can function to modulate serine 392 phosphorylation in cells

The proliferation of normal human fibroblasts is dependent upon negative regulation of p53 function by mdm2

Loss of function of the tumour suppressor gene p53 is a key event in most human cancers. Although usually occurring through mutation, in some tumour types this appears to be achieved via an indirect mechanism involving inappropriate expression of a functional inhibitor, mdm2, which binds to the transactivation domain of p53. This interaction offers an ideal potential target for novel cancer therapies. However, therapeutic specificity may depend on the extent to which this p53-inhibitory action of mdm2 is also required by normal cells. Transgenic data have already established that mdm2 is needed to prevent embryonic lethality, but the situation in adult cells is still unclear. Here we show that micro-injection of normal human fibroblasts with an antibody directed against the p53-binding domain of mdm2 induces expression of p53-responsive genes, and furthermore results in p53-dependent growth arrest. We conclude that normal cell proliferation can be dependent on negative regulation of p53 by mdm2, a finding which raises an important note of caution for mdm2-directed cancer therapies

Loss of responsiveness to transforming growth factor beta (TGFbeta) is tightly linked to tumorigenicity in a model of thyroid tumour progression

It has been suggested that an important step in the progression of some epithelial tumours is the loss of responsiveness to the growth-inhibitory effects of transforming growth factor beta (TGFbeta). Here we describe the use of a model of thyroid tumorigenesis to investigate this question. Seven genetically closely related epithelial cell lines were derived following infection of primary cultures of rat thyroid epithelium with retroviral vectors encoding mutant ras. A strong negative correlation (p &lt; 0.001) was found between the responsiveness of the lines to TGFbeta growth inhibition in vitro and their tumorigenicity in nude mice. Whereas TGFbeta-unresponsive and TGFbeta-stimulated lines formed rapidly growing, poorly differentiated tumours at all injection sites, cells that retained a partial inhibitory response formed much more slowly growing tumours, which showed a high degree of glandular differentiation. A line which retained full inhibition by TGFbeta formed slowly growing tumours at only 30% of injection sites, and cells explanted from these tumours subsequently showed a much reduced TGFbeta response in vitro. Our data using thyroid cells thus greatly strengthen the suggestion from previous studies that loss of growth inhibition by TGFbeta is associated with malignant progression of epithelial tumours. We also present an experimental model of papillary thyroid cancer which may prove useful in identifying the molecular changes involved in progression to the anaplastic form of the disease

Activation of p53 protein function in response to cellular irradiation

p53 protein is a key regulatory component of a stress-inducible cell-cycle checkpoint pathway in mammalian cells, which can promote either cell-growth arrest or apoptosis, depending on the type of cell and damaging agent utilized. Environmental insults that can activate the p53 pathway are quite distinct, and include ionizing and nonionizing radiation (1–5), antimetabolites which inhibit ribonucleotide biosynthesis (6), inhibitors of spindle formation (7), microtubule-affecting drugs (8), factors inducing differentiation (9), signaling pathways activated in transformed cells during anchorage-independent growth (10), heat shock (11), and hypoxia (12)

Interaction between p53 and TGF beta 1 in control of epithelial cell proliferation

Although loss of sensitivity to transforming growth factor beta (TGF beta) may be a key step in the escape of epithelial tumours from normal growth control, the intracellular signals determining responsiveness remain controversial, particularly the role of p53. We have investigated this question using thyroid epithelial lines as a model. We analysed (i) human thyroid cancer cell lines having either wild-type (wt) or mutant p53; (ii) rat thyroid lines derived by spontaneous immortalisation following introduction of mutant H-ras, which exhibit high levels of wt p53 but loss of p53-mediated cell-cycle control. Loss of response to TGF beta 1 was found in all human lines bearing mutant p53, and in the majority of the functionally equivalent rat lines, consistent with a role of wt p53 in mediating response. However, introduction of a dominant negative p53 mutant into TGF beta 1 responsive human lines containing wt p53 did not reduce responsiveness, demonstrating that p53 function is not necessary for TGF beta 1 response. On the other hand, expression of a temperature-sensitive (ts) p53 gene in a partially-responsive rat line demonstrated a highly significant modulation of TGF beta response, which fell from 65% inhibition of 3H-thymidine labelling index at 32.5 degrees C (wt p53 conformation) to only 14% at 37.5 degrees C (mutant conformation). The results suggest that p53 and TGF beta generate separate but interacting inhibitory signals, i.e. that p53 modulates but does not mediate TGF beta response. This conclusion explains previous conflicting data and is consistent with current models of cell cycle control by multiple inhibitors of cyclin-dependent kinases