The Bag-1 inhibitor, Thio-2, reverses an atypical 3D morphology driven by Bag-1L overexpression in a MCF-10A model of ductal carcinoma in situ.

Mammary MCF-10A cells seeded on reconstituted basement membrane form spherical structures with a hollow central lumen, termed acini, which are a physiologically relevant model of mammary morphogenesis. Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional protein overexpressed in breast cancer and ductal carcinoma in situ. When present in the nucleus Bag-1 is predictive of clinical outcome in breast cancer. Bag-1 exists as three main isoforms, which are produced by alternative translation initiation from a single mRNA. The long isoform of Bag-1, Bag-1L, contains a nuclear localisation sequence not present in the other isoforms. When present in the nucleus Bag-1L, but not the other Bag-1 isoforms, can interact with and modulate the activities of estrogen-, androgen- and vitamin D-receptors. Overexpression of Bag-1 mRNA in MCF-10A is known to produce acini with luminal filling reminiscent of ductal carcinoma in situ. As this mRNA predominantly overexpresses the short isoform of Bag-1, Bag-1S, we set out to examine whether the nuclear Bag-1L isoform is sufficient to drive premalignant change by developing a Bag-1L-overexpressing MCF-10A model. Two clones differentially overexpressing Bag-1L were grown in two-dimensional (2D) and three-dimensional (3D) cultures and compared with an established model of HER2-driven transformation. In 2D cultures, Bag-1L overexpression reduced proliferation but did not affect growth factor responsiveness or clonogenicity. Acini formed by Bag-1L-overexpressing cells exhibited reduced luminal clearing when compared with controls. An abnormal branching morphology was also observed which correlated with the level of Bag-1L overexpression, suggesting further malignant change. Treatment with Thio-2, a small-molecule inhibitor of Bag-1, reduced the level of branching. In summary, 3D cultures of MCF-10A mammary epithelial cells overexpressing Bag-1L demonstrate a premalignant phenotype with features of ductal carcinoma in situ. Using this model to test the small-molecule Bag-1 inhibitor, Thio-2, reveals its potential to reverse the atypical branched morphology of acini that characterizes this premalignant change.

CtBPs promote mitotic fidelity through their activities in the cell nucleus.

CtBPs form NADH-sensitive chromatin-modifying complexes, which link cellular metabolism to gene transcription. They also function in the cytoplasm to regulate Golgi fissioning; their inhibition can consequently cause a Golgi-dependent checkpoint in G(2). We have recently identified a novel role of CtBPs in the maintenance of mitotic fidelity; inhibition of CtBP synthesis resulting in reduced association of aurora B with mitotic chromatin and aberrant segregation of chromosomes. Here, we demonstrate that it is the interaction of CtBPs with transcriptional regulators and/or chromatin-modifying enzymes in the cell nucleus, rather than their role in Golgi fission, which is critical for the maintenance of mitotic fidelity.

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 3' 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.

C-terminal binding proteins: emerging roles in cell survival and tumorigenesis.

Within a cell, the levels and activity of multiple pro- and anti-apoptotic molecules act in concert to regulate commitment to apoptosis. Whilst the balance between survival and death can be tipped by the effects of single molecules, cellular apoptosis control pathways very often incorporate key transcription factors that co-ordinately regulate the expression of multiple apoptosis control genes. C-terminal binding proteins (CtBPs), which were originally identified through their binding to the Adenovirus E1A oncoprotein, have been described as such transcriptional regulators of the apoptosis program. Specifically, CtBPs function as transcriptional co-repressors, and have been demonstrated to promote cell survival by suppressing the expression of several pro-apoptotic genes. In this review we summarize the evidence supporting a key role for CtBP proteins in cell survival. We also describe the known mechanisms of transcriptional control by CtBPs, and review the multiplicity of intracellular signaling and transcriptional control pathways with which they are known to be involved. Finally we consider these findings in the context of additional known roles of CtBP molecules, and the potential implications that this combined knowledge may have for our comprehension of diseases of cell survival, notably cancer.

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.

Stoichiometric phosphorylation of human p53 at Ser315 stimulates p53-dependent transcription.

p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser(315) phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser(315) can be detected in vivo, whether steady-state Ser(315) phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser(315) where one, two, three, or four phosphates/tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser(315) phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser(315) site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser(315) after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.

Synergistic activation of p53-dependent transcription by two cooperating damage recognition pathways.

High level activation of p53-dependent transcription occurs following cellular exposure to genotoxic damaging agents such as UV-C, while ionizing radiation damage does not induce a similarly potent induction of p53-dependent gene expression. Reasoning that one of the major differences between UV-C and ionizing radiation damage is that the latter does not inhibit general transcription, we attempted to reconstitute p53-dependent gene expression in ionizing irradiated cells by co-treatment with selected transcription inhibitors that alone do not activate p53. p53-dependent transcription can be dramatically enhanced by the treatment of ionizing irradiated cells with low doses of DRB, which on its own does not induce p53 activity. The mechanism of ionizing radiation-dependent activation of p53-dependent transcription using DRB is more likely due to inhibition of gene transcription rather than prolonged DNA damage, as the non-genotoxic and general transcription inhibitor Roscovitine also synergistically activates p53 function in ionizing irradiated cells. These results identify two distinct signal transduction pathways that cooperate to fully activate p53-dependent gene expression: one responding to lesions induced by ionizing radiation and the second being a kinase pathway that regulates general RNA Polymerase II activity.

Posttranslational modifications of p53 in replicative senescence overlapping but distinct from those induced by DNA damage.

Replicative senescence in human fibroblasts is absolutely dependent on the function of the phosphoprotein p53 and correlates with activation of p53-dependent transcription. However, no evidence for posttranslational modification of p53 in senescence has been presented, raising the possibility that changes in transcriptional activity result from upregulation of a coactivator. Using a series of antibodies with phosphorylation-sensitive epitopes, we now show that senescence is associated with major changes at putative regulatory sites in the N and C termini of p53 consistent with increased phosphorylation at serine-15, threonine-18, and serine-376 and decreased phosphorylation at serine-392. Ionizing and UV radiation generated overlapping but distinct profiles of response, with increased serine-15 phosphorylation being the only common change. These results support a direct role for p53 in signaling replicative senescence and are consistent with the generation by telomere erosion of a signal which shares some but not all of the features of DNA double-strand breaks.

Dephosphorylation of p53 at Ser20 after cellular exposure to low levels of non-ionizing radiation.

Induction of the transactivation function of p53 after cellular irradiation was studied under conditions in which upstream signaling events modulating p53 activation were uncoupled from those regulating stabilization. This investigation prompted the discovery of a novel radiation-responsive kinase pathway targeting Ser20 that results in the masking of the DO-1 epitope in undamaged cells. Unmasking of the DO-1 epitope via dephosphorylation occurs in response to low doses of non-ionizing radiation. Our data show that phosphorylation at Ser20 reduces binding of the mdm2 protein, suggesting that a function of the Ser20-kinase pathway may be to produce a stable pool of inactive p53 in undamaged cells which can be readily activated after cellular injury. Phospho-specific monoclonal antibodies were used to determine whether the Ser20 signaling pathway is coupled to the Ser15 and Ser392 radiation-responsive kinase pathways. These results demonstrated that: (1) dephosphorylation at Ser20 is co-ordinated with an increased steady-state phosphorylation at Ser392 after irradiation, without p53 protein stabilization, and (2) stabilization of p53 protein can occur without Ser15 phosphorylation at higher doses of radiation. These data show that the Ser20 and Ser392 phosphorylation sites are both targeted by an integrated network of signaling pathways which is acutely sensitive to radiation injury.

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.

Tolerance of high levels of wild-type p53 in transformed epithelial cells dependent on auto-regulation by mdm-2.

A significant proportion of human cancers express high levels of p53 protein in the absence of an underlying mutation in the gene. Using transformed (Vh1) and non-transformed (FRTL-5) rat thyroid epithelial cell lines as a model, we have examined the mechanisms by which high levels of wild-type p53 may be tolerated. Stable transfection with p53-dependent reporter constructs demonstrated that the 'excess' wild-type p53 in Vh1 cells is not associated with a comparable increase in p53-dependent transcription (though the response to u.v. irradiation is retained). Mdm-2, which binds p53 and inhibits its transactivation activity, is overexpressed in Vh1 cells in the absence of gene amplification and in a p53-dependent manner. Furthermore disruption of p53-mdm-2 complex formation in Vh1 cells by microinjection of an antibody to the p53-binding domain of mdm-2 resulted in a dramatic increase in p53-dependent transcription. Since only a small proportion of the p53 in Vh1 cells was found to be in complex with mdm-2 (the majority of unbound protein being in a latent form), this suggests that mdm-2 selectively binds a pool of p53 that would otherwise be active as a sequence-specific activator of transcription. We suggest that, in some types of tumour, the 'sensitivity' of the p53-driven mdm-2 feedback loop may be sufficient to prevent free, active p53 reaching the level required for growth arrest or apoptosis, making them an ideal target for therapies designed to disrupt p53-mdm-2 interactions.

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 < 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.

Mutant p53 rescues human diploid cells from senescence without inhibiting the induction of SDI1/WAF1.

Although the cyclin-dependent kinase inhibitor p21SDI1 (WAF1/CIP1) has been proposed as the mediator of p53-induced cell cycle arrest following DNA damage, several stimuli now appear to induce SDI1 independent of p53 function. We have examined the behavior of p53 and SDI1 in an isogeneic model by manipulating p53 status in normal diploid human fibroblasts using an amphotropic retroviral vector. Following DNA strand break damage induced by bleomycin, both SDI1 induction and G1-S cell cycle arrest are p53 dependent, consistent with SDI1 being the key mediator. In contrast, in cellular senescence (and following UV irradiation), induction of SDI1 occurs independent of p53 function yet growth arrest is still p53 dependent. We conclude (a) that redundant pathways exist for induction of SDI1, but that (b) SDI1, while perhaps necessary, is not sufficient for inhibition of cell cycle progression, requiring the cooperation of an additional factor (possibly another cyclin-dependent kinase inhibitor) whose expression, at least in the case of senescence, is strictly p53 dependent.

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.

Evasion of p53-mediated growth control occurs by three alternative mechanisms in transformed thyroid epithelial cells.

Using the thyroid as a model of multistep epithelial tumorigenesis, we have used representative cell lines to correlate the degree of malignant transformation with the functional status of p53 and the integrity of cell-cycle check-points. Three distinct phenotypes were observed: Type I lines, derived from poorly-differentiated human thyroid cancers, expressed high levels of mutant p53 protein; Type II, also poorly-differentiated but derived from rat, showed over-expression of wild-type (wt) p53 with marked cell-cell heterogeneity: Type III, from well-differentiated human cancers, contained uniformly low levels of wt p53. All cell lines containing wt p53 retained a near-normal induction of p53 by DNA damage. However, the ability to undergo growth arrest differed strikingly. Whereas Type I and II lines had lost both G2/M and G1/S check points, Type III cells retained both. In Type III cells, as in diploid human fibroblasts, mutant p53 expression specifically abrogated G1/S check-point function with no other change in phenotype. These data demonstrate 3 mechanisms for evasion of p53 growth control: (i) direct mutation (ii) indirect inactivation, or (iii) 'avoidance' of activation, most probably due to failure to reach a critical threshold of DNA damage.

Stepwise transformation of primary thyroid epithelial cells by a mutant Ha-ras oncogene: an in vitro model of tumor progression.

Activating mutations of the ras oncogene family occur at high frequency in all stages of thyroid tumorigenesis, both human and experimental. To test the causal nature of this association, and to investigate the biological role of ras mutation, we introduced a mutant c-Ha-ras gene into normal rat thyroid follicular cells using an ecotropic retroviral vector. The major immediate effect was to greatly extend the proliferative lifespan of these cells in culture from less than 3 to more than 15 doublings, without any observable loss of growth-factor dependence or differentiated functions. This in vitro phenotype strongly supports an initiating role for ras mutation in the genesis of benign thyroid tumors (adenomas) in vivo. Spontaneous transformation was observed at low frequency on continuous culture of mutant ras-expressing cells, giving rise to fully immortalized, growth factor-independent, highly tumorigenic lines. Transformation was associated with (i) loss of responsiveness to the growth inhibitor TGF-beta 1, and (ii) greatly increased nuclear levels of p53 protein, which unexpectedly was not due to point mutation in the conserved regions of the p53-coding sequence. We postulate that these two phenomena are causally related to each other and to the transformed phenotype.