Sequestration Of P53 Research Articles

Sequestration of p27 within the cytoplasm of cardiac myocytes in chronic ischemic heart disease: pathogenic implications for ischemic cardiomyopathy

A number of recent studies have suggested that cardiac myocytes, previously considered post-mitotic, re-enter the cell cycle and possess the ability to proliferate with certain pathogenic stimuli. To study this further, we examined cellular proliferation in myocardial tissue from subjects with chronic ischemic heart disease-associated myocardial infarction and subsequent congestive heart failure. We found striking increases in cytoplasmic phospho-p27, a well-known mitotic regulator, compared to controls by both immunocytochemical and immunoblot analyses. However, we found no evidence for cardiac myocyte proliferation in either disease or control subjects using both mitotic counting (no mitotic figures were observed) and Ki-67 immunocytochemistry, which demonstrated a 0% proliferation index. That increased cytoplasmic phospho-p27 is not accompanied by division prompts us to speculate that ectopic cell cycle activation occurs in the face of minimal to absent myocyte proliferation per se. Based on these findings, and the parallel findings in post-mitotic neurons in neurodegenerative disease, we suggest that cell-cycle activation in ischemic heart disease is a deleterious event that perpetuates disease pathogenesis culminating in myocardial failure.

An intact sequence-specific DNA-binding domain is required for human cytomegalovirus-mediated sequestration of p53 and may promote in vivo binding to the viral genome during infection

The p53 protein is stabilized during infection of primary human fibroblasts with human cytomegalovirus (HCMV). However, the p53 in HCMV-infected cells is unable to activate its downstream targets. HCMV accomplishes this inactivation, at least in part, by sequestering p53 into viral replication centers within the cell's nucleus soon after they are established. In order to better understand the interplay between HCMV and p53 and the mechanism of sequestration, we constructed a panel of mutant p53-GFP fusion constructs for use in transfection/infection experiments. These mutants affected several post-translational modification sites and several sites within the central sequence-specific DNA-binding domain of the protein. Two categories of p53 sequestration were observed when the mutant constructs were transfected into primary fibroblasts and then infected at either high or low multiplicity. The first category, including all of the post-translational modification mutants, showed sequestration comparable to a wild-type (wt) control, while the second category, mutants affecting the DNA-binding core, were not specifically sequestered above control GFP levels. This suggested that the DNA-binding ability of the protein was required for sequestration. When the HCMV genome was analyzed for p53 consensus binding sites, 21 matches were found, which localized either to the promoters or the coding regions of viral proteins involved in DNA replication and processing as well as structural proteins. An analysis of in vivo binding to these identified sites via chromatin immunoprecipitation assays revealed differential binding to several of the sites over the course of infection.

Overexpression of the p65 Subunit of NF-kB and IkB Mediated Nuclear Sequestration of p53 as Common Events in Philadelphia Positive and Negative Chronic Myeloid Leukemia.

Introduction. Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder caused by the Philadelphia translocation. Rare patients with a clinical presentation of CML are negative for the Ph chromosome. The pathogenesis of Ph negative CML is still unknown. Different reports have demonstrated that the transcription factor NF-kappaB is essential for Bcr-Abl mediated transformation. NF-kappaB is composed of two subunits (mainly p65 and p50) which are retained into the cytoplasm by the inhibitory protein IkappaB-alpha. Different stimuli trigger IkappaB degradation and nuclear translocation of NF-kappaB, where it mediates the transcription of different genes involved in cellular proliferation, transformation and resistance to apoptosis.Aim of the work. We have evaluated the role of NF-kappaB in primary chronic myeloid leukemia samples both positive and negative for the Philadelphia chromosome.Methods. Bone marrow samples of 10 chronic myeloproliferative disorders (6 Ph positive and 4 Ph negative CML), 1 CML blast phase, 2 Acute Myeloid Leukemia and 3 healthy donor have been collected at diagnosis. Each sample has been lysed to obtain cytosolic and nuclear extracts. Western blot have been performed to evaluate the expression of p65 and the regulatory protein IkappaB-alpha. DNA binding activity of NF-kappaB has been measured with an ELISA method (TransAM). To inhibit NF-kB, primary cells have been incubated with 1 microM MG-132, 90 microM Resveratrol and 5 microM Bay11-7082 or have been infected with a lentivirus construct containing dsRNA directed against the p65 subunit of NF-kB and a lentivirus expressing a mutated IKB which retains NF-kB inactive in the cytoplasm.Results. Ph+ and Ph- CML samples express both in the cytosol and in the nucleus higher levels of p65 respect to normal peripheral blood and normal bone marrow samples. In normal samples IkB-alpha is detectable only in the cytosol while in Ph+ and Ph- CML samples it is predominately expressed in the nucleus. DNA binding activity of NF-kappaB is not particularly increased in CML respect to normal bone marrow samples. Only CML blast phase and Acute Myeloid Leukemia show markedly increased DNA binding activity of NF-kappaB. Immunoprecipitates of nuclear and cytosolic IkappaB-alpha have been performed. The pro-apoptotic p53 co-immunoprecipitates with IkappaB-alpha. Treatment with NF-kappaB inhibitors MG-132, Resveratrol and Bay11-7082 disrupts p53-IkBalpha dimer rendering cells susceptible to the apoptotic stimulation induced by Doxorubicine. To confirm these data, primary CML samples have also been infected with a lentivirus construct containing dsRNA directed against p65 and a lentivirus expressing a mutated IKB which retains NF-kB inactive in the cytoplasm. In both cases cells becomes more sensitive to Doxorubicin-induced apoptosis, due to disruption of IkB - p53 dimer.Conclusion. Nuclear and cytosolic IkappaB-alpha mediated p53 sequestration is a common event in both Ph+ and Ph- CML, which may contribute to the pathogenesis of this disorder due to impairment of p53 pro-apoptotic functions. NF-kB inhibition may represent a powerful strategy to render CML cells more susceptible to apotosis.

Toll-like Receptor Signaling Stimulates Cell Cycle Entry and Progression in Fibroblasts

Toll-like receptors (TLRs) are proteins involved in recognition of foreign pathogen-associated molecular patterns and activation of processes leading to innate immune recognition. We show that stimulation of fibroblasts with a TLR5 ligand, flagellin, can induce proliferation of serum-starved cells or prevent cell cycle exit upon serum withdrawal independently of autologous growth factor secretion. Other TLR ligands, such as poly(I:C) and lipopolysaccharide, can have a similar effect only if the action of type I interferons is blocked. Flagellin stimulation can prevent cell cycle arrest induced by overexpression of exogenous cyclin-dependent kinase inhibitor p27. Stimulation of TLR5 and overexpression of MyD88, but not TRIF, TIRAP, or TRAM, result in p27 degradation, which can be suppressed by dominant negative Akt and mutation of the p27 C-terminal Thr(187) site. These data provide evidence for a nonimmune and cell autonomous role of TLR signaling, whereby TLR stimulation provides a positive signal for cell division.

The Ews/Fli-1 Fusion Gene Changes the Status of p53 in Neuroblastoma Tumor Cell Lines

One hallmark of Ewing's sarcoma/peripheral neuroectodermal tumors is the presence of the Ews/Fli-1 chimeric oncogene. Interestingly, infection of neuroblastoma tumor cell lines with Ews/Fli-1 switches the differentiation program of neuroblastomas to Ewing's sarcoma/peripheral neuroectodermal tumors. Here we examined the status of cytoplasmically sequestered wt-p53 in neuroblastomas after stable expression of Ews/Fli-1. Immunofluorescence revealed that in the neuroblastoma-Ews/Fli-1 infectant cell lines, p53 went from a punctate-pattern of cytoplasmic sequestration to increased nuclear localization. Western blot analysis revealed that PARC was down-regulated in one neuroblastoma cell line but not expressed in the second. Therefore, decreased PARC expression could not fully account for relieving p53 sequestration in the neuroblastoma tumor cells. Neuroblastoma-Ews/Fli-1 infectant cell lines showed marked increases in p53 protein expression without transcriptional up-regulation. Interestingly, p53 was primarily phosphorylated, without activation of its downstream target p21(WAF1). Western blot analysis revealed that whereas MDM2 gene expression does not change, p14(ARF), a negative protein regulator of MDM2, increases. These observations suggest that the downstream p53 pathway may be inactivated as a result of abnormal p53. We also found that p53 has an extended half-life in the neuroblastoma-Ews/Fli-1 infectants despite the retention of a wild-type sequence in neuroblastoma-Ews/Fli-1 infectant cell lines. We then tested the p53 response pathway and observed that the neuroblastoma parent cells responded to genotoxic stress, whereas the neuroblastoma-Ews/Fli-1 infectants did not. These results suggest that Ews/Fli-1 can directly abrogate the p53 pathway to promote tumorigenesis. These studies also provide additional insight into the relationship among the p53 pathway proteins.

Expression of hepatitis C virus non-structural 5A protein in the liver of transgenic mice

Hepatitis C virus (HCV) is a major etiologic agent for chronic hepatitis worldwide often leading to the development of cirrhosis and hepatocellular carcinoma. However, the mechanism for development of chronic hepatitis or hepatocarcinogenesis by HCV remains unclear. HCV NS5A protein possesses many intriguing properties, including sequestration of p53 in the cytoplasm, downregulation of p21 protein, activation of STAT3, and inhibition of tumor necrosis factor-α-mediated apoptosis. Thus, we investigated whether this viral protein has oncogenic property in vivo. In the absence of an efficient cell culture system for virus growth and a suitable small animal model for HCV infection, transgenic FVB mice were generated by targeting the HCV NS5A genomic region cloned under the control of a liver-specific apoE promoter or mouse major urinary promoter (MUP). The apoE promoter is constitutively expressed in liver, on the other hand, the MUP is developmentally regulated and expressed in the liver after birth. Reverse transcription polymerase chain reaction and Western blot analysis indicated establishment of HCV NS5A transgene expression in several lines from both groups of mice. Immunohistochemical studies suggested the presence of NS5A in the cytoplasm of hepatocytes. The transgenic animals were phenotypically similar to their normal littermates and did not exhibit a major histological change within the liver up to 24 months of age. Our results suggested HCV NS5A protein is not directly cytopathic or oncogenic in this FVB transgenic mouse model, although this viral protein promotes cell growth in vitro. These animals will be a valuable model of HCV immunopathology as well as for evaluation of siRNA, interferon and other cytokine therapies.

Sequestration of p53 in the cytoplasm by adenovirus type 12 E1B 55-kilodalton oncoprotein is required for inhibition of p53-mediated apoptosis.

The adenovirus E1B 55-kDa protein is a potent inhibitor of p53-mediated transactivation and apoptosis. The proposed mechanisms include tethering the E1B repression domain to p53-responsive promoters via direct E1B-p53 interaction. Cytoplasmic sequestration of p53 by the 55-kDa protein would impose additional inhibition on p53-mediated effects. To investigate further the role of cytoplasmic sequestration of p53 in its inhibition by the E1B 55-kDa protein we systematically examined domains in both the Ad12 55-kDa protein and p53 that underpin their colocalization in the cytoplasmic body and show that the N-terminal transactivation domain (TAD) of p53 is essential for retaining p53 in the cytoplasmic body. Deletion of amino acids 11 to 27 or even point mutation L22Q/W23S abolished the localization of p53 to the cytoplasmic body, whereas other parts of TAD and the C-terminal domain of p53 are dispensable. This cytoplasmic body is distinct from aggresome associated with overexpression of some proteins, since it neither altered vimentin intermediate filaments nor associated with centrosome or ubiquitin. Formation of this structure is sensitive to mutation of the Ad12 55-kDa protein. Strikingly, mutation S476/477A near the C terminus of the Ad12 55-kDa protein eliminated the formation of the cytoplasmic body. The equivalent residues in the Ad5 55-kDa protein were shown to be critical for its ability to inhibit p53. Indeed, Ad12 55-kDa mutants that cannot form a cytoplasmic body can no longer inhibit p53-mediated effects. Conversely, the Ad12 55-kDa protein does not suppress p53 mutant L22Q/W23S-mediated apoptosis. Finally, we show that E1B can still sequester p53 that contains the mitochondrial import sequence, thereby potentially preventing the localization of p53 to mitochondria. Thus, cytoplasmic sequestration of p53 by the E1B 55-kDa protein plays an important role in restricting p53 activities.

P27 and cyclin E/D2 associations in testicular germ cell tumors: implications for tumorigenesis.

The cyclins are key regulators of cell cycle progression and cellular proliferation. We have previously shown that in testicular germ cell tumors, cyclin E expression correlates with more aggressive tumors, higher clinical stage, and the presence of pulmonary metastases. Here, we have examined the association between cyclin activation and the proliferative rate of the pluripotential testicular tumor cell. We have shown that in a panel of 30 testicular germ cell tumors, 15 cases (50%) expressed the cyclin dependent kinase inhibitor p27; of note, 13 of 14 embryonal carcinomas (93%) coexpressed cyclin E and p27, suggesting inhibition of this cyclin. We show that 25 of 30 (83%) of the testicular germ cell tumors express cyclin D2. Using immunoprecipitation assays from the embryonal carcinoma cell line NTera2 or from tumor cell extracts, we have shown that cyclin D2 is complexed with p27, consistent with its known ability to sequester and block the cyclin E inhibitory function of p27. From these results, we propose a model in testicular germ cell tumors, in particular embryonal carcinomas, whereby the overexpression of cyclin D2, a gene localized on chromosome 12p--a region of DNA amplification in germ cell tumors--leads to the functional sequestration of p27 in the presence of cyclin E and cyclin D2, thus favoring cellular proliferation.

The nuclear localization of NFkappaB and p53 is positively correlated with HPV16 E7 level in laryngeal squamous cell carcinoma.

The interaction between the HPV (human papilloma virus) 16 E7 and other cell growth factors, such as p53 and NFkappaB in laryngeal cancer is not clearly understood. The aim of this study was to examine the expression of these three proteins in tumor and non-tumor laryngeal tissues from patients with laryngeal squamous cell carcinoma. These three proteins were dominantly expressed in the nucleus and their levels were higher in the tumor tissue than in the non-tumor tissue, although the comparison between the tumor and non-tumor tissues of p53 staining did not reach significance. The intensity of the nuclear stain of E7 and p53 was stronger than that of p65, a subunit of NFkappaB. Correlation analysis revealed that there was a positive relationship between the level of HPV16 E7 and the expression of p65. The correlation between E7 and p53 was also significant, although to a lesser degree. The finding of nuclear localization of p65 suggests that NFkappaB is constantly activated in the laryngeal cancer cells, whereas the sequestration of p53 in the nucleus may represent a mutated form of p53, which is probably inactivated by HPV16 oncoproteins. In conclusion, this study suggests that the nuclear localization of NFkappaB and p53 may play a role in the development of human laryngeal squamous cell carcinoma infected with HPV16.

Changes in expression and immunolocalization of protein associated with toxic bile salts-induced apoptosis in rat hepatocytes

Cholestatic liver injury results from the accumulation of toxic bile salts within the liver. The aim of the present study was to examine the temporal changes in expression and immunolocalization of protein associated with apoptosis in cholestatic rat liver. Rats were anesthetized and cholestasis was induced by double ligation of the common bile duct and sectioning between the ligatures. The animals were euthanized at day 3 and at weeks 1, 2, 4, and 6 after bile duct ligation (BDL). Apoptotic cell death was increased fivefold after 3 days of BDL, decreased over 2 weeks, and remained constant thereafter as has been demonstrated by TUNEL staining. Western blot analysis for Bax, Bcl-2, cytochrome c, and p53 were performed. Results show that total cellular Bax protein was increased 3 days after BDL and decreased over time thereafter. We observed the translocation of Bax to mitochondria and subsequent release of cytochrome c. According to our immunohistochemical data, nuclear p53 increased 3 days after BDL, but cytoplasmic sequestration of p53 was observed after 1 week. The expression of c-Myc was inhibited by 3 days, but increased at later stages following BDL. Bcl-2 was increased over time in BDL rats. Our data suggest toxic bile salts-induced hepatocellular apoptosis is related to differential expression of Bcl-2 family member protein and release of cytochrome c. Cellular localization of p53 plays an important role in apoptotic death of hepatocytes in BDL rats.

Salvia miltiorrhiza Inhibits Biliary Obstruction-Induced Hepatocyte Apoptosis by Cytoplasmic Sequestration of p53

Cholestatic liver injury is caused by hepatocellular apoptosis induced by toxic bile salts. We have studied the effects of a traditional Chinese herbal medicine, Salvia miltiorrhiza, on apoptotic cell death in bile duct-ligated (BDL) rats. We also attempted to clarify the molecular mechanisms of the hepatoprotective effects of S. miltiorrhiza in this animal model. A water extract of S. miltiorrhiza (Sm-X; 200 mg/kg; po) was administered to BDL rats for 10 days. Rats were euthanized and apoptosis was detected in liver tissue by TUNEL staining. Western blot analysis and immunostaining for α-smooth muscle actin (α-SMA), Bax, Bcl-2, and p53 were performed. Results show that the treatment of BDL rats with Sm-X significantly improved the liver function parameters, although the expression of α-SMA, a marker of hepatic stellate cell activation, was not affected. Treatment with Sm-X significantly reduced the number of apoptotic cells. A time-dependent decrease in Bax protein level and an increase in Bcl-2 protein level were observed in BDL rats treated with Sm-X. Immunohistochemical analysis demonstrated that p53 was strongly positive in hepatocyte nuclei of BDL rats but that treatment with Sm-X induced a cytoplasmic sequestration of p53. Taken together, hepatoprotective effects of Sm-X partially ascribe to the antiapoptotic property in hepatocytes. Treatment of Sm-X-induced cytoplasmic sequestration of p53, downregulation of Bax, and upregulation of Bcl-2 protein. This study identifies and delineates signaling factors involved in the antiapoptotic properties of Sm-X and suggests a potential application of S. miltiorrhiza in the clinical management of hepatic disease induced by toxic bile salts following biliary obstruction.

Removal of Cdk inhibitors through both sequestration and downregulation in zearalenone-treated MCF-7 breast cancer cells.

Treatment of MCF 7 cells with the fungal estrogen zearalenone induced cyclin E-associated kinase activity transiently within 9-12 h; total cyclin-dependent kinase (Cdk) 2 activity was elevated for 24 h and beyond. This increased cyclin E/Cdk2 activity was associated with sequestration of the Cdk inhibitor p27 Cdk inhibitor 1B (p27(KIP1)) by newly formed cyclin D1/Cdk4 complexes and with downregulation of p27(KIP1) expression. The activation of cyclin A/Cdk2 activity corresponded with virtual elimination of p27(KIP1). The activity of cyclin E/Cdk2 complexes from zearalenone-treated lysates was inhibited in vitro by recombinant p27(KIP1), and this inhibition was relieved by the addition of recombinant cyclin D1/Cdk4 complexes. Thus, sequestration of p27(KIP1) by cyclin D1/Cdk4 resulted in activation of Cdk2 in vitro. Cdk inhibitory activity in lysates of zearalenone-treated cells was depleted by anti-p27(KIP1) and anti-Cdc2 interacting protein (p21(CIP1)) antibodies. Overexpression of the Cdk4/6-specific Cdk inhibitor of Cdk4 p16(INK4A) was associated with increased association of p27(KIP1) with Cdk2, concomitant with disruption of D cyclin/Cdk4 complexes. The proteasome inhibitor 2-leu-leu-leu-H aldehyde (MG-132) was relatively ineffective in inhibiting the initial, sequestration-dependent activation of cyclin E/Cdk2 yet was as effective as p16(INK4A) in inhibiting activation of cyclin A/Cdk2 later in G(1). Downregulation of p27(KIP1) proceeded in p16(INK4A)-expressing cells after zearalenone treatment, and G(1) arrest afforded by p16(INK4A) expression was reversible upon prolonged treatment with zearalenone. Zearalenone treatment of MCF-7 cells elicited expression of F-box protein S phase kinase-associated protein 2 (p45(SKP2)), a substrate-specific component of the ubiquitin-ligase complex that targets p27(KIP1) for degradation in the proteasome. These studies suggest that both sequestration of Cdk inhibitors by cyclin D1/Cdk4 complexes and downregulation of p27(KIP1) play major roles in the induction of Cdk2 activity and S phase entry elicited by estrogens in MCF-7 cells.

Cyclin D1 Overexpression Induces Progestin Resistance in T-47D Breast Cancer Cells Despite p27Kip1 Association with Cyclin E-Cdk2

Long-term growth inhibition, arrest in G(1) phase and reduced activity of both cyclin D1-Cdk4 and cyclin E-Cdk2 are elicited by progestin treatment of breast cancer cells in culture. Decreased cyclin expression, induction of p18(INK4c) and increased association of the CDK inhibitors p21(WAF1/Cip1) and p27(Kip1) with cyclin E-Cdk2 have been implicated in these responses. To determine the role of decreased cyclin expression, T-47D human breast cancer cells constitutively expressing cyclin D1 or cyclin E were treated with the progestin ORG 2058. Overexpression of cyclin E had only a modest effect on growth inhibition. Although cyclin E expression was maintained during progestin treatment, cyclin E-Cdk2 activity decreased by approximately 60%. This was accompanied by p27(Kip1) association with cyclin E-Cdk2, indicating that both cyclin E down-regulation and p27(Kip1) recruitment contribute to the decrease in activity. In contrast, overexpression of cyclin D1 induced progestin resistance and cell proliferation continued despite decreased cyclin E-Cdk2 activity. Progestin treatment of cyclin D1-overexpressing cells was associated with increased p27(Kip1) association with cyclin E-Cdk2. Thus the ability of cyclin D1 to confer progestin resistance does not depend on sequestration of p27(Kip1) away from cyclin E-Cdk2, providing evidence for a critical function of cyclin D1 other than as a high-capacity "sink" for p27(Kip1). These data indicate that regulation of cyclin D1 is a critical element of progestin inhibition in breast cancer cells and suggest that breast cancers overexpressing cyclin D1 may respond poorly to progestin therapy.

NEW EMBO MEMBERS' REVIEW: Hsp70 interactions with the p53 tumour suppressor protein

The heat shock proteins (HSPs) are encoded by genes whose expression is substantially increased during stress conditions, such as heat shock, alcohol, inhibitors of energy metabolism, heavy metals, oxidative stress, fever or inflammation. During these conditions, HSPs increase cell survival by protecting and disaggregating stress‐labile proteins (Skowyra et al ., 1990), as well as the proteolysis of the damaged proteins (Wickner et al ., 1999). Under non‐stress conditions, HSPs have multiple housekeeping functions, such as folding and translocating newly synthesized proteins, activation of specific regulatory proteins, including transcription factors, replication proteins and kinases, protein degradation, protein signalling, including steroid hormone activation and tumour immunogenicity, and antigen presentation (for reviews see Helmbrecht et al ., 2000; Jolly and Morimoto, 2000). This broad spectrum of functions gave rise to the term ‘molecular chaperone’, an entity that acts to assist other proteins folding and maturating in the cell. It should also be emphasized that not all HSPs are molecular chaperones and not all chaperones are HSPs (Ellis and Hartl, 1999). HSPs are designated nomenclature according to their approximate molecular weight, e.g. the 70 kDa HSP is known as the molecular chaperone Hsp70. The 70 kDa heat shock‐related proteins comprise a family of highly conserved molecular chaperones that regulate a wide variety of cellular processes during normal and stress conditions (Boorstein et al ., 1994). Hsp70 is one of the most abundant of these proteins, accounting for as much as 1–2% of total cellular protein (Herendeen et al ., 1979). In humans, there are at least 11 distinct genes that code for Hsp70 isoforms, which are located on several different chromosomes (Tavaria et al ., 1996). The major, constitutively expressed hsp70 isoform is called hsc70 (gene product known as the clathrin‐uncoating ATPase or Hsp73) (Welch, 1992). The transcription of inducible forms of hsp70 or hsp72 are under …

Constitutive MEK/MAPK activation leads to p27(Kip1) deregulation and antiestrogen resistance in human breast cancer cells.

Antiestrogens, such as the drug tamoxifen, inhibit breast cancer growth by inducing cell cycle arrest. Antiestrogens require action of the cell cycle inhibitor p27(Kip1) to mediate G1 arrest in estrogen receptor-positive breast cancer cells. We report that constitutive activation of the mitogen-activated protein kinase (MAPK) pathway alters p27 phosphorylation, reduces p27 protein levels, reduces the cdk2 inhibitory activity of the remaining p27, and contributes to antiestrogen resistance. In two antiestrogen-resistant cell lines that showed increased MAPK activation, inhibition of the MAPK kinase (MEK) by addition of U0126 changed p27 phosphorylation and restored p27 inhibitory function and sensitivity to antiestrogens. Using antisense p27 oligonucleotides, we demonstrated that this restoration of antiestrogen-mediated cell cycle arrest required p27 function. These data suggest that oncogene-mediated MAPK activation, frequently observed in human breast cancers, contributes to antiestrogen resistance through p27 deregulation.

The SV40 small t-antigen prevents mammary gland differentiation and induces breast cancer formation in transgenic mice; truncated large T-antigen molecules harboring the intact p53 and pRb binding region do not have this effect.

We report here for the first time, that the SV40 small t-antigen inhibits mammary gland differentiation during mid-pregnancy and that about 10% of multiparous WAP-SVt transgenic animals develop breast tumors with latencies ranging from 10-17 months. Cyclin D1 is deregulated and over expressed in the small t-antigen positive mammary gland epithelial cells (ME-cells) and in the breast tumor cells. SV40 small t-antigen immortalized ME-cells (t-ME-cells) exhibit a strong intranuclear cyclin D1 staining, also in the absence of external growth factors and the cells continue to divide for several days without serum. In addition, the expression rate of cyclin E and p21(Waf1) but not of p53 is increased. Coimmunoprecipitation experiments revealed that p21(Waf1) is mainly associated with the cyclin D/CDK4 but not with the cyclin E/CDK2 complex. WAP-SVT transgenic animals exhibit an almost regular mammary gland development until late pregnancy but the majority of the ME-cells are eliminated by apoptosis during the early lactation period. Tumor formation is delayed and less efficient than in T/t-antigen positive animals. Sequestration of p53 and pRb by the N-terminal truncated T-antigen molecules (T1-antigen and T2-antigen) does not affect mammary gland differentiation and the transgenic animals (WAP-SVBst-Bam) do not develop breast tumors.

Hepatitis C virus NS5A physically associates with p53 and regulates p21/waf1 gene expression in a p53-dependent manner.

We have previously demonstrated that hepatitis C virus (HCV) NS5A protein promotes cell growth and transcriptionally regulates the p21/waf1 promoter, a downstream effector gene of p53. In this study, we investigated the molecular mechanism of NS5A-mediated transcriptional repression of p21/waf1. We observed that transcriptional repression of the p21/waf1 gene by NS5A is p53 dependent by using p53 wild-type (+/+) and null (-/-) cells. Interestingly, p53-mediated transcriptional activation from a synthetic promoter containing multiple p53 binding sites (PG13-LUC) was abrogated following expression of HCV NS5A. Additional studies using pull-down experiments, in vivo coimmunoprecipitation, and mammalian two-hybrid assays demonstrated that NS5A physically associates with p53. Confocal microscopy revealed sequestration of p53 in the perinuclear membrane and colocalization with NS5A in transfected HepG2 and Saos-2 cells. Together these results suggest that an association of NS5A and p53 allows transcriptional modulation of the p21/waf1 gene and may contribute to HCV-mediated pathogenesis.

Modulation of p27/Cdk2 complex formation through 4D5-mediated inhibition of HER2 receptor signaling

The molecular mechanisms mediating the anti-proliferative effects of the murine anti-HER2 monoclonal antibody (4D5) were investigated in HER2-overexpressing human carcinoma cell lines. Treatment with 4D5 resulted in a dramatic accumulation of BT-474 breast carcinoma cells in G1; concomitant with reduced expression of proteins involved in sequestration of the cyclin E/Cdk2 inhibitor protein p27, increased association of p27 with Cdk2 complexes and Cdk2 inactivation. No equivalent effects were observed in BT-474 cells treated with a control, non-inhibitory HER2 monoclonal antibody (FRP5) or in a HER2-overexpressing cell line insensitive to 4D5 treatment (MKN7 gastric carcinoma cells), confirming the relationship between these molecular changes and 4D5-mediated inhibition of proliferation. Increased p27 expression was also observed in 4D5-treated BT-474 cells; however an antisense approach demonstrated that this increase was not required for Cdk2 inactivation or establishment of the G1 block. These data suggest that 4D5 interferes with HER2 receptor signaling, resulting in downregulation of proteins involved in p27 sequestration. This causes release of p27, allowing binding and inhibition of cyclin E/Cdk2 complexes and inhibition of G1/S progression. This model was confirmed using a second 4D5-sensitive. HER2-overexpressing breast tumor line (SKBR3), and suggests that the dependency of a given tumor cell on elevated HER2-receptor signaling for the maintenance of p27 sequestration proteins may determine the clinical response to treatment with the humanized anti-HER2 monoclonal antibody Herceptin (trastuzumab).

Retinoic acid-induced apoptosis of the CHP134 neuroblastoma cell line is associated with nuclear accumulation of p53 and is rescued by the GDNF/Ret signal.

Neuroblastoma (NBL) is one of the most common solid malignancies in childhood and is derived from the sympathetic precursor cells. Although p53, a tumor suppressor, has been reported to be rarely mutated in NBLs, it is sequestered abnormally in the cytoplasm of the NBL cell. The mechanism and functional role of the abnormal intracellular localization of p53 remain unclear. Here, we established an in vitro system of apoptosis model using a NBL cell line CHP134 which also showed a cytoplasmic sequestration of p53. The treatment of the cells with 1 or 5 microM all-trans retinoic acid (RA) induced moderate neurite outgrowth followed by massive death of CHP134 cells by days 5 to 6. TUNEL staining showed that the cell death was due to apoptosis. Immunofluorescent stain demonstrated that p53 was strongly positive in the nucleus on day 5, which was accompanied with induction of p21WAF1. In addition, expression of caspase-3 was also increased during the cell death. Intriguingly, the RA treatment induced expression of Ret tyrosine kinase receptor in CHP134 cells. The addition of ligands, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN), inhibited apoptosis as well as nuclear accumulation of p53 in the cell. The present results suggest that the RA-induced apoptosis of NBL cells is associated with activation of both the caspase cascade and the p53-mediated pathway with its nuclear translocation. The neurotrophic signal through the GDNF-Ret system may prevent the neuronal cell death.

Disruption of TGF-beta growth inhibition by oncogenic ras is linked to p27Kip1 mislocalization.

Expression of oncogenic Ras in epithelial tumor cells is linked to the loss of transforming growth factor-beta (TGF-beta) anti-proliferative activity, and was proposed to involve inhibition of Smad2/3 nuclear translocation. Here we studied several epithelial cell lines expressing oncogenic N-RasK61 and show that TGF-beta-induced nuclear translocation of and transcriptional activation by Smad2/3 were unaffected. In contrast, oncogenic Ras mediated nuclearto-cytoplasmic mislocalization of p27KiP1 (p27) and of the cyclin-dependent kinase (CDK) CDK6, but not CDK2. Concomitantly, oncogenic Ras abrogated the ability of TGF-beta to release p27 from CDK6, to enhance its binding to CDK2 and to inhibit CDK2 activity. Inactivation of Ras by a specific antagonist restored the growth inhibitory response to TGF-beta with concurrent normalization of p27 and CDK6 localization. Therefore, the disruption of TGF-beta-mediated growth inhibition by oncogenic Ras appears to be due to lack of inhibition of CDK2, caused by the sequestration of p27 and CDK2 in different subcellular compartments and by the loss of TGF-beta-induced partner switching of p27 from CDK6 to CDK2.