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Biomedical subjects

M Oren

Publications and source records attributed to M Oren.

At least 37 records · Page 2Linked to original sources
PubMed2001

Down-regulation of beta-catenin by activated p53.

beta-Catenin is a cytoplasmic protein that participates in the assembly of cell-cell adherens junctions by binding cadherins to the actin cytoskeleton. In addition, it is a key component of the Wnt signaling pathway. Activation of this pathway triggers the accumulation of beta-catenin in the nucleus, where it activates the transcription of target genes. Abnormal accumulation of beta-catenin is characteristic of various types of cancer and is caused by mutations either in the adenomatous polyposis coli protein, which regulates beta-catenin degradation, or in the beta-catenin molecule itself. Aberrant accumulation of beta-catenin in tumors is often associated with mutational inactivation of the p53 tumor suppressor. Here we show that overexpression of wild-type p53, by either transfection or DNA damage, down-regulates beta-catenin in human and mouse cells. This effect was not obtained with transcriptionally inactive p53, including a common tumor-associated p53 mutant. The reduction in beta-catenin level was accompanied by inhibition of its transactivation potential. The inhibitory effect of p53 on beta-catenin is apparently mediated by the ubiquitin-proteasome system and requires an active glycogen synthase kinase 3beta (GSK3beta). Mutations in the N terminus of beta-catenin which compromise its degradation by the proteasomes, overexpression of dominant-negative DeltaF-beta-TrCP, or inhibition of GSKbeta activity all rendered beta-catenin resistant to down-regulation by p53. These findings support the notion that there will be a selective pressure for the loss of wild-type p53 expression in cancers that are driven by excessive accumulation of beta-catenin.

Animals↗
PubMed2001

Sonohysterography for the diagnosis of residual trophoblastic tissue.

OBJECTIVE: To assess the efficacy, safety, and associated complications of sonohysterography for the diagnosis of residual trophoblastic tissue. METHODS: We conducted a prospective study of 23 consecutive patients admitted to our ultrasonography unit with clinical and ultrasonographic signs of retained intrauterine tissue. RESULTS: Twelve patients had hydrosonographic features suggestive of residual trophoblastic tissue (i.e., an intrauterine lesion not detachable from the uterine wall after instillation of saline), whereas in 11 cases the hydrosonographic findings were negative for retained tissue. Blood flow was detected within abnormal intrauterine masses in 4 of 12 patients with trophoblastic tissue, whereas it was not detected in any patient without retained tissue (P = .093). No complications were encountered during the procedure or the postprocedure period. None of the patients had anesthetic complications, perforation of the uterus, fluid overload, or any other surgical complication. All 12 patients underwent hysteroscopic removal of the suspected residual trophoblastic tissue, and histologic confirmation of residual trophoblastic tissue was obtained in all cases. CONCLUSIONS: Sonohysterography for detection and diagnosis of residual trophoblastic tissue is an accurate and safe procedure. Further studies comparing the efficacy of sonohysterography with that of diagnostic hysteroscopy are warranted.

Abortion, Induced↗
PubMed2000

Degradation of the E7 human papillomavirus oncoprotein by the ubiquitin-proteasome system: targeting via ubiquitination of the N-terminal residue.

The E7 oncoprotein of the high risk human papillomavirus type 16 (HPV-16), which is etiologically associated with uterine cervical cancer, is a potent immortalizing and transforming agent. It probably exerts its oncogenic functions by interacting and altering the normal activity of cell cycle control proteins such as p21WAF1, p27KIP1 and pRb, transcriptional activators such as TBP and AP-1, and metabolic regulators such as M2-pyruvate kinase (M2-PK). Here we show that E7 is a short-lived protein and its degradation both in vitro and in vivo is mediated by the ubiquitin-proteasome pathway. Interestingly, ubiquitin does not attach to any of the two internal Lysine residues of E7. Substitution of these residues with Arg does not affect the ability of the protein to be conjugated and degraded; in contrast, addition of a Myc tag to the N-terminal but not to the C-terminal residue, stabilizes the protein. Also, deletion of the first 11 amino acid residues stabilizes the protein in cells. Taken together, these findings strongly suggest that, like MyoD and the Epstein Barr Virus (EBV) transforming Latent Membrane Protein 1 (LMPI), the first ubiquitin moiety is attached linearly to the free N-terminal residue of E7. Additional ubiquitin moieties are then attached to an internal Lys residue of the previously conjugated molecule. The involvement of E7 in many diverse and apparently unrelated processes requires tight regulation of its function and cellular level, which is controlled in this case by ubiquitin-mediated proteolysis.

Adenosine Triphosphate↗
PubMed2000

Opposing effects of Ras on p53: transcriptional activation of mdm2 and induction of p19ARF.

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. Here, we show that the mdm2 gene is also regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2.

Animals↗
PubMed2000

Generation of oscillations by the p53-Mdm2 feedback loop: a theoretical and experimental study.

The intracellular activity of the p53 tumor suppressor protein is regulated through a feedback loop involving its transcriptional target, mdm2. We present a simple mathematical model suggesting that, under certain circumstances, oscillations in p53 and Mdm2 protein levels can emerge in response to a stress signal. A delay in p53-dependent induction of Mdm2 is predicted to be required, albeit not sufficient, for this oscillatory behavior. In line with the predictions of the model, oscillations of both p53 and Mdm2 indeed occur on exposure of various cell types to ionizing radiation. Such oscillations may allow cells to repair their DNA without risking the irreversible consequences of continuous excessive p53 activation.

3T3 Cells↗
PubMed2000

Physical and functional interaction between p53 mutants and different isoforms of p73.

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro and in vivo with p73 alpha, beta, gamma, and delta. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

Breast Neoplasms↗
PubMed2000

Unmasking of phosphorylation-sensitive epitopes on p53 and Mdm2 by a simple Western-phosphatase procedure.

Monoclonal antibodies are widely used for the assessment of protein expression levels, protein-protein interactions and protein localization. Phosphorylation of one or more residues within an epitope recognized by a particular antibody may compromise the ability of that antibody to bind the target protein. Inhibition of immunoreactivity by phosphorylation has been reported for many antibody/protein pairs. Here we describe a simple convenient protocol for assessing the effect of phosphorylation on immunoreactivity, employing phosphatase treatment of Western blotted membranes. The efficacy of this protocol is demonstrated for p53 and for Mdm2. This method is useful for obtaining more uniform protein quantification, as well as for rapid assessment of changes in the extent of phosphorylation within a given epitope in response to defined signals.

Alkaline Phosphatase↗
PubMed2000

Behavioral alterations associated with apoptosis and down-regulation of presenilin 1 in the brains of p53-deficient mice.

Presenilin 1 (PS1) expression is repressed by the p53 tumor suppressor. As shown herein, wild-type PS1 is an effective antiapoptotic molecule capable of significantly inhibiting p53-dependent and p53-independent cell death. We analyzed, at the functional and molecular levels, the brains of p53 knockout mice. Surprisingly, we found that lack of p53 expression induces apoptotic brain lesions, accompanied by learning deficiency and behavioral alterations. p53-deficient mice show an unexpected overexpression of p21(waf1) with subsequent down-regulation of PS1 in their brains. This process is progressive and age-dependent. These data indicate that the p53 pathway, besides affecting tumor suppression, may play a major role in regulating neurobehavioral function and cell survival in the brain.

Animals↗
PubMed2000

The loss of mdm2 induces p53-mediated apoptosis.

The p53 tumor suppressor gene product is negatively regulated by the product of its downstream target, mdm2. The deletion of mdm2 in the mouse results in embryonic lethality at 5.5 days post coitum (d.p.c.) which can be overcome by simultaneous loss of the p53 tumor suppressor, substantiating the importance of the negative regulatory function of MDM2 on p53 function in vivo. Hence, the loss of MDM2 allowed the unregulated p53 protein to continuously exert its growth-suppressing activity, which either led to a complete G1 arrest or induced the p53-dependent apoptotic pathway, resulting in the death of the mdm2-/- embryos. To determine which of these possibilities is occurring, mouse embryo fibroblasts (MEFs) from p53 null and p53/mdm2 double null embryos were transfected with a retroviral vector carrying a temperature-sensitive p53 (tsp53) cDNA. Shifting of single-cell clonal populations to the permissive temperature caused the p53-/-mdm2-/- fibroblasts expressing tsp53 to undergo apoptosis in a dose-dependent manner. This phenotype was not observed in the tsp53 expressing p53-/- clones nor the parental cell lines. Thus, our data indicate that the simple loss of mdm2 can induce the p53-dependent apoptotic pathway in vivo.

Animals↗
PubMed2000

The Werner syndrome protein contributes to induction of p53 by DNA damage.

Mutations in the p53 tumor-suppressor gene promote increased genomic instability and cancer. Mutations in the WRN gene, encoding a DNA helicase, underlie the segmental progeroid Werner syndrome (WS). WS is also associated with increased genomic instability and elevated cancer risk. The p53 and WRN proteins can engage in direct protein-protein interactions. We report that excess WRN elicits increased cellular p53 levels and potentiates p53-mediated apoptosis. Importantly, cells derived from WS patients exhibit an attenuated and delayed induction of p53 by UV or by the topoisomerase I inhibitor camptothecin. These results suggest that WRN may participate in the activation of p53 in response to certain types of DNA damage. Furthermore, the failure to induce p53 effectively may contribute to enhanced genomic instability and elevated cancer risk in WS patients.

Cells, Cultured↗
PubMed2000

Sonographic and clinical characteristics of struma ovarii.

Our objective was to evaluate the preoperative clinical, laboratory, and sonographic characteristics of struma ovarii in comparison to ovarian dermoid cysts. A retrospective review of gynecologic patients operated on for mature cystic teratoma over a 10 year period identified 12 cases of struma ovarii. These cases, combined with 4 additional cases from an earlier report, were the subject of this study. Results were compared to 32 cases of ovarian dermoid cysts. Ovarian struma ovarii occurred in 12 (4.8%) of 251 cases of ovarian dermoid cysts. Most patients were premenopausal, and the mean lesion diameter was 57.3 mm (range, 30-95 mm). Struma ovarii occurred more frequently (68.8%) in the right adnexa and was seen with a normal CA-125 level. Blood flow, assessed by Doppler ultrasonography, was located in the center of the lesion in all cases of struma ovarii. Rare cases were seen with elevated tumor markers and low resistance blood flow. With regard to dermoid cysts, blood flow had a higher resistive index. In addition, no blood flow could be detected from the center of the echoic lesion in dermoid cysts (P < 0.0001). In summary, it is difficult to distinguish between struma ovarii and dermoid cysts on the basis of their sonographic appearance. Nevertheless, Doppler flow may aid in the preoperative diagnosis of struma ovarii. Blood flow signals, detected from the center of the echoic lesion, and low resistance to flow may be more common in struma ovarii.

Adult↗
PubMed1999

Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage.

The p53 tumor-suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. This process is associated with posttranslational modifications of p53, some of which are mediated by the ATM protein kinase. However, these modifications alone may not account in full for p53 stabilization. p53's stability and activity are negatively regulated by the oncoprotein MDM2, whose gene is activated by p53. Conceivably, p53 function may be modulated by modifications of MDM2 as well. We show here that after treatment of cells with ionizing radiation or a radiomimetic chemical, but not UV radiation, MDM2 is phosphorylated rapidly in an ATM-dependent manner. This phosphorylation is independent of p53 and the DNA-dependent protein kinase. Furthermore, MDM2 is directly phosphorylated by ATM in vitro. These findings suggest that in response to DNA strand breaks, ATM may promote p53 activity and stability by mediating simultaneous phosphorylation of both partners of the p53-MDM2 autoregulatory feedback loop.

Alkaline Phosphatase↗
PubMed1999

Physical and functional interaction between p53 and the Werner's syndrome protein.

Werner's syndrome is a human autosomal recessive disorder leading to premature aging. The mutations responsible for this disorder have recently been localized to a gene (WRN) encoding a protein that possesses DNA helicase and exonuclease activities. Patients carrying WRN gene mutations exhibit an elevated rate of cancer, accompanied by increased genomic instability. The latter features are also characteristic of the loss of function of p53, a tumor suppressor that is very frequently inactivated in human cancer. Moreover, changes in the activity of p53 have been implicated in the onset of cellular replicative senescence. We report here that the WRN protein can form a specific physical interaction with p53. This interaction involves the carboxyl-terminal part of WRN and the extreme carboxyl terminus of p53, a region that plays an important role in regulating the functional state of p53. A small fraction of WRN can be found in complex with endogenous p53 in nontransfected cells. Overexpression of WRN leads to augmented p53-dependent transcriptional activity and induction of p21(Waf1) protein expression. These findings support the existence of a cross-talk between WRN and p53, which may be important for maintaining genomic integrity and for preventing the accumulation of aberrations that can give rise to premature senescence and cancer.

Aging, Premature↗
PubMed1999

SIAH-1 promotes apoptosis and tumor suppression through a network involving the regulation of protein folding, unfolding, and trafficking: identification of common effectors with p53 and p21(Waf1).

We have previously described biological model systems for studying tumor suppression in which, by using H-1 parvovirus as a selective agent, cells with a strongly suppressed malignant phenotype (KS or US) were derived from malignant cell lines (K562 or U937). By using cDNA display on the K562/KS cells, 15 cDNAs were now isolated, corresponding to genes differentially regulated in tumor suppression. Of these, TSAP9 corresponds to a TCP-1 chaperonin, TSAP13 to a regulatory proteasome subunit, and TSAP21 to syntaxin 11, a vesicular trafficking molecule. The 15 cDNAs were used as a molecular fingerprint in different tumor-suppression models. We found that a similar pattern of differential regulation is shared by activation of p53, p21(Waf1), and the human homologue of Drosophila seven in absentia, SIAH-1. Because SIAH-1 is differentially expressed in the various models, we characterized it at the protein and functional levels. The 32-kDa, mainly nuclear protein encoded by SIAH-1, can induce apoptosis and promote tumor suppression. These results suggest the existence of a common mechanism of tumor suppression and apoptosis shared by p53, p21(Waf1), and SIAH-1 and involving regulation of the cellular machinery responsible for protein folding, unfolding, and trafficking.

Animals↗
PubMed1999

Interaction of c-Abl and p73alpha and their collaboration to induce apoptosis.

c-Abl, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of c-Abl. p73, a member of the p53 family of tumour-suppressor proteins, can also induce apoptosis. Here we show that the apoptotic activity of p73alpha requires the presence of functional, kinase-competent c-Abl. Furthermore, p73 and c-Abl can associate with each other, andthis binding is mediated by a PxxP motif in p73 and the SH3 domain of c-Abl. We find that p73 is a substrate of the c-Abl kinase and that the ability of c-Abl to phosphorylate p73 is markedly increased by gamma-irradiation. Moreover, p73 is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signalling pathway involving p73 and c-Abl.

3T3 Cells↗
PubMed1999

Excess beta-catenin promotes accumulation of transcriptionally active p53.

beta-catenin is a multifunctional protein, acting both as a structural component of the cell adhesion machinery and as a transducer of extracellular signals. Deregulated beta-catenin protein expression, due to mutations in the beta-catenin gene itself or in its upstream regulator, the adenomatous polyposis coli (APC) gene, is prevalent in colorectal cancer and in several other tumor types, and attests to the potential oncogenic activity of this protein. Increased expression of beta-catenin is an early event in colorectal carcinogenesis, and is usually followed by a later mutational inactivation of the p53 tumor suppressor. To examine whether these two key steps in carcinogenesis are interrelated, we studied the effect of excess beta-catenin on p53. We report here that overexpression of beta-catenin results in accumulation of p53, apparently through interference with its proteolytic degradation. This effect involves both Mdm2-dependent and -independent p53 degradation pathways, and is accompanied by augmented transcriptional activity of p53 in the affected cells. Increased p53 activity may provide a safeguard against oncogenic deregulation of beta-catenin, and thus impose a pressure for mutational inactivation of p53 during the later stages of tumor progression.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Mutations in serines 15 and 20 of human p53 impair its apoptotic activity.

Phosphorylation of the p53 tumor suppressor protein is likely to play an important role in regulating its activity. To study the regulatory role of potential phosphorylation sites within the N-terminal transactivation domain of human p53 (hp53), a series of p53 serine mutants were evaluated for transcriptional transactivation and sequence specific DNA binding. The role of these mutations in regulating p53-mediated growth suppression and programmed cell death was examined. This mutational analysis comprised serine residues located at positions 6, 9, 15, 20, 33 and 37 of human p53. Substitution of serine for alanine, either at individual residues or at all six residues together, did not affect the suppression of cell growth and cell transformation, or the ability to bind DNA specifically and to transactivate different promoters, nor did it alter p53 expression. However, the ability of p53 to induce apoptosis was impaired by specific serine substitutions. Mutations in all six N-terminal serines together reduced the apoptotic activity of p53 in H1299 cells by 50%. Analysis of individual mutants revealed that mutations in serine 15 and 20 are primarily responsible for this impairment. Our results suggest that these serines play a role in the regulation of p53-mediated apoptosis.

Alanine↗