E Moallem

Publications (10)48.99 Total impact

• Article: Transactivation-deficient p73alpha (p73Deltaexon2) inhibits apoptosis and competes with p53.

  I Fillippovich, N Sorokina, M Gatei, Y Haupt, K Hobson, E Moallem, K Spring, M Mould, M A McGuckin, M F Lavin, K K Khanna
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  ABSTRACT: p73 has recently been identified as a structural and functional homolog of the tumor suppressor protein p53. Overexpression of p53 activates transcription of p53 effector genes, causes growth inhibition and induced apoptosis. We describe here the effects of a tumor-derived truncated transcript of p73alpha (p73Deltaexon2) on p53 function and on cell death. This transcript, which lacks the acidic N-terminus corresponding to the transactivation domain of p53, was initially detected in a neuroblastoma cell line. Overexpression of p73Deltaexon2 partially protects lymphoblastoid cells against apoptosis induced by anti-Fas antibody or cisplatin. By cotransfecting p73Deltaexon2 with wild-type p53 in the p53 null line Saos 2, we found that this truncated transcript reduces the ability of wild-type p53 to promote apoptosis. This anti-apoptotic effect was also observed when p73Deltaexon2 was co-transfected with full-length p73 (p73alpha). This was further substantiated by suppression of p53 transactivation of the effector gene p21/Waf1 in p73Deltaexon2 transfected cells and by inhibition of expression of a reporter gene under the control of the p53 promoter. Thus, this truncated form of p73 can act as a dominant-negative agent towards transactivation by p53 and p73alpha, highlighting the potential implications of these findings for p53 signaling pathway. Furthermore, we demonstrate the existence of a p73Deltaexon2 transcript in a very significant proportion (46%) of breast cancer cell lines. However, a large spectrum of normal and malignant tissues need to be surveyed to determine whether this transdominant p73 variant occurs in a tumor-specific manner.
  Oncogene 02/2001; 20(4):514-22. · 6.37 Impact Factor
• Article: Mutations in serines 15 and 20 of human p53 impair its apoptotic activity.

  T Unger, R V Sionov, E Moallem, C L Yee, P M Howley, M Oren, Y Haupt
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  ABSTRACT: 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.
  Oncogene 06/1999; 18(21):3205-12. · 6.37 Impact Factor
• Source

  Available from: nih.gov

  Article: Critical role for Ser20 of human p53 in the negative regulation of p53 by Mdm2.

  T Unger, T Juven-Gershon, E Moallem, M Berger, R Vogt Sionov, G Lozano, M Oren, Y Haupt
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  ABSTRACT: In response to environmental stress, the p53 phosphoprotein is stabilized and activated to inhibit cell growth. p53 stability and activity are negatively regulated by the murine double minute (Mdm2) oncoprotein in an autoregulatory feedback loop. The inhibitory effect of Mdm2 on p53 has to be tightly regulated for proper p53 activity. Phosphorylation is an important level of p53 regulation. In response to DNA damage, p53 is phosphorylated at several N-terminal serines. In this study we examined the role of Ser20, a potential phosphorylation site in human p53, in the regulation of p53 stability and function. Substitution of Ser20 by Ala (p53-Ala20) significantly increases the susceptibility of human p53 to negative regulation by Mdm2 in vivo, as measured by apoptosis and transcription activation assays. Mutation of Ser20 to Ala renders p53 less stable and more prone to Mdm2-mediated degradation. While the in vitro binding of p53 to Mdm2 is not increased by the Ala20 mutation, the same mutation results in a markedly enhanced binding in vivo. This is consistent with the conclusion that phosphorylation of Ser20 in vivo attenuates the binding of wild-type p53 to Mdm2. Peptides bearing non-phosphorylated Ser20 or Ala20 compete with p53 for Mdm2 binding, while a similar peptide with phosphorylated Ser20 does not. This implies a critical role for Ser20 in modulating the negative regulation of p53 by Mdm2, probably through phosphorylation-dependent inhibition of p53-Mdm2 interaction.
  The EMBO Journal 05/1999; 18(7):1805-14. · 9.20 Impact Factor
• Article: c-Abl neutralizes the inhibitory effect of Mdm2 on p53.

  R V Sionov, E Moallem, M Berger, A Kazaz, O Gerlitz, Y Ben-Neriah, M Oren, Y Haupt
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  ABSTRACT: Upon exposure to stress signals, the p53 tumor suppressor protein is stabilized and induces growth suppression. p53 activities are efficiently inhibited by the Mdm2 oncoprotein through an autoregulatory feedback loop. In addition, Mdm2 promotes p53 degradation, thereby terminating its growth inhibitory signal. Hence, p53 exerts its effects during the interval between p53 activation and the subsequent inhibition by Mdm2. Modulation of this interval by regulatory proteins may determine the extent and duration of p53 activity. Recent studies have shown that the c-Abl protein-tyrosine kinase binds p53 and enhances its transcriptional activity. Here we provide an explanation for the cooperation between these proteins. We demonstrate that c-Abl increases the expression level of the p53 protein. The enhanced expression is achieved by inhibiting Mdm2-mediated degradation of p53. This provides a likely mechanistic explanation for the findings that c-Abl overcomes the inhibitory effects of Mdm2 on p53-mediated transcriptional activation and apoptosis. These results suggest that c-Abl modulates the time window within which p53 remains active. The ability of c-Abl to neutralize the inhibitory effects of Mdm2 on p53 may be important for its growth inhibitory function.
  Journal of Biological Chemistry 04/1999; 274(13):8371-4. · 4.77 Impact Factor
• Article: RNA-Protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate.

  E Moallem, R Kilav, J Silver, T Naveh-Many
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  ABSTRACT: Parathyroid hormone (PTH) regulates serum calcium and phosphate levels, which, in turn, regulate PTH secretion and mRNA levels. PTH mRNA levels are markedly increased in rats fed low calcium diets and decreased after low phosphate diets, and this effect is post-transcriptional. Protein-PTH mRNA binding studies, with parathyroid cytosolic proteins, showed three protein-RNA bands. This binding was to the 3'-untranslated region (UTR) of the PTH mRNA and was dependent upon the terminal 60 nucleotides. Parathyroid proteins from hypocalcemic rats showed increased binding, and proteins from hypophosphatemic rats decreased binding, correlating with PTH mRNA levels. There is no parathyroid cell line; however, a functional role was provided by an in vitro degradation assay. Parathyroid proteins from control rats incubated with a PTH mRNA probe led to an intact transcript for 40 min; the transcript was intact with hypocalcemic proteins for 180 min and with hypophosphatemic proteins only for 5 min. A PTH mRNA probe without the 3'-UTR, or just the terminal 60 nucleotides, incubated with hypophosphatemic proteins, showed no degradation at all, indicating that the sequences in the 3'-UTR determine PTH mRNA degradation. Hypocalcemia and hypophosphatemia regulate PTH gene expression post-transcriptionally. This correlates with binding of proteins to the PTH mRNA 3'-UTR, which determines its stability.
  Journal of Biological Chemistry 03/1998; 273(9):5253-9. · 4.77 Impact Factor
• Article: Regulation of the parathyroid hormone gene by calcium, phosphate and 1,25-dihydroxyvitamin D.

  J Silver, E Moallem, R Kilav, A Sela, T Naveh-Many
  Nephrology Dialysis Transplantation 01/1998; 13 Suppl 1:40-4. · 3.40 Impact Factor
• Source

  Available from: oxfordjournals.org

  Article: New insights into the regulation of parathyroid hormone synthesis and secretion in chronic renal failure.

  J Silver, E Moallem, R Kilav, E Epstein, A Sela, T Naveh-Many
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  ABSTRACT: The main factors which regulate parathyroid hormone (PTH) production are calcium, phosphate, vitamin D and the sex steroids, estrogens and progestagins. Hypocalcaemia leads to increased PTH secretion in seconds and minutes, gene expression in hours and parathyroid cell number in weeks and months. Hypercalcaemia leads to a decrease in PTH secretion by its action on the parathyroid cell calcium receptor and no decrease in PTH mRNA concentrations. There is now convincing evidence that phosphate regulates the parathyroids independent of its effect on serum calcium and 1,25-dihydroxyvitamin D3. (1,25(OH)2D3). In vivo in rats hypophosphataemia markedly decreases PTH mRNA and serum PTH independent of its effect on serum calcium and 1,25(OH)2D3. Clinical studies also indicate that phosphate regulates the parathyroids independent of its effect on serum calcium and 1,25(OH)2D3 1,25(OH)2D3 itself has a marked effect on the parathyroids where it decreases PTH gene transcription by a direct action. Parathyroid cell proliferation is regulated by dietary calcium and phosphate with hypocalcaemia markedly increasing and hypophosphataemia markedly decreasing the number of proliferating cells. The application of basic science findings of how calcium, phosphate and 1,25(OH)2D3 regulate the parathyroids has led to an efficient and safe prescription for the management of the secondary hyperparathyroidism of chronic renal failure which is the maintenance of a normal serum calcium and phosphate and the careful use of bolus doses of 1,25(OH)2D3.
  Nephrology Dialysis Transplantation 02/1996; 11 Suppl 3:2-5. · 3.40 Impact Factor
• Article: Effects of passive immunization against parathyroid hormone-related protein: PTHrP is the responsible factor in mediating hypercalcemia in the Walker carcinosarcoma 256 rat model.

  T Schilling, E Blind, R Baier, H P Sinn, E Moallem, J Silver, R Ziegler, F Raue
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  ABSTRACT: The Walker carcinosarcoma (WCS) 256 is a well-characterized rat model of humoral hypercalcemia of malignancy (HHM). We addressed the question of whether parathyroid hormone-related protein (PTHrP) is the factor responsible for mediating HHM in this model. WCS 256 cells were subcutaneously implanted in female rats. We examined the plasma at days 0, 2, 4, 6, and 8. The midregional PTHrP measured by radioimmunoassay (RIA) and the plasma calcium increased significantly. Measuring PTHrP by a two-site immunoradiometric assay (IRMA) showed comparable results. There was a strong positive correlation between plasma calcium and midregional PTHrP (r = 0.85, p < 0.0001). A strong positive correlation between tumor weight and both midregional PTHrP (r = 0.83, p < 0.0001) and plasma calcium (r = 0.87, p < 0.0001) was also found. After surgical removal of the tumor at day 5, both plasma calcium and plasma PTHrP levels fell to within the normal range. Ip administration of native polyclonal antiserum against PTHrP(53-84) led to a significant decrease of plasma calcium. Extracted WCS 256 tumor showed 5-fold increased levels of midregional PTHrP compared with liver. Immunohistochemistry and Western blot were positive for PTHrP. RNA from the WCS 256 tumor was positive for PTHrP whereas liver tissue RNA was negative. WCS 256 cells grown in vitro also secreted PTHrP into the medium. We conclude that PTHrP is synthesized and secreted by WCS 256 and that PTHrP is the factor responsible for mediating hypercalcemia in the WCS 256 rat model.
  Journal of Bone and Mineral Research 01/1995; 10(1):7-16. · 6.37 Impact Factor
• Article: New aspects in the control of parathyroid hormone secretion.

  J Silver, E Moallem, E Epstein, R Kilav, T Naveh-Many
  [show abstract] [hide abstract]
  ABSTRACT: Ca2+ binds to a parathyroid cell Ca2+ receptor, which is G protein-coupled and activates inositol triphosphate production. Mutations in the Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Chronic hypocalcemia increases parathyroid hormone messenger RNA levels and parathyroid cell hyperplasia. Parathyroid cells in vitro are heterologous in their response to Ca2+. The concept of a higher Ca2+ set-point in secondary hyperparathyroidism is controversial. Calcitriol is more effective than the less hypercalcemia analogues in decreasing parathyroid hormone messenger RNA and immunoreactive parathyroid hormone levels, and its kinetics are well established. Phosphate and estrogens regulate the parathyroid independently of 1,25 dihydroxyvitamin D3 and Ca2+. The physiology of the effects of endothelin and insulin-like growth factors on the parathyroid need to be established. Important advances are being made in understanding the regulation of parathyroid hormone synthesis and secretion, which are relevant to both normal physiology and the pathogenesis and treatment of diseases such as the secondary hyperparathyroidism of renal failure and osteoporosis.
  Current Opinion in Nephrology and Hypertension 08/1994; 3(4):379-85. · 4.33 Impact Factor
• Article: Transactivation-deficient p73 alpha (p73 Delta exon2) inhibits apoptosis and competes with p53

  I Fillippovich, N Sorokina, M Gatei, Y Haupt, K Hobson, E Moallem, K Spring, M Mould, MA McGuckin, MF Lavin, KK Khanna
  [show abstract] [hide abstract]
  ABSTRACT: p73 has recently been identified as a structural and functional homolog of the tumor suppressor protein p53. Overexpression of p53 activates transcription of p53 effector genes, causes growth inhibition and induced apoptosis. We describe here the effects of a tumor-derived truncated transcript of p73 alpha (p73 Delta exon2) on p53 function and on cell death. This transcript, which lacks the acidic N-terminus corresponding to the transactivation domain of p53, was initially detected in a neuroblastoma cell line. Overexpression of p73 Delta exon2 partially protects lymphoblastoid cells against apoptosis induced by anti-Fas antibody or cisplatin. By cotransfecting p73 Delta exon2 with wild-type p53 in the p53 null line Saos 2, we found that this truncated transcript reduces the ability of wild-type p53 to promote apoptosis. This anti-apoptotic effect was also observed when p73 Delta exon2 was co-transfected with full-length p73 (p73 alpha). This was further substantiated by suppression of p53 transactivation of the effector gene p21-Waf1 in p73 Delta exon2 transfected cells and by inhibition of expression of a reporter gene under the control of the p53 promoter. Thus, this truncated form of p73 can act as a dominant-negative agent towards transactivation by p53 and p73 alpha, highlighting the potential implications of these findings for p53 signaling pathway. Furthermore, we demonstrate the existence of a p73 Delta exon2 transcript in a very significant proportion (46%) of breast cancer cell lines. However, a large spectrum of normal and malignant tissues need to be surveyed to determine whether this transdominant p73 variant occurs in a tumor-specific manner.