From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 1998 92: 2977-2979 A Functional Wild-Type p53 Gene Is Expressed in Human Acute Myeloid Leukemia Cell Lines Trenna Sutcliffe, Loning Fu, Jacinth Abraham, Homayoun Vaziri and Samuel Benchimol Updated information and services can be found at: http://www.bloodjournal.org/content/92/8/2977.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on October 21, 2014. For personal use only. CORRESPONDENCE 2977 REFERENCES 1. Ashkin A, Dziedzic JM, Bjorkholm JE, Chu S: Observation of a single-beam gradient force optical trap for dielectric particles. Optic Lett 11:288, 1986 2. Ashkin A, Dziedzic JM: Optical trapping and manipulation of viruses and bacteria. Science 235:1517, 1987 3. Bronkhorst PJ, Streekstra GJ, Grimbergen J, Nijhof EJ, Sixma JJ, Brakenhoff GJ: A new method to study shape recovery of red blood cells using multiple optical trapping. Biophys J 69:1666, 1995 4. Huruta RR, Barjas-Castro ML, Saad STO, Costa FF, Cezar CL: A new method to study mechanical properties of red blood cells using optical tweezers. Blood 90:6a, 1997 (abstr, suppl 1) 5. Sutera SP, Gardner RA, Boylan CW, Carroll GL, Chang KC, Marvel JS, Kilo C, Gonen B, Williamson JR: Age related changes in deformability of human erythrocytes. Blood 65:275, 1985 6. Beutler E, West C: The storage of hard-packed red blood cells in citrate-phosphate-dextrose (CPD) and CPD-adenine (CPDA-1). Blood 54:280, 1970 7. Card RT, Mohandas N, Perkins HA, Shohet SB: Deformability of stored red cells. Relationship to degree of packing. Transfusion 22:96, 1982 8. Wolf LC: The membrane and lesions of storage in preserved red cells. Transfusion 25:185, 1985 A Functional Wild-Type p53 Gene Is Expressed in Human Acute Myeloid Leukemia Cell Lines To the Editor: p53 mutations have been detected in about 10% of all acute myeloid leukemia (AML) patients, mostly in patients with 17p monosomy.1-3 The scarcity of p53 mutations in AML could mean that, in the vast majority of AML patients, loss of p53 protein function is not required for the development of this disease. Alternatively, it is possible that inactivation of the p53 growth regulatory pathway is important and that this can occur either through disruption of downstream effector molecules or through epigenetic mechanisms that regulate p53 protein function. It has been suggested, for example, that inactivation of wild-type p53 protein in AML occurs through a mechanism involving conformational change of the protein4,5 or through binding to MDM2 protein.6-8 We have examined the functional status of the wild-type p53 protein expressed in cell lines derived from AML blasts on the basis of site-specific DNA binding activity, transactivation of p53-responsive genes, and ability to promote cell cycle arrest in G1 in response to g-irradiation.9 The first two properties of p53 protein are strongly associated with its tumor suppressor function.10,11 A Nucleotide sequence analysis of the entire p53 coding region in four p53-expressing AML cell lines (OCI/AML-2, -3, -4, and -5) 12 showed wild-type sequence. The site-specific DNA binding activity of p53 protein expressed in OCI/AML-3 and OCI/AML-5 cells was examined using an electrophoretic mobility shift assay (EMSA). Nuclear protein extracts were prepared from g-irradiated or untreated cells and mixed with a 32P-labeled double-stranded oligonucleotide containing a p53 binding consensus sequence, p53CON.13 DNA damage increases the intracellular concentration of p53 protein and is also believed to activate the latent, sequence-specific DNA binding activity of p53. Whereas little, if any, DNA binding activity was detected in the nonirradiated extracts, the formation of a p53:DNA complex was evident when extracts were prepared from irradiated cells (Fig 1). Inclusion of the p53-specific monoclonal antibody PAb421 in the binding reaction resulted in a supershifted p53:DNA complex and served to confirm the presence of p53 protein in the protein:DNA complex. DNA binding was not observed when an extract from the p53-negative cell line Lan1 was used in the EMSA. B Fig 1. DNA binding activity of p53 protein in AML cell lines. Nuclear extracts prepared from untreated or g-irradiated OCI/AML-5 (A) and OCI/AML-3 (B) cells were incubated with a 32P-labeled double-stranded oligonucleotide containing the p53 consensus sequence (p53CON) with (1) or without (2) the p53-specific monoclonal antibody PAb421 and analyzed by EMSA. Lan1 cells, which lack p53 protein, were used as a negative control. The OCI/AML-5 and Lan1 extracts were prepared 3 hours after g-irradiation with a dose of 6 Gy. The OCI/AML-3 extracts were prepared at the times indicated after g-irradiation with a dose of 2 Gy. The arrow labeled B points to the p53:DNA complex, and the arrow labeled A points to the supershifted antibody:p53:DNA complex. From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 2978 CORRESPONDENCE Activation of p21WAF1 gene transcription after g-irradiation depends on wild-type p53 protein, and the p21WAF1 gene has been proposed to be a critical downstream effector in the p53-specific pathway of growth control in mammalian cells.14-17 Northern blot analysis (Fig 2) indicated that the basal level of p21WAF1 mRNA was ninefold higher in OCI/AML-5 than in the mutant p53-expressing human erythroleukemia cell line OCIM2. Furthermore, 3.5 hours after irradiation with 6 Gy, p21WAF1 mRNA levels increased ninefold in OCI/AML-5 and about threefold in OCIM2 cells. Irradiated OCI/AML-5 cells contained about 30-fold more p21WAF1 mRNA than did irradiated OCIM2 cells. No further increase in p21WAF1 mRNA levels was noted at later times after irradiation. p21WAF1 induction was also observed in irradiated OCI/ AML-3 and OCI/AML-4 cell lines.18 The mRNA levels for GADD45 and MDM2, two other genes known to be transcriptionally regulated by p53 in response to DNA damage, also increased after g-irradiation of AML cell lines (data not shown). OCI/AML-3, OCI/AML-5, and OCIM2 cells were irradiated with a dose of 6 Gy and cell proliferation was assessed 16 hours later by propidium iodide staining and flow cytometry. Both OCI/AML-3 and OCI/AML-5 cells were blocked in the G1 and G2 phases of the cell cycle with little, if any, cells present in S phase. In contrast, the mutant p53-expressing OCIM2 cells accumulated in G2 and showed no evidence of a block in G1 (Fig 3). The failure of OCIM2 cells to arrest in G1 after g-irradiation suggests that the G1 cell cycle block observed in irradiated OCI/AML-3 and OCI/AML-5 cells is likely to be dependent on functional p53 protein. Irradiation-induced G1 arrest was confirmed by dual-parameter flow cytometry after pulse labeling cells with BrdU and staining for DNA content with propidium iodide and for BrdU incorporation with a fluorescein isothiocyanate (FITC)-conjugated antibody for BrdU. OCI/AML-3 and OCI/AML-5 showed a ninefold and sixfold increase in the G1:S ratio 16 hours after g-irradiation (6 Gy), respectively. An increase in the G1:S ratio provides a good indicator of G1 delay. Our results indicate that p53 function (DNA binding, transactivation, and G1 checkpoint) is not lost during the development of AML or in the establishment of these AML cell lines. Functional p53 protein has also been demonstrated in human neuroblastoma,19 non-Hodgkin’s lymphoma,20 and even in certain HPV-positive cancer Fig 3. Cell cycle changes in AML cells after exposure to g-irradiation (6 Gy). The DNA content was determined by staining the cells with propidium iodide and the resulting profiles resulting from propidium iodide fluorescence are shown. For the irradiated cells, the cell cycle analyses were performed 16 hours after irradiation. OCIM2 cells were used as a control. cell lines21 that contain wild-type p53 alleles. Hence, loss of p53 function or inactivation of the p53-dependent growth arrest pathway is not required for the development of certain malignancies, including AML. A B Fig 2. Expression of p21WAF1 mRNA in g-irradiated OCI/AML-5 cells. Samples of total RNA (20 mg) prepared from cells at different times after exposure to 6 Gy of g-irradiation were fractioned on an agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized sequentially with 32P-labeled probes for human p21WAF1 cDNA (A) and 18S ribosomal RNA (B). OCIM2 cells, which express mutant p53 protein, were used as a control. Signal intensities were quantitated on a phosphorimager. The ratio of the p21WAF1 RNA signal to the 18S ribosomal RNA signal in the OCIM2 sample (0 hours) was arbitrarily set to 1.0 and the normalized values of p21WAF1 mRNA are shown at the bottom of (A). From www.bloodjournal.org by guest on October 21, 2014. For personal use only. CORRESPONDENCE 2979 ACKNOWLEDGMENT Supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada. Trenna Sutcliffe Loning Fu Jacinth Abraham Homayoun Vaziri Samuel Benchimol Division of Cellular/Molecular Biology Ontario Cancer Institute Princess Margaret Hospital Department of Medical Biophysics University of Toronto Toronto, Ontario, Canada REFERENCES 1. Lai JL, Preudhomme C, Zandecki M, Flactif M, Vanrumbeke M, Lepelley P, Wattel E, Fenaux P: Myelodysplastic syndromes and acute myeloid leukemia with 17p deletion. An entity characterized by specific dysgranulopoiesis and a high incidence of p53 mutation. Leukemia 9:370, 1995 2. Fenaux P, Jonveaux P, Quiquandon I, Lai JL, Pignon JM, LoucheuxLefebvre MH, Bauters F, Berger R, Kerckaert JP: p53 gene mutations in acute myeloid leukemia with 17p monosomy. Blood 78:1652, 1991 3. Soenen V, Preudhomme C, Rournier C, Daudignon A, Lai JL, Fenaux P: 17p deletion in acute myeloid leukemia with myelodysplastic syndrome. Analysis of breakpoints and deleted segments by fluorescence in situ. Blood 91:1008, 1998 4. Zhu YM, Bradbury D, Russell N: Expression of different conformations of p53 in the blast cells of acute myeloblastic leukaemia is related to in vitro growth characteristics. Br J Cancer 68:851, 1993 5. Zhang W, Hu G, Estey E, Hester J, Deisseroth A: Altered conformation of the p53 protein in myeloid leukemia cells and mitogen-stimulated normal blood cells. Oncogene 7:1645, 1992 6. Bueso-Ramos CE, Yang Y, deLeon E, McCowan P, Stass SA, Albitar M: The human MDM-2 oncogene is overexpressed in leukemias. Blood 82:2617, 1993 7. Watanabe T, Ichikawa A, Saito H, Hotta T: Overexpression of the MDM2 oncogene in leukemia and lymphoma. Leuk Lymphoma 21:391, 1996 8. Seliger B, Papadileris S, Vogel D, Hess G, Brendel C, Storkel S, Ortel J, Kolbe K, Huber C, Huhn D, Neubauer A: Analysis of the p53 and MDM-2 gene in acute myeloid leukemia. Eur J Haematol 57:230, 1996 9. Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB: Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci USA 89:7491, 1992 10. Pietenpol JA, Tokino T, Thiagalingam S, El-Deiry WS, Kinzler KW, Vogelstein B: Sequence-specific transcriptional activation is essential for growth suppression by p53. Proc Natl Acad Sci USA 91:1998, 1994 11. Crook T, Marston NJ, Sara EA, Vousden KH: Transcriptional activation by p53 correlates with suppression of growth but not transformation. Cell 79:817, 1994 12. Yang GS, Minden MD, McCulloch EA: Influence of schedule on regulated sensitivity of AML blasts to cytosine arabinoside. Leukemia 7:1012, 1993 13. Funk WD, Pak DT, Karas RH, Wright WE, Shay JW: A transcriptionally active DNA-binding site for human p53 protein complexes. Mol Cell Biol 12:2866, 1992 14. Di Leonardo A, Linke SP, Clarkin K, Wahl GM: DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev 8:2540, 1994 15. El-Deiry WS, Harper JW, O’Connor PM, Velculescu VE, Canman CE, Jackman J, Pietenpol JA, Burrell M, Hill DE, Wang Y, Wiman KG, Mercer WE, Kastan MB, Kohn KW, Elledge SJ, Kinzler KW, Vogelstein B: WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 54:1169, 1994 16. Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, Elledge SJ, Reed SI: p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76:1013, 1994 17. Slebos RJ, Lee MH, Plunkett BS, Kessis TD, Williams BO, Jacks T, Hedrick L, Kastan MB, Cho KR: p53-dependent G1 arrest involves pRB-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein. Proc Natl Acad Sci USA 91:5320, 1994 18. Fu L, Benchimol S: Participation of the human p53 38UTR in translational repression and activation following g-irradiation. EMBO J 16:4117, 1997 19. Goldman SC, Chen CY, Lansing TJ, Gilmer TM, Kastan MB: The p53 signal transduction pathway is intact in human neuroblastoma despite cytoplasmic localization. Am J Pathol 148:1381, 1996 20. Maestro R, Gloghini A, Doglioni C, Piccinin S, Vukosavljevic T, Gasparotto D, Carbone A, Boiocchi M: Human non-Hodgkin’s lymphomas overexpress a wild-type form of p53 which is a functional transcription activator of the cyclin-dependent kinase inhibitor p21. Blood 89:2523, 1997 21. Butz K, Shahabeddin L, Geisen C, Spitkovsky D, Ullmann A, Hoppe-Seyler F: Functional p53 protein in human papillomaviruspositive cancer cells. Oncogene 10:927, 1995 Juvenile Genetic Hemochromatosis Is Clinically and Genetically Distinct From the Classical HLA-Related Disorder To the Editor: Genetic hemochromatosis (GH) is a common HLA-linked recessive disorder characterized by progressive parenchymal iron loading and the appearance of clinical manifestations in the fifth decade of life, predominantly in males. HFE has been recently identified as the candidate gene, with most patients being homozygous for a Cys-282 = Tyr (C282Y) mutation and others being compound heterozygotes for C282Y and a second mutation, His-63 = Asp (H63D).1 Homozygosity for C282Y is found in more than 90% of North European patients,2 but in only 64% of severely iron-loaded Italian individuals.3 This finding may suggest that various genetic iron overload syndromes exist in addition to the HFE-related one. Fifteen years ago, we described cases of juvenile GH suggesting that this was a distinct disease entity.4 In the juvenile condition, males and females appear to be equally affected. Patients present with hypogonadotropic hypogonadism and, unless proper treatment is started, die early because of cardiac dysfunction. We now provide further evidence that the juvenile condition is clinically and genetically distinct from the classical adult disorder. The pedigrees of our two Italian families with juvenile GH are shown in Fig 1. The clinical features of family 1 were reported in 1983,4 whereas family 2 has never been described. Of the four affected individuals, three presented with hypogonadotropic hypogonadism at 14 to 21 years of age. The affected male of family 2 presented with cardiac failure at 20 years of age and died at 21 years of age of congestive cardiomyopathy.
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