The Chimeric E2A-HLF Transcription Factor Abrogates p53-Induced Apoptosis in Myeloid Leukemia Cells

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1397-1405 ◽  
Author(s):  
Rachel A. Altura ◽  
Takeshi Inukai ◽  
Richard A. Ashmun ◽  
Gerard P. Zambetti ◽  
Martine F. Roussel ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
B Cells ◽  
Growth Factor ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Wild Type ◽  
Wild Type P53 ◽  
Induced Apoptosis

Abstract Leukemic lymphoblasts expressing the E2A-HLF oncoprotein possess wild-type p53 genes, but do not undergo apoptosis in response to DNA damage. Experimentally, E2A-HLF prevents apoptosis due to growth factor deprivation or γ-irradiation in interleukin-3 (IL-3)–dependent murine pro-B cells. To directly test the chimeric protein’s ability to abrogate p53-mediated cell death, we used mouse myeloid leukemia cells (M1p53tsval) that constitutively express a temperature-sensitive (ts) mutant p53 gene and undergo apoptosis when p53 assumes an active wild-type configuration. This effect is blocked by treatment with IL-6, which allows the cells to survive in culture despite wild-type p53 activation. We introduced E2A-HLF into M1p53tsval cells and found that they were resistant to p53-mediated apoptosis and that E2A-HLF effectively substituted for the survival functions of IL-6. The expression of p53-responsive genes such as p21 and Bax was upregulated normally, suggesting that E2A-HLF acts downstream of p53 to block execution of the p53-induced apoptotic program. NFIL3, a growth factor-regulated bZIP protein that binds to the same DNA-consensus site as E2A-HLF, delays apoptosis in IL-3–dependent pro-B cells deprived of growth factor. By contrast, in the present study, enforced expression of NFIL3 failed to protect M1p53tsval cells from p53-dependent apoptosis and actively antagonized the ability of IL-6 to rescue cells from that fate, consistent with its role as either a transcriptional repressor or activator, depending on the cell type in which it is expressed. We conclude that the E2A-HLF chimera abrogates p53-induced apoptosis in leukemic cells, possibly through the transcriptional modulation of cell death pathways that are activated by p53 in response to DNA damage. © 1998 by The American Society of Hematology.

The Chimeric E2A-HLF Transcription Factor Abrogates p53-Induced Apoptosis in Myeloid Leukemia Cells

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1397-1405
Author(s):  
Rachel A. Altura ◽  
Takeshi Inukai ◽  
Richard A. Ashmun ◽  
Gerard P. Zambetti ◽  
Martine F. Roussel ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
B Cells ◽  
Growth Factor ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Wild Type ◽  
Wild Type P53 ◽  
Induced Apoptosis

Leukemic lymphoblasts expressing the E2A-HLF oncoprotein possess wild-type p53 genes, but do not undergo apoptosis in response to DNA damage. Experimentally, E2A-HLF prevents apoptosis due to growth factor deprivation or γ-irradiation in interleukin-3 (IL-3)–dependent murine pro-B cells. To directly test the chimeric protein’s ability to abrogate p53-mediated cell death, we used mouse myeloid leukemia cells (M1p53tsval) that constitutively express a temperature-sensitive (ts) mutant p53 gene and undergo apoptosis when p53 assumes an active wild-type configuration. This effect is blocked by treatment with IL-6, which allows the cells to survive in culture despite wild-type p53 activation. We introduced E2A-HLF into M1p53tsval cells and found that they were resistant to p53-mediated apoptosis and that E2A-HLF effectively substituted for the survival functions of IL-6. The expression of p53-responsive genes such as p21 and Bax was upregulated normally, suggesting that E2A-HLF acts downstream of p53 to block execution of the p53-induced apoptotic program. NFIL3, a growth factor-regulated bZIP protein that binds to the same DNA-consensus site as E2A-HLF, delays apoptosis in IL-3–dependent pro-B cells deprived of growth factor. By contrast, in the present study, enforced expression of NFIL3 failed to protect M1p53tsval cells from p53-dependent apoptosis and actively antagonized the ability of IL-6 to rescue cells from that fate, consistent with its role as either a transcriptional repressor or activator, depending on the cell type in which it is expressed. We conclude that the E2A-HLF chimera abrogates p53-induced apoptosis in leukemic cells, possibly through the transcriptional modulation of cell death pathways that are activated by p53 in response to DNA damage. © 1998 by The American Society of Hematology.

Bcl-2 Protects against p53-Induced Apoptosis through Hdm2

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5333-5333
Author(s):  
Line Wergeland ◽  
Kevin B. Spurgers ◽  
Eystein Oveland ◽  
Torill Høiby ◽  
Manel Cascallo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Acute Myeloid Leukemia ◽  
Protein Level ◽  
Myeloid Leukemia ◽  
Proteasome Inhibitors ◽  
Wild Type ◽  
Inhibitory Molecule ◽  
Wild Type P53 ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Hdm2 is up-regulated in several malignancies including sarcomas and acute myeloid leukemia, where it counteracts the anti-proliferative and pro-apoptotic effect of wild type p53. The anti-apoptotic protein Bcl-2 is often elevated in many tumors with wild type p53 and serves to block p53-induced apoptosis. We demonstrate that the protein level of Hdm2 positively correlates with the level of Bcl-2 and follows the Bcl-2 level in different cell systems. Over-expression of Bcl-2 protects Hdm2 from DNA-damage induced degradation in a dose dependant manner. In addition, modulation of Bcl-2 by shRNA knockdown reduced the Hdm2 protein level in parallel. Consequently, treatment of AML cells with the Bcl-2 small inhibitory molecule HA14-1 attenuated the level of Hdm2. The Bcl-2 level, but not the DNA damage induced Hdm2 degradation, was affected by disruption of the E3 ubiquitin ligase activity of Hdm2. In addition, the DNA-damage induced Hdm2 down-regulation was blocked by disrupted E1 ubiquitin-activation, defect polyubiquitination and by proteasome inhibitors. Finally, we show that Bcl-2 protection from p53-induced cell death requires co-expression of Hdm2 in double null p53/mdm2 mouse embryonic fibroblasts. Our results indicate that Bcl-2 regulates the Hdm2 level and that Hdm2 is a key mediator in Bcl-2 inhibition of p53-induced apoptosis. This is of particular therapeutic interest for cancers displaying elevated Hdm2 and Bcl-2, like sarcoma and acute myeloid leukemia.

scholarly journals Induced Apoptosis Investigation in Wild-type and FLT3-ITD Acute Myeloid Leukemia Cells by Nanochannel Electroporation and Single-cell qRT-PCR

2016 ◽  
Vol 24 (5) ◽  
pp. 956-964 ◽  
Author(s):  
Keliang Gao ◽  
Xiaomeng Huang ◽  
Chi-Ling Chiang ◽  
Xinmei Wang ◽  
Lingqian Chang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Single Cell ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Wild Type ◽  
Qrt Pcr ◽  
Acute Myeloid Leukemia Cells ◽  
Induced Apoptosis ◽  
Acute Myeloid

Inhibition of p53-MDM2 Interaction by Small-Molecule Antagonist of MDM2 Effectively Induces Apoptosis in Leukemias.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2536-2536
Author(s):  
Kensuke Kojima ◽  
Marina Konopleva ◽  
Masato Shikami ◽  
Maria Cabreira-Hansen ◽  
C. Ellen Jackson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Annexin V ◽  
Cell Number ◽  
Leukemia Cells ◽  
P53 Mutations ◽  
Wild Type ◽  
Cycle Arrest ◽  
Wild Type P53 ◽  
Induced Apoptosis

Abstract Alteration of the p53 gene is one of the most frequent events in human tumorigenesis and about 50% of all solid tumors have been reported to carry p53 mutations. The inactivation of p53 in cancer has been associated with poor survival, refractory disease and chemoresistance. p53 mutations rarely occur in hematopoietic malignancies. Instead, MDM2 which is a principal cellular antagonist of p53, is overexpressed in the majority of leukemias. Recently, potent and selective small-molecule antagonists of MDM2, Nutlins, have been identified (Science303:844–888, 2004). Nutlins bind MDM2 in the p53-binding pocket and activate the p53 pathway in human cancer cells with wild-type p53, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice. In this study, we investigated the potential antileukemic activity of the MDM2 antagonist. Treatment of wild-type p53 OCI-AML-3 cells with 5 μM of an active compound (Nutlin-3a) induced cell cycle arrest and apoptosis as evidenced by flow-cytometric analysis (51% reduction of S-phase at 12 h, 27% sub-G1 DNA content and 57% Annexin V positivity at 48 h). Similar proapoptotic effects were observed in MOLM-13 cells which have wild-type p53, but not in p53-null (HL-60 and U937) or mutant p53 (Raji, Jurkat and NB-4) cells. Nutlin-3a induced apoptosis in a dose- and time-dependent manner, and induced maximal effect on cell cycle arrest at 1 μM. Western blot analysis showed that in OCI-AML-3 cells, wild-type p53 accumulated at 1 h after exposure to Nutlin-3a. Increased levels of MDM2, p21 and Noxa proteins were observed at 1 to 3h. This resulted in cleavage of caspase-9 followed by cleavage of caspase-3. A pharmacologic interaction study between MDM2 inhibitor and Ara-C using a fixed-ratio (1:1) experimental design showed highly synergistic cell growth inhibition (CI = 0.44) and induction of apoptosis (CI = 0.83) in OCI-AML-3 cells. Initial studies conducted in primary leukemia cells demonstrated that Nutlin-3a induced apoptosis in 4 of 5 AML samples tested (68–97% Annexin V induction and 65–93% cell number reduction) and 2 CLL samples (>50% Annexin V induction and 37% and 58% cell number reduction). Since MDM2 protein is overexpressed and p53 is not mutated in the majority of primary leukemia cells, this approach may have therapeutic utility in leukemias.

G2/M Arrest Sensitizes Chronic Myelogenous Leukemia Cells to TRAIL-Induced Apoptosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4465-4465
Author(s):  
David Devlin ◽  
Eva Szegezdi ◽  
Paavilainen Tanja ◽  
Orsolya Orosz ◽  
Michael O'Dwyer ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Cell Cycle Progression ◽  
Cell Cycle Checkpoint ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Induced Apoptosis ◽  
Cycle Dependent ◽  
Low Sensitivity

Abstract Abstract 4465 The death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receives great interest as it targets and kills cancerous cells, but not non-transformed cells. While it is in phase I/II clinical trials for a range of solid tumours, the generally low sensitivity of leukemia cells to TRAIL makes it a less attractive therapeutic for these cancers. We found that doxorubicin and cytarabine, agents that induce DNA damage and impair cell cycle progression, can sensitize CML cells to TRAIL with CI<1 at Fa of ED25 and ED50 (based on median-effect method using the isobologram equation). Inhibition of the cell cycle checkpoint kinases Chk1/2 with UCN-01 did not influence TRAIL-induced apoptosis nor could it abolish the sensitizing effect of doxorubicin. Interestingly, inhibition of Ataxia Telangiectasia Mutated (ATM), a key DNA damage response kinase, with KU-55933 induced a G2/M arrest and enhanced TRAIL-induced apoptosis. Inhibition of ATM alone induced 22±3.1% apoptosis and increased TRAIL-induced apoptosis from 27.2±4.7% to 68±7.2%. Cell cycle analysis revealed that while the proportion of cells in the G0/G1 and S phases slightly increased, the proportion of the cells in the G2/M phase dropped by 31.6±3.2% (p<0.05) indicating that G2/M arrested cells were more sensitive to TRAIL than cells in G0/G1 and S phases. TRAIL-induced CML cell death was also synergistically enhanced by arresting the cells in G2/M using the microtubule disrupting drugs, nocodazole or colcemide. Cells were treated with a concentration of nocodazole or colcemide that induced above 90% G2/M arrest for 16 h (0.3 mM and 0.1 mg/ml, respectively) followed by treatment with 250 ng/ml of TRAIL for 24 h. Nocodazole, colcemide and TRAIL individually induced 19±3.7% 26.3±4.4% and 27.2±4.7% cell death, while combination of nocodazole or colcemide with TRAIL resulted 89±6.8% and 93±5.9% cell death, respectively. In summary, we found that induction of DNA damage sensitizes CML cells to TRAIL and that TRAIL-sensitivity of CML cells is cell cycle-dependent. Disclosures: O'Dwyer: Novartis: Honoraria.

scholarly journals Simultaneous activation of p53 and inhibition of XIAP enhance the activation of apoptosis signaling pathways in AML

Blood ◽  
2010 ◽  
Vol 115 (2) ◽  
pp. 306-314 ◽  
Author(s):  
Bing Z. Carter ◽  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Erich Koller ◽  
Clemencia Pinilla ◽  
...  
Keyword(s):  
Cell Death ◽  
Myeloid Leukemia ◽  
Wild Type ◽  
Combination Strategy ◽  
Mdm2 Antagonist ◽  
P53 Activation ◽  
Simultaneous Activation ◽  
Murine Double Minute ◽  
Caspase 6 ◽  
Induced Apoptosis

Abstract Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a–induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic.

Ubenimex Enhances the Gemtuzumab Ozogamicin-Induced Cytotoxicity Against Myeloid Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4370-4370
Author(s):  
Yasushi Takamatsu ◽  
Shiro Jimi ◽  
Yuka Yoshimura ◽  
Junji Suzumiya ◽  
Kazuo Tamura
Keyword(s):  
Cell Death ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Myeloid Cell ◽  
Quantitative Relationship ◽  
K562 Cells ◽  
Gemtuzumab Ozogamicin ◽  
Leukemia Cells ◽  
Induced Apoptosis

Abstract Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to a derivative of a tumoricidal antibiotic calicheamicin. GO alone induces complete remission in 26% of AML patients in first relapse. One way to improve the response rate is to combine GO with other agents. A quantitative relationship has been shown between rates and degrees of CD33 expression and GO-induced cytotoxicity in vitro by using gene transfer to manipulate CD33 expression in myeloid cell lines. We first studied CD33 expression on myeloid leukemia cells by stimulating with several agents in vitro, and found that ubenimex, but not G-CSF, M-CSF, or ATRA, increased CD33 expression on both HL-60 and K562 cells. Ubenimex is an aminopeptidase inhibitor isolated from Streptomyces olivoreticuli and commercially available as an oral agent for clinical use for AML treatment. To investigate whether ubenimex enhances the cytotoxicity of GO, HL-60 and K562 cells were cultured with GO and/or ubenimex for 3 days. When GO was administered, cell viabilities of HL-60 and K562 were reduced to 31.5% and 90.3% as compared with control, respectively. Treatment with ubenimex alone did not influence viabilities of either HL-60 or K562. However, when cells were preincubated with ubenimex and then cultured with GO, cell viabilities decreased to 18.5% and 81.4% for HL-60 and K562, respectively, indicating that pretreatment with ubenimex enhanced GO-induced myeloid leukemia cell death in vitro. We next assessed the mechanism of cell death. The treatment with GO alone and ubenimex alone induced apoptosis in 39.2% and 2.9% of HL-60 cells, respectively. When HL-60 cells were preincubated with ubenimex and then cultured with GO, the number of apoptotic cells increased to 62.9%, demonstrating that ubenimex augments GO-induced apoptosis. Our data suggest that the priming of AML cells with ubenimex should improve the clinical efficacy of GO.

Level of Myeloperoxidase Expression as a Crucial Determinant of Sensitivity of Myeloid Leukemia Cells to Parthenolide-Induced Apoptosis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2945-2945
Author(s):  
Soo Jeong Kim ◽  
Ju In Eom ◽  
Hye Won Lee ◽  
Hoi Kyung Jeung ◽  
Jin Seok Kim ◽  
...  
Keyword(s):  
Cell Death ◽  
Myeloid Leukemia ◽  
Prognostic Significance ◽  
Flow Cytometric Analysis ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Apoptotic Cells ◽  
Strong Inhibitor ◽  
Induced Apoptosis

Abstract Parthenolide (PTL) is a sesquiterpene lactone found as the major active component in Feverfew (Tanacetum parthenium). PTL is a strong inhibitor of NF-kB activation and STAT transcriptional activity, resulting in a downregulation of antiapoptotic gene transcription. Recently, PTL was demonstrated to have promising anti-cancer effects through inhibition of DNA synthesis and reactive oxygen species (ROS)-associated intrinsic apoptosis. However, the sensitivity to PTL-induced cell death was different according to leukemia cells. Therefore, it will be important to identify parameters predicting response to PTL-induced cell death. Myeloperoxidase (MPO), a typical lineage marker for acute myeloid leukemia (AML), was also shown to have a prognostic significance in this disorder. Since it was shown that MPO is a potential regulator of oxidative stress, we examined the effect of MPO expression upon the PTL-induced leukemia cell death. First we compared the extent and mechanism of PTL-induced cell death between parental K562 and MPO-overexpressing K562 (K562/MPO) cells. K562/MPO cells were kindly provided by Dr. Sawayama (Nagasaki University, Japan). Annexin V labeling evaluation revealed that PTL induced apoptosis in K562/MPO cells in a dosedependent manner. The fraction of apoptotic cells after 24 hours treatment with 10mM of PTL was significantly higher in K562/MPO cells (52.2 ± 0.4%) compared to parental K562 cells (13.1 ± 4.8%, p&lt;0.001). When the cells were treated with PTL, the population that lost mitochondrial membrane disruption (MMP) was 45.1 ± 12.4% in K562/MPO cells, which was significantly higher than K562 cells (0.7 ± 0.1%, p&lt;0.001). Cleavage of caspase-3, -8, -9, and PARP was observed in K562/MPO cells after PTL treatment, whereas it was not shown in K562 cells. We next examined the possible involvement of ROS in PTL-induced cell death by flow cytometric analysis using dihydroethidium fluorescent probe. PTL drastically increased relative ROS levels in K562/MPO cells (4.1 ± 0.1). However, the increase in ROS levels induced by PTL was not demonstrated in K562 cells (1.5 ± 0.1, p&lt;0.01). Marked downregulation of Bcl-2, Bcl-xL, and NF-kB was demonstrated preferentially in K562/MPO cells. c-Jun N-terminal kinase phosphorylation was remarkably increased only in K562/MPO cells. We next evaluated the PTL-induced cell death in primary leukemic blasts obtained from patients with AML. Quantitative measurement of MPO expression was done using flow cytometry analysis. Interestingly, the fraction of apoptotic cells induced by 24 hours treatment of PTL was significantly higher in AML specimens consisting of higher than 50% of MPO-positive cells (MPOhi cases; 44.5 ± 1.6%, n = 6) compared to AML specimens consisting of lower than 20% of MPO-positive cells (MPOlo cases; 1.4 ± 1.1%, n = 4, p&lt;0.001). Our findings indicate that PTL induces apoptosis in myeloid leukemia cells is associated with increased ROS and the extent of MPO expression is a crucial determinant of their sensitivity to PTL-induced cell death.

scholarly journals Activated H-ras rescues E1A-induced apoptosis and cooperates with E1A to overcome p53-dependent growth arrest.

1995 ◽  
Vol 15 (8) ◽  
pp. 4536-4544 ◽  
Author(s):  
H J Lin ◽  
V Eviner ◽  
G C Prendergast ◽  
E White
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Dna Synthesis ◽  
Growth Arrest ◽  
Mutant P53 ◽  
Permissive Temperature ◽  
Wild Type ◽  
Wild Type P53 ◽  
Dependent Growth ◽  
Induced Apoptosis

The adenovirus E1A oncogene products stimulate DNA synthesis and cell proliferation but fail to transform primary baby rat kidney (BRK) cells because of the induction of p53-mediated programmed cell death (apoptosis). Overexpression of dominant mutant p53 (to abrogate wild-type p53 function) or introduction of apoptosis inhibitors, such as adenovirus E1B 19K or Bcl-2 oncoproteins, prevents E1A-induced apoptosis and permits transformation of BRK cells. The ability of activated Harvey-ras (H-ras) to cooperate with E1A to transform BRK cells suggests that H-ras is capable of overcoming the E1A-induced, p53-dependent apoptosis. We demonstrate here that activated H-ras was capable of suppressing apoptosis induced by E1A and wild-type p53. However, unlike Bcl-2 and the E1B 19K proteins, which completely block apoptosis but not p53-dependent growth arrest, H-ras expression permitted DNA synthesis and cell proliferation in the presence of high levels of wild-type p53. The mechanism by which H-ras regulates apoptosis and cell cycle progression is thereby strikingly different from that of the E1B 19K and Bcl-2 proteins. BRK cells transformed with H-ras and the temperature sensitive murine mutant p53(val 135), which lack E1A, underwent growth arrest at the permissive temperature for wild-type p53. p53-dependent growth arrest, however, could be relieved by E1A expression. Thus, H-ras alone was insufficient and cooperation of H-ras and E1A was required to override growth suppression by p53. Our data further suggest that two complementary growth signals from E1A plus H-ras can rescue cell death and thus permit transformation.

scholarly journals Expression of the p53 tumor suppressor gene induces differentiation and promotes induction of differentiation by 1,25-dihydroxycholecalciferol in leukemic U-937 cells

Blood ◽  
1996 ◽  
Vol 87 (3) ◽  
pp. 1064-1074 ◽  
Author(s):  
M Ehinger ◽  
G Bergh ◽  
T Olofsson ◽  
B Baldetorp ◽  
I Olsson ◽  
...  
Keyword(s):  
Cell Death ◽  
Flow Cytometric Analysis ◽  
Negative Control ◽  
Leukemic Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
Fourfold Increase ◽  
Induction Of Differentiation ◽  
Wild Type P53

Leukemic U-937 cells, which lack normal p53, were stably transfected with a temperature-sensitive mutant of p53 to investigate the consequences for growth and differentiation. On induction of wild-type p53 activity at the permissive temperature, some of these cells underwent maturation as judged by the capacity for oxidative burst and the appearance of monocyte related cell surface molecules. Moreover, wild-type p53-expressing cells were more sensitive than p53-negative control cells to induction of differentiation by 1,25- dihydroxycholecalciferol; a twofold to fourfold increase of the fraction of cells showing signs of terminal maturation was observed when wild-type p53-expressing cells were incubated with 1,25- dihydroxycholecalciferol at concentrations that only slightly affected control cells. Whereas wild-type p53 activity per se induced maturation of certain cells, other underwent cell death judging from the reduced capability to exclude trypan blue and the appearance of fragmented DNA in flow cytometric analysis. The p53-induced cell death could be inhibited by incubation with 1,25-dihydroxy-cholecalciferol, but not all-trans retinoic acid. Thus, 1,25-dihydroxycholecalciferol, seemed to increase the survival of wild-type p53-expressing cells and to cooperate with wild-type p53 to induce differentiation. The data imply that p53-mediated maturation in U-937 cells depends on optimal regulation of signals for differentiation, survival and proliferation, and suggest a role for p53 in the differentiation induction of leukemic cells.

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