Proteins In Leukemic Cells Research Articles

Accumulation of hypoxia-inducible factor-1 protein and its role in the differentiation of myeloid leukemic cells induced by all-trans retinoic acid

The clinical activities of all-trans retinoic acid in the treatment of acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia, have triggered extensive studies aimed at defining the mechanisms by which this compound induces differentiation of leukemic cells. Recent studies show that hypoxia-inducible factor-1 alpha (HIF-1 alpha) contributes to the differentiation of acute myeloid leukemia cells via transcriptional activity-independent mechanisms. We investigated whether all-trans retinoic acid affects HIF-1 alpha protein and whether this has a role in all-trans retinoic acid-induced differentiation. The acute myeloid leukemia cell lines NB4 and U937 were treated with all-trans retinoic acid, and HIF-1 alpha/HIF-1 beta mRNA and proteins were measured respectively by real-time quantitative reverse transcriptase polymerase chain reaction and western blotting. To investigate the role of HIF-1 alpha in all-trans retinoic acid-induced differentiation, NB4 cells, U937 cells, U937 cells in which HIF-1 alpha was induced by the withdrawal of tetracycline and U937 cells with stable expression of specific short hairpin RNA against HIF-1 alpha, Runx1, C/EBP alpha and PU.1, were treated with all-trans retinoic acid and/or the hypoxiamimetic agent cobalt chloride (CoCl(2)). Cellular differentiation was evaluated by morphological criteria and myeloid differentiation antigens. all-trans retinoic acid rapidly increased endogenous and inducible expressed or CoCl(2)-stabilized HIF-1 alpha protein in leukemic cells under normoxia. Importantly, suppression of HIF-1 alpha expression by specific short hairpin RNA partially but significantly inhibited all-trans retinoic acid-induced differentiation of the U937 cell line. Reciprocally, the differentiation induced by all-trans retinoic acid was significantly enhanced by conditional HIF-1 alpha induction and HIF-1 alpha-stabilizing CoCl(2) treatment. Furthermore, knock-down of PU.1, Runx1 and C/EBP alpha, three transcriptional factors crucial for normal hematopoiesis, greatly inhibited the differentiation cooperation of all-trans retinoic acid and HIF-1 alpha induction. This work provides the first demonstration that HIF-1 alpha, a protein rapidly responsive to all-trans retinoic acid, plays a role in all-trans retinoic acid-induced differentiation of leukemic cells. These observations shed new light on the molecular mechanisms underlying all-trans retinoic acid-induced differentiation of acute myeloid leukemia cells.

Roles of Phosphorylated p210bcr/abl and Hsp90 Protein in Apoptosis Induced by Berbamine in K562 Cells.

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disorder characterized by accumulation of mature and immature granulocytes in peripheral blood and bone marrow due to uncontrolled growth and resistance to apoptosis. The dysregulated activity of the bcr/abl oncoprotein tyrosine kinase, which is encoded by the bcr-abl fusion gene generated from the chromosomal translocation of t(9; 22) and present in approximately 95% of CML patients, has been shown to be responsible for these malignant phenotypes. Numerous studies have demonstrated that only being phosphorylated that p210bcr/abl oncoprotein can promote cell proliferation and survival, and block apoptosis of tumor cells. Bcr/abl tyrosine kinase has chaperone association with heat shock protein 90 (Hsp90), which plays an essential role in stabilization of bcr/abl protein. Here we describe the activity of a natural small molecular compound, berbamine from plant Berberis amurensis that can selectively induce cell death of both Gleevecsensitive and -resistant Ph+ CML cells, but little is known about its exact mechanisms. We investigated the the expression levels of phosphorylated p210bcr/abl protein and Hsp90 protein in apoptosis induced by berbamine in K562 cells by means of various technologies, including cell culture system, flow cytomerty, western blot and immunoprecipitation (IP).Methods: Human Ph+ CML leukemia K562 cells, which express endogenous p210bcr/abl protein, were cultured in RPMI 1640 and treated with berbamine as indicated time and dose. Flow cytometry (FCM) and Annexin V-FLUOS/PI staining kit were used to evaluate apoptosis of leukemic cells; FCM and cytoperm/cytofix plus Caspase-3-McAb-PE were employed to measure leukemic cells with activated Caspase-3. Phosphorylation of p210bcr/abl protein in leukemic cells were assessed by a combination of immunoprecipitation (IP) with c-abl antibody and Western blot with p-Tyr(pY99)antibody. The protein levels of P210bcr/abl, Hsp90 and Hsp70 in leukemic cells were determined by Western blot with antibodies to c-abl, Hsp90 and Hsp70, respectively.Results:After treatment with berbamine at 8 μg/ml for 48 h, the percentages of leukemic cells expressing activated caspase-3 and apoptotic cells were 45.69% and 48.43%, respectively.IP and WB results showed that berbamine at low concentration markedly inhibited phosphorylation of p210bcr/abl protein in leukemia cells, and the amount of phosphorylated p210bcr/abl in leukemia cells exposured to berbamine at 8 μg/ml for 6 h were only 8.41% of that of untreated leukemia cells without the protein levels of P210bcr/abl down-regulated.Berbamine also down-regulated chaperone Hsp90 protein, and the amount of Hsp90 protein in leukemia cells treated with berbamine at 8 μg/ml for 48 h accounted for 18.37% of that of untreated leukemia cells. Interestingly, berbamine at 8 μg/ml had no obvious effects on chaperone Hsp70 protein expression associated with the resistance of leukemia cells to apoptosis.Conclusions:Berbamine could induce caspase-3-mediated apoptosis of Ph+ leukemia cells through inhibiting phosphorylation of p210bcr/abl protein and down-regulating its chaperone Hsp90 protein.Unlike Hsp90 inhibitor GA that could upregulate Hsp70, berbamine had no obvious effects on chaperone Hsp70 protein expression in leukemia cells, suggesting that berbamine may be a novel class of Hsp90 inhibitor, and further study is required.

Differential Targets and Subcellular Localization of Antitumor Alkyl-lysophospholipid in Leukemic Versus Solid Tumor Cells

Synthetic alkyl-lysophospholipids represent a family of promising anticancer drugs that induce apoptosis in a variety of tumor cells. Here we have found a differential subcellular distribution of the alkyl-lysophospholipid edelfosine in leukemic and solid tumor cells that leads to distinct anticancer responses. Edelfosine induced rapid apoptosis in human leukemic cells, including acute T-cell leukemia Jurkat and Peer cells, but promoted a late apoptotic response, preceded by G(2)/M arrest, in human solid tumor cells such as cervix epitheloid carcinoma HeLa cells and lung carcinoma A549 cells. c-Jun amino-terminal kinase (JNK) and caspase-3 were accordingly activated at earlier times in edelfosine-treated Jurkat cells as compared with drug-treated HeLa cells. Both leukemic and solid tumor cells took up this alkyl-lysophospholipid and expressed the two putative edelfosine targets, namely cell surface Fas death receptor (also known as APO-1 or CD95) and endoplasmic reticulum CTP: phosphocholine cytidylyltransferase. However, edelfosine was mainly located to plasma membrane lipid rafts in Jurkat and Peer leukemic cells and to endoplasmic reticulum in solid tumor HeLa and A549 cells. Edelfosine induced translocation of Fas, Fas-associated death domain-containing protein, and JNK into membrane rafts in Jurkat cells, but not in HeLa cells. In contrast, edelfosine inhibited phosphatidylcholine biosynthesis in both HeLa and A549 cells, but not in Jurkat or Peer leukemic cells, before the triggering of apoptosis. These data indicate that edelfosine targets two different subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts in leukemic cells and endoplasmic reticulum in solid tumor cells.

Mechanisms and Activity of PPARγ-Active Triterpenoids CDDO and CDDO-Me in Leukemias.

First-line therapy of acute myeloid leukemia (AML) consists of combinations of cytarabine and an anthracycline. While initial complete remissions are frequent, most patients succumb to resistant disease underlining the need for novel, more effective agents. The most striking progress in AML therapy was achieved by targeting the nuclear receptor RARα with ATRA. Research in our laboratory has demonstrated that the novel synthetic triterpenoid CDDO (2-cyano-3,12- dioxooleana-1,9-dien-28-oic acid) and its more active C28 methyl ester derivative CDDO-Me inhibit growth and induce apoptosis in a variety of cancers including AML, CLL and blast crisis CML. CDDO and to a much higher degree CDDO-Me are potent activators of the nuclear transcription factor Peroxisome Proliferator-Activated Receptor gamma (PPARγ). In a mammalian two-hybrid assay, the CDDO and CDDO-Me induced activation of PPARγ was associated with a marked increase in multiple coactivator recruitment (SRC-1, SRC-2, SRC-3, TRAP220/DRIP205, CARM-1 and PGC-1) that is qualitatively different from that induced by other PPARγ ligands. CDDO induced a higher degree of myelo-monocytic differentiation in DRIP205-overexpressing HL-60 cells suggesting that high cellular levels of DRIP205 co-activator modulate differentiation response to PPARγ ligation. CDDO induced p21 mRNA and protein in leukemic cells and transactivation of the p21 promoter in a p53-independent fashion. We have recently identified the PPARγ-independent depletion of mitochondrial glutathione (GSHm) as a novel mechanism of action resulting in redox disbalance and mitochondrial damage as mechanisms of pro-apoptotic effects of CDDO and CDDO-Me. Gene expression studies using cDNA arrays demonstrate that CDDO induces genes involved in the antioxidant response (AR) including phase II detoxifying enzymes (glutamate cysteine ligase, GSH transferase, etc.) and antioxidant enzymes (heme oxygenase 1, thioredoxin reductase). Cotreatment with the GSH precursor, n-acetyl cystein prevented apoptosis and loss of viability induced by CDDOs, whereas alkylation of intracellular thiols by diethylmaleate decreased the accumulation of a biotinylated derivative of CDDO, TP-301, in U937 leukemic cells suggesting that intracellular reduced thiols are functional targets of CDDO and its derivatives. The in vivo studies using liposomal CDDO-Me in a conditional leukemia model demonstrated significant reduction of leukemia burden as measured by bioluminescence imaging and prolongation of survival. Based on the ample pre-clinical evidence of anti-leukemia effects and on the favorable PK/toxicity profile of the parental compound, CDDO will enter Phase I clinical trials in hematologic malignancies in 3Q 2005 and CDDO-Me in 1Q 2006.

Prognostic Value of ZAP-70 Expression Detected by Immunohistochemistry on Bone Marrow Biopsies in Early Phase Chronic Lymphocytic Leukaemia.

New biological prognostic factors have been developed over the last years with the aim of predicting at presentation the heterogeneous clinical course of B chronic lymphocytic leukaemia (B-CLL) and of planning a risk-adapted treatment strategy. Among them ZAP-70 expression on leukemic cells as evaluated by molecular analysis or flow cytometry, initially proposed as surrogate of IgVH mutational status, appears a strong prognostic marker in B-CLL. However the optimal methodological approach to immunophenotipical demonstration of ZAP-70 expression as still matter of debate. We evaluated the cytoplasmic expression of ZAP-70 protein in leukemic cells by immunohistochemical method on bone marrow trephine biopsies taken at diagnosis of 84 patients with B-CLL. We used a mouse anti-human monoclonal antibody to ZAP-70 (clone 2F3.2, 1/200, Upstate, Lake Placid NY) with a polymeric labelling two-step method (Dako cytomation EnVision+, HRP). The results were correlated with age, sex, Binet stage, pattern of bone marrow infiltration, survival and clinical outcome. They were 54 males (64%), aged 34 to 80 years (median 62.5). At presentation 69 (82%) were Binet stage A, 9 (11%) stage B and 6 (7%) stage C. Among the 60 survivors, the median follow-up period from diagnosis was 78.5 months (range 22 – 236 months) Thirty-nine cases (46%) of B-CLL patients had cytoplasmic expression of ZAP-70. This group of patients presented higher percentage of advanced Binet stage (B–C) (p= 0.001). The ZAP-70 positivity was significantly related to inferior OS (55% vs 90% at 7 years)(HR 4.67 CI 1.95–11.14) and PFS (15% vs 57% at 7 years) (HR 5.52 CI 2.57–11.86), confirmed in multivariate analysis. ZAP-70 expression was correlated to poorer outcome also when we evaluated only the 69 stage A patients and the 56 cases with non-diffuse bone marrow infiltration, whereas in patients with diffuse pattern ZAP-70 expression had no prognostic significance. In conclusion, immunohistological detection of ZAP-70 on bone marrow samples at diagnosis appears a useful methodological approach to identify patients with different prognosis in B-CLL.

Absence or Low Expression of Fas-Associated Protein with Death Domain in Acute Myeloid Leukemia Cells Predicts Resistance to Chemotherapy and Poor Outcome

In acute myeloid leukemia (AML), coexpression of death receptors and ligands of the tumor necrosis factor (TNF) receptor/TNF-alpha superfamily on leukemic cells after chemotherapy is not always accompanied by apoptosis, suggesting that the apoptotic death receptor signaling pathway is disrupted. Because Fas-associated protein with death domain (FADD) is the main adaptor for transmitting the Fas, TNF-related apoptosis-inducing ligand receptors, and TNF receptor 1 death signal, expression of FADD was analyzed by Western blot and immunocytochemistry in leukemic cells of 70 de novo AML patients treated with the European Organization of Research and Treatment of Cancer AML-10 randomized trial before initiation of induction chemotherapy. Thirty seven percent of patients (17 of 46) with FADD negative/low (FADD(-/low)) leukemic cells had a primary refractory disease compared with 12% of FADD(+) patients (3 of 24; P = 0.05). FADD(-/low) expression was significantly associated with a worse event-free survival [EFS (P = 0.04)] and overall survival (P = 0.04). In multivariate analysis, FADD(-/low) protein expression was independently associated with a poor EFS and overall survival (P = 0.002 and P = 0.026, respectively). Importantly, FADD(-/low) protein expression predicted poor EFS even in patients with standard- or good-risk AML (P = 0.009). Thus, we identified low or absent expression of the FADD protein in leukemic cells at diagnosis as a poor independent prognostic factor that can predict worse clinical outcome even for patients with standard- or good-risk AML.

Proteomic Analysis of Human Acute Leukemia Cells

French-American-British (FAB) classification of acute leukemia with genetic heterogeneity is important for treatment and prognosis. However, the distinct protein profiles that contribute to the subtypes and facilitate molecular definition of acute leukemia classification are still unclear. The proteins of leukemic cells from 61 cases of acute leukemia characterized by FAB classification were separated by two-dimensional electrophoresis, and the differentially expressed protein spots were identified by both matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and tandem electrospray ionization MS (ESI-MS/MS). The distinct protein profiles of acute leukemia FAB types or subtypes were successfully explored, including acute myeloid leukemia (AML), its subtypes (M2, M3, and M5) and acute lymphoid leukemia (ALL), which were homogeneous within substantial samples of the respective subgroups but clearly differed from all other subgroups. We found a group of proteins that were highly expressed in M2 and M3, rather than other subtypes. Among them, myeloid-related proteins 8 and 14 were first reported to mark AML differentiation and to differentiate AML from ALL. Heat shock 27 kDa protein 1 and other proteins that are highly expressed in ALL may play important roles in clinically distinguishing AML from ALL. Another set of proteins up-regulated was restricted to granulocytic lineage leukemia. High-level expression of NM23-H1 was found in all but the M3a subtype, with favorable prognosis. These data have implications in delineating the pathways of aberrant gene expression underlying the pathogenesis of acute leukemia and could facilitate molecular definition of FAB classification. The extension of the present analysis to currently less well-defined acute leukemias will identify additional subgroups.

Expression of the candidate MCT-1 oncogene in B- and T-cell lymphoid malignancies

AbstractOur laboratory has recently discovered a novel candidate oncogene, MCT-1, amplified in human T-cell lymphoma and mapped to chromosome Xq22-24. This region is amplified in a subset of primary B-cell non-Hodgkin lymphoma (NHL), suggesting that increased copy number of a gene(s) located in this region confers a growth advantage to some primary human lymphomas. We examined a diverse panel of lymphoid malignancies for the expression of MCT-1. We demonstrated that there are significantly increased levels of MCT-1 protein in a panel of T-cell lymphoid cell lines and in non-Hodgkin lymphoma cell lines. Furthermore, we identified a subset of primary diffuse large B-cell lymphomas that exhibited elevated levels of MCT-1 protein. Interestingly, all transformed follicular lymphomas in our study demonstrated elevated protein levels of MCT-1. There was no detectable MCT-1 protein in leukemic cells from patients with chronic lymphocytic leukemia or in any healthy lymphoid tissue examined. Lymphoid cell lines overexpressing MCT-1 exhibited increased growth rates and displayed increased protection against apoptosis induced by serum starvation when compared with matched controls. We found that MCT-1—overexpressing cells show constitutively higher levels of phosphorylated PKB/Akt protein, especially under serum starvation. Activation of survival pathways may be an additional function of the MCT-1 gene. Our data suggest that high levels of MCT-1 protein may be associated with a high-risk subset of lymphoid neoplasms and may further support the potential role of MCT-1 in promoting human lymphoid tumor development. (Blood. 2003;102: 297-302)

Effects of Interferon-α on Cell Cycle Regulatory Proteins in Leukemic Cells

One prominent activity of Interferons (IFNs) is their ability to induce cell cycle arrest, and this effect has furthermore been proposed to be of major importance in mediating the clinical antitumor activity of IFNs. In several IFN sensitive established cell lines, a rapid upregulation of the cyclin dependent kinase inhibitor p21 occurs following IFN- α treatment, and is thought to play a major role as an effector for this phenomenon by triggering further events. The aim of this study was to investigate how these previous findings in established cells lines correlate with clinical material. We therefore, analyzed how IFN- α influences the cell cycle distribution, by analysis of cellular DNA content, and the level of various cell cycle regulatory proteins by Western blot analysis, in primary leukemic cells. In 5 of 10 examined acute myeloid leukemia samples and in 1 of 6 chronic lymphocytic leukemia sample a clear increase in p21 protein levels was detected following treatment with IFN- α, while p21 protein levels were unaffected by IFN treatment in any of the examined acute lymphoblastic leukemia samples. In our total material consisting of 21 patient samples all other cell cycle regulatory proteins studied (p27, Cyclin E, Cdk2), were largely unaffected by IFN treatment. These results confirm that IFN- α can act as a potent regulator of Cdk-inhibitor expression, and that the induction of p21 seems to be a primary event in IFN- α mediated cell cycle regulation.

A Link between Apoptosis and Degree of Phosphorylation of High Mobility Group A1a Protein in Leukemic Cells

Nuclear phosphoprotein HMGA1a, high mobility group A1a, (previously HMGI) has been investigated during apoptosis. A change in the degree of phosphorylation of HMGA1a has been observed during apoptosis induced in four leukemic cell lines (HL60, K562, NB4, and U937) by drugs (etoposide, camptothecin) or herpes simplex virus type-1. Both hyper-phosphorylation and de-phosphorylation of HMGA1a have been ascertained by liquid chromatography-mass spectrometry. Hyper-phosphorylation (at least five phosphate groups/HMGA1a molecule) occurs at the early apoptotic stages and is probably related to HMGA1a displacement from DNA and chromatin release from the nuclear scaffold. De-phosphorylation (one phosphate or no phosphate groups/HMGA1a molecule) accompanies the later formation of highly condensed chromatin in the apoptotic bodies. We report for the first time a direct link between the degree of phosphorylation of HMGA1a protein and apoptosis according to a process that involves the entire amount of HMGA1a present in the cells and, consequently, whole chromatin. At the same time we report that variously phosphorylated forms of HMGA1a protein are also mono-methylated.

MDM2 AND p53 IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA: Higher Expression in Childhood Leukemias with Poor Prognosis Compared to Long-Term Survivors

In previous studies the authors have found increased expression of p53 and MDM2 proteins in leukemic cells in a majority of children eligible for bone marrow transplantation (BMT) due to relapse orprognostically unfavorable features. In this study theimmunohistochemical expression of p53, MDM2, and p21 Cip1 was investigatedin bone marrow samples from the time of diagnosis in 30 children with acute lymphoblastic leukemia (ALL) surviving disease-free at least 5 years. This group was compared with 15 advanced ALL patients, admitted for BMT. In 7 of the BMT patients original diagnostic marrow samples were also available for analysis. Four out of 30 ALL patients in the relapse-free group expressed p53 in theoriginal leukemic cell population, while 8/15 advanced ALL patients did before BMT (p = .014 ). Four out of 30 cases in the relapse-free group expressed MDM2, while 10/15 in the BMT group did (p = .0011 ). In retrospect, MDM2 over expression at the time of diagnosis was also more common (p = .0098 ) in the BMT group as well as p53 over expression (p = .054 ) compared to nonrelapsed patients.

Peptides derived from the whole sequence of BCR-ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes.

Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210(bcr-abl) fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210(bcr-abl) is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210(bcr-abl) breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.

Subcellular localization of Bcr, Abl, and Bcr-Abl proteins in normal and leukemic cells and correlation of expression with myeloid differentiation.

We used specific antisera and immunohistochemical methods to investigate the subcellular localization and expression of Bcr, Abl, and Bcr-Abl proteins in leukemic cell lines and in fresh human leukemic and normal samples at various stages of myeloid differentiation. Earlier studies of the subcellular localization of transfected murine type IV c-Abl protein in fibroblasts have shown that this molecule resides largely in the nucleus, whereas transforming deletion variants are localized exclusively in the cytoplasm. Here, we demonstrate that the murine type IV c-Abl protein is also found in the nucleus when overexpressed in a mouse hematopoietic cell line. However, in both normal and leukemic human hematopoietic cells, c-Abl is discerned predominantly in the cytoplasm, with nuclear staining present, albeit at a lower level. In contrast, normal endogenous Bcr protein, as well as the aberrant p210BCR-ABL and p190BCR-ABL proteins consistently localize to the cytoplasm in both cell lines and fresh cells. The results with p210BCR-ABL were confirmed in a unique Ph1-positive chronic myelogenous leukemia (CML) cell line, KBM5, which lacks the normal chromosome 9 and hence the normal c-Abl product. Because the p210BCR-ABL protein appears cytoplasmic in both chronic phase and blast crisis CML cells, as does the p190BCR-ABL in Ph1-positive acute leukemia, a change in subcellular location of Bcr-Abl proteins between cytoplasm and nucleus cannot explain the different spectrum of leukemias associated with p210 and p190, nor the transition from the chronic to the acute leukemia phenotype seen in CML. Further analysis of fresh CML and normal hematopoietic bone marrow cells reveals that p210BCR-ABL, as well as the normal Bcr and Abl proteins, are expressed primarily in the early stages of myeloid maturation, and that levels of expression are reduced significantly as the cells mature to polymorphonuclear leukocytes. Similarly, a decrease in Bcr and Abl levels occurs in HL-60 cells induced by DMSO to undergo granulocytic differentiation. The action of p210BCR-ABL and its normal counterparts may, therefore, take place during the earlier stages of myeloid development.

Detection of p53 protein in leukemic cells

Detection of p53 protein in leukemic cells