Detection of p53 gene mutations by single strand conformational polymorphism (SSCP) in human acute myeloid leukemia-derived cell lines

Fleckenstein, Diana; Uphoff, Cord C.; Drexler, Hans G.; Quentmeier, Hilmar

doi:10.1016/s0145-2126(01)00107-2

Cited by 26 publications

(19 citation statements)

References 40 publications

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“…19 Among the 6 AML cell lines, various p53 mutations have been reported for KG1, HL60 and U937 which result in lack of p53 protein and any p53-mediated transcriptional activity. 20,21 Accordingly, we could not detect p53 in Western blot analysis in these cells (Fig. 6c).…”

Section: G 1 Cycle Arrest Of Aml Cells After Treatment With 5-aza-cdrmentioning

confidence: 79%

5‐Aza‐2′‐deoxycytidine induces p21^WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

Schmelz

Wagner

Dörken

et al. 2005

Intl Journal of Cancer

106

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The DNA methylation inhibitor 5-Aza-2 -deoxycytidine (5-AzaCdR) has significant therapeutic value for the treatment of patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The demethylating effect of 5-Aza-CdR has been well characterized. In contrast, less is known about the molecular events downstream of the methylation inhibition. Here, 5-Aza-CdR induced apoptosis in AML cells (both p53 mutant and wild-type) but not in epithelial or normal PBMCs. Cell death was accompanied by activation of the mitochondrial apoptosis pathway, as shown by release of cytochrome c and AIF and loss of mitochondrial membrane potential (⌬⌿m). Activation of caspase-3 (but not ؊6 and ؊8) was detectable using Western blot analysis and measurement of caspase enzymatic activity. 5-Aza-CdR treatment resulted in the induction of p21, which correlated with the arrest of AML cells in the G 1 cell cycle phase. Induction of p21 expression was independent of its promoter methylation status but mediated by 5-Aza-CdR-induced reexpression of the tumor-suppressor p73, a known upstream regulator of p21. The p73 promoter was hypermethylated in AML cell lines and in primary AML cells but not in epithelial cells, which were resistant toward 5-Aza-CdR. Therefore, 5-Aza-CdRmediated specific killing of myeloid cells might be dependent on its ability to revert p73 promoter methylation and to reexpress p73 mRNA. In addition, exogenous expression of p73 rendered epithelial cells sensitive to apoptosis induced by 5-Aza-CdR or other cytostatic drugs. We therefore conclude that p73 is a relevant target for methylation-dependent efficacy of 5-Aza-CdR in AML cells.Key words: 5-aza-2Ј-deoxycytidine; p21 WAF ; p73; p53; myeloid leukemia; DNA methylation DNA methylation is an epigenetic mechanism of gene regulation that plays a crucial role in carcinogenesis due to silencing of cancer-related genes. 1 Transcriptional blockage occurs as a consequence of methylated CpG islands in the 5Ј region of genes, which is mediated by DNMTs during DNA replication. 2 In contrast to gene mutation, DNA methylation is a reversible event open to attack by DNA methylation inhibitors like 5-Aza-CdR. As a nucleoside analogue, 5-Aza-CdR is integrated into DNA and inhibits DNA methylation by trapping DNMTs. While other methylation inhibitors like zebularine are known, most data are available for 5-Aza-CdR in terms of its clinical utility. Two phase II clinical studies have reported promising therapeutic activity of 5-Aza-CdR (decitabine) for patients with MDS 3 or CML. 4 Two other studies, enrolling mostly patients with AML, reported good response rates after decitabine treatment. 5,6 5-Aza-CdR effectively reverts methylation-dependent gene repression, as shown, e.g., for p15 INK4b in vitro and in vivo. 6 In AML cells, promoter hypermethylation has been reported for several other genes, including the estrogen receptor, E-cadherin 7 HIC1, calcitonin 8 and MYOD. 9 The molecular effects of 5-Aza-CdR can include histone deacetylation a...

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Section: G 1 Cycle Arrest Of Aml Cells After Treatment With 5-aza-cdrmentioning

confidence: 79%

5‐Aza‐2′‐deoxycytidine induces p21^WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

Schmelz

Wagner

Dörken

et al. 2005

Intl Journal of Cancer

106

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“…The promyelocytic cell line NB4 is characterized by mutated TP53 and non-functional p53 protein [22,23] as well as wild type FLT3 [24]. DNA damaging 25 Gy IR of NB4 cells resulted in increased apoptosis, but no modulation of FLT3 or HDM2 mRNA was observed (Fig.…”

Section: Resultsmentioning

confidence: 99%

Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

et al. 2007

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Background: Acute myeloid leukemia (AML) cells are characterized by non-mutated TP53, high levels of Hdm2, and frequent mutation of the Flt3 receptor tyrosine kinase. The juxtamembrane mutation of FLT3 is the strongest independent marker for disease relapse and is associated with elevated Bcl-2 protein and p53 hyper-phosphorylation in AML. DNA damage forms the basic mechanism of cancer cell eradication in current therapy of AML.

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“…OCI-AML-3 and MOLM-13 cells have wild-type p53, whereas p53 is disabled in HL-60 and NB4 cell lines by either deletion (HL-60) or missense mutation (NB4) of the p53 gene. [28][29][30] Bone marrow and peripheral blood samples were obtained from patients with AML (Ͼ 60% blasts) and healthy donors after informed consent, according to institutional guidelines and the Declaration of Helsinki. Mononuclear cells were purified by Ficoll-Hypaque (Sigma Chemical, St Louis, MO) density-gradient centrifugation.…”

Section: Cell Lines Primary Samples and Cell Culturesmentioning

confidence: 99%

MDM2 antagonists induce p53-dependent apoptosis in AML: implications for leukemia therapy

Kojima

Konopleva

Samudio

et al. 2005

Blood

356

403

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Detection of p53 gene mutations by single strand conformational polymorphism (SSCP) in human acute myeloid leukemia-derived cell lines

Cited by 26 publications

References 40 publications

5‐Aza‐2′‐deoxycytidine induces p21^WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

5‐Aza‐2′‐deoxycytidine induces p21^WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

MDM2 antagonists induce p53-dependent apoptosis in AML: implications for leukemia therapy

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Detection of p53 gene mutations by single strand conformational polymorphism (SSCP) in human acute myeloid leukemia-derived cell lines

Cited by 26 publications

References 40 publications

5‐Aza‐2′‐deoxycytidine induces p21WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

5‐Aza‐2′‐deoxycytidine induces p21WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

MDM2 antagonists induce p53-dependent apoptosis in AML: implications for leukemia therapy

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5‐Aza‐2′‐deoxycytidine induces p21^WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

5‐Aza‐2′‐deoxycytidine induces p21^WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia