scholarly journals DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis.

1997 ◽  
Vol 17 (7) ◽  
pp. 4070-4079 ◽  
Author(s):  
M Stanulla ◽  
J Wang ◽  
D S Chervinsky ◽  
S Thandla ◽  
P D Aplan
Keyword(s):  
Dna Cleavage ◽  
Cellular Response ◽  
Topoisomerase Ii ◽  
Apoptotic Cell ◽  
Chemotherapeutic Agents ◽  
Higher Order ◽  
Site Specific ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Topo Ii

A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML.

scholarly journals Site-specific DNA cleavage within the MLL breakpoint cluster region induced by topoisomerase II inhibitors [see comments]

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2649-2658 ◽  
Author(s):  
PD Aplan ◽  
DS Chervinsky ◽  
M Stanulla ◽  
WC Burhans
Keyword(s):  
Dna Cleavage ◽  
Topoisomerase Ii ◽  
Model Systems ◽  
Site Specific ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Topoisomerase Ii Inhibitors ◽  
Mll Gene ◽  
Secondary Leukemias ◽  
Double Strand Dna

The MLL gene located at 11q23 is frequently disrupted by chromosomal translocation in a wide spectrum of newly diagnosed acute leukemias. Recently, it has become apparent that the MLL gene is very frequently disrupted by chromosomal translocations in patients with secondary leukemias associated with chemotherapeutic regimens incorporating topoisomerase II inhibitors. These secondary leukemias associated with topoisomerase II inhibitors (most commonly teniposide, etoposide, or doxorubicin) have distinct clinical and biologic features which have led to the speculation that they are induced by treatment with topoisomerase II inhibitors. We have identified a site within the MLL breakpoint cluster region (bcr) that is highly sensitive to double- strand DNA cleavage induced by topoisomerase II inhibitors. This finding is quite specific and highly reproducible. Although it was initially discovered in malignant lymphoblasts isolated from a patient receiving multiagent chemotherapy, this site-specific double-strand DNA cleavage can be induced in tissue culture using malignant cell lines as well as peripheral blood from normal individuals. Site-specific cleavage occurs in a significant fraction of cells using a variety of model systems, is both time and dose dependent, and can be induced with either doxorubicin or etoposide. This site-specific cleavage maps to the same region as a consensus topoisomerase II cleavage site within the MLL bcr. These results suggest that site specific cleavage within the MLL bcr induced by topoisomerase II inhibitors may be an early step leading to MLL translocations and secondary leukemia.

scholarly journals Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia: correlation with scaffold attachment regions and topoisomerase II consensus binding sites

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1912-1922 ◽  
Author(s):  
PL Broeker ◽  
HG Super ◽  
MJ Thirman ◽  
H Pomykala ◽  
Y Yonebayashi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Topoisomerase Ii ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Topo Ii ◽  
Acute Myeloid

Abstract A major unresolved question for 11q23 translocations involving MLL is the chromosomal mechanism(s) leading to these translocations. We have mapped breakpoints within the 8.3-kb BamHI breakpoint cluster region in 31 patients with acute lymphoblastic leukemia and acute myeloid leukemia (AML) de novo and in 8 t-AML patients. In 23 of 31 leukemia de novo patients, MLL breakpoints mapped to the centromeric half (4.57 kb) of the breakpoint cluster region, whereas those in eight de novo patients mapped to the telomeric half (3.87 kb). In contrast, only two t-AML breakpoints mapped in the centromeric half, whereas six mapped in the telomeric half. The difference in distribution of the leukemia de novo breakpoints is statistically significant (P = .02). A similar difference in distribution of breakpoints between de novo patients and t-AML patients has been reported by others. We identified a low- or weak-affinity scaffold attachment region (SAR) mapping just centromeric to the breakpoint cluster region, and a high-affinity SAR mapping within the telomeric half of the breakpoint cluster region. Using high stringency criteria to define in vitro vertebrate topoisomerase II (topo II) consensus sites, one topo II site mapped adjacent to the telomeric SAR, whereas six mapped within the SAR. Therefore, 74% of leukemia de novo and 25% of t-AML breakpoints map to the centromeric half of the breakpoint cluster region map between the two SARs; in contrast, 26% of the leukemia de novo and 75% of the t-AML patient breakpoints map to the telomeric half of the breakpoint cluster region that contains both the telomeric SAR and the topo II sites. Thus, the chromatin structure of the MLL breakpoint cluster region may be important in determining the distribution of the breakpoints. The data suggest that the mechanism(s) leading to translocations may differ in leukemia de novo and in t-AML.

scholarly journals Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia: correlation with scaffold attachment regions and topoisomerase II consensus binding sites

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1912-1922 ◽  
Author(s):  
PL Broeker ◽  
HG Super ◽  
MJ Thirman ◽  
H Pomykala ◽  
Y Yonebayashi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Topoisomerase Ii ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Topo Ii ◽  
Acute Myeloid

A major unresolved question for 11q23 translocations involving MLL is the chromosomal mechanism(s) leading to these translocations. We have mapped breakpoints within the 8.3-kb BamHI breakpoint cluster region in 31 patients with acute lymphoblastic leukemia and acute myeloid leukemia (AML) de novo and in 8 t-AML patients. In 23 of 31 leukemia de novo patients, MLL breakpoints mapped to the centromeric half (4.57 kb) of the breakpoint cluster region, whereas those in eight de novo patients mapped to the telomeric half (3.87 kb). In contrast, only two t-AML breakpoints mapped in the centromeric half, whereas six mapped in the telomeric half. The difference in distribution of the leukemia de novo breakpoints is statistically significant (P = .02). A similar difference in distribution of breakpoints between de novo patients and t-AML patients has been reported by others. We identified a low- or weak-affinity scaffold attachment region (SAR) mapping just centromeric to the breakpoint cluster region, and a high-affinity SAR mapping within the telomeric half of the breakpoint cluster region. Using high stringency criteria to define in vitro vertebrate topoisomerase II (topo II) consensus sites, one topo II site mapped adjacent to the telomeric SAR, whereas six mapped within the SAR. Therefore, 74% of leukemia de novo and 25% of t-AML breakpoints map to the centromeric half of the breakpoint cluster region map between the two SARs; in contrast, 26% of the leukemia de novo and 75% of the t-AML patient breakpoints map to the telomeric half of the breakpoint cluster region that contains both the telomeric SAR and the topo II sites. Thus, the chromatin structure of the MLL breakpoint cluster region may be important in determining the distribution of the breakpoints. The data suggest that the mechanism(s) leading to translocations may differ in leukemia de novo and in t-AML.

Site-Specific DNA Cleavage byChlorellaVirus Topoisomerase II†

Biochemistry ◽  
2002 ◽  
Vol 41 (39) ◽  
pp. 11761-11769 ◽  
Author(s):  
John M. Fortune ◽  
Jennifer S. Dickey ◽  
Oleg V. Lavrukhin ◽  
James L. Van Etten ◽  
R. Stephen Lloyd ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Topoisomerase Ii ◽  
Site Specific

scholarly journals Attenuation of drug-stimulated topoisomerase II–DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIα

1998 ◽  
Vol 336 (3) ◽  
pp. 727-733 ◽  
Author(s):  
Masako AOYAMA ◽  
Dale R. GRABOWSKI ◽  
George R. DUBYAK ◽  
Andreas I. CONSTANTINOU ◽  
Lisa A. RYBICKI ◽  
...  
Keyword(s):  
Dna Damage ◽  
Complex Formation ◽  
Topoisomerase Ii ◽  
Wild Type ◽  
Acetoxymethyl Ester ◽  
Free System ◽  
Site Specific ◽  
Calcium Buffer ◽  
Topo Ii ◽  
Cleavable Complex

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIα phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II–DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II–DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N´,N´-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II–DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIα. Tryptic phosphopeptide mapping of topo IIα protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746–29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIα are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.

Stimulation of Site-Specific Topoisomerase II-Mediated DNA Cleavage by an N-Methylpyrrolecarboxamide-anilinoacridine Conjugate: Relation to DNA Binding

Biochemistry ◽  
1994 ◽  
Vol 33 (33) ◽  
pp. 9865-9874 ◽  
Author(s):  
Philippe Fosse ◽  
Brigitte Rene ◽  
Jean-Marie Saucier ◽  
Jean-Pierre Henichart ◽  
Michael J. Waring ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Cleavage ◽  
Topoisomerase Ii ◽  
Site Specific ◽  
Stimulation Of

MLL-Aberrations, Associated to T-AML, Are Induced by Chemotherapy In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3004-3004
Author(s):  
Jorg Basecke ◽  
Edda Seiffert ◽  
Kamran Karim ◽  
Martina Podleschny ◽  
Annegret Becker ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Early Recognition ◽  
Alkylating Agents ◽  
Inverse Pcr ◽  
High Dose ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Topo Ii

Abstract Background: Therapy related acute myelogenous leukemias (t-AML) are frequently observed following previous chemotherapy with alkylating agents and topoisomerase II-inhibitors (topo II-i.). Their incidence ranges between 5 – 15% in non-myeloablative and myeloablative treatment protocols respectively. T-AML after topo II-i. therapy often exhibit balanced MLL/11q23 translocations, e.g. t(9;11) and t(11;19). It is thought that the first step to induce MLL translocations is breakage and illegitimate recombination at the MLL breakpoint cluster region (bcr), which can be detected by genomic MLL-inverse PCR in vitro (Libura et al. 2004, Blood). The aim of this study was to detect the induction of MLL-aberrations in vivo after topo II-i. containing treatment regimens, using RT- and genomic inverse PCR (iPCR) and evaluate this information for early recognition of t-AML. Material and Methods: Patients with non-Hodgkin’s lymphoma, treated by intermediate or high dose chemotherapy, (MegaCHOEP protocol of the DSNHL) were enrolled. 216 samples of 75 patients were taken as peripheral blood (PB) after informed consent according to the convention of Helsinki. Peripheral blood of healthy adult donors were used as control. Blood samples were subjected either to a nested t(9;11) and t(11;19) RT- PCR or the genomic MLL-iPCR allowing detection of non specific aberrations in the MLL breakpoint cluster region. iPCR was carried out as described (Libura et al.). Processing of samples was carried out according to GLP guidelines for PCR. Results: 215 samples of 76 patients, taken at different time points of therapy have been investigated. The incidence of the t(9;11) and t(11,19) ranges between 0,5% (1/216) and 9,7% (21/216) of all samples and 1,3% (1/76) and 27,6% (21/76) of all patients. There was a tendency towards a higher incidence of these aberrations in patients which have completed therapy. Pretherapeutic samples were always PCR-negative and none of the patients has developed a t-AML so far. A total of 36 samples (18 healthy subjects, 18 patients) were subjected to MLL-iPCR. Surprisingly, the incidence of MLL-aberrations (deletions, insertions) in the healthy cohort was comparable to that of the patients’ samples (14/18 vs.12/18). We speculated that MLL-aberrations ex vivo might correlate with spontaneous apoptosis. We studied 10 samples of healthy donors at 0, 24 and 48h after having taken the sample. MLL-aberrations were not observed at 0 hrs, but emerged in 5/10 and 7/10 samples after 24 and 48h, respectively. Conclusion: These results show, that chemotherapy seems to induce the emergence of the t-AML associated MLL-translocations t(9;11) and t(11;19). Since their incidence exceeds the incidence of t-AML, characterized by these translocations, most of the positive PCR-results seem to represent a transient chemotherapy induced genetic instability and not a t-AML in a preleukemic phase. The genomic MLL iPCR is currently used for the detection of chemotherapy-induced MLL-genomic aberrations. Our results indicate, that apoptosis might induce aberrations in the MLL bcr, which need to be discerned from those which are provoked by chemotherapy.

Stimulation by γ-carboline derivatives (simplified analogues of antitumor ellipticines) of site specific DNA cleavage by calf DNA topoisomerase II

1990 ◽  
Vol 39 (4) ◽  
pp. 669-676 ◽  
Author(s):  
Philippe Fossé ◽  
Brigitte René ◽  
Jean-Marie Saucier ◽  
Chi Hung Nguyen ◽  
Emile Bisagni ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Site Specific

Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus

1990 ◽  
Vol 10 (6) ◽  
pp. 2774-2786
Author(s):  
M Reitman ◽  
G Felsenfeld
Keyword(s):  
Dna Cleavage ◽  
Topoisomerase Ii ◽  
Dnase I ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Beta Globin ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
Topo Ii ◽  
Erythroid Development

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5'-rho-beta H-beta A-epsilon-3'). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of rho to just downstream of epsilon. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the rho-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of rho, beta H, and epsilon. (v) A site 3' of beta A and 5' of epsilon was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo II sites. These data are consistent with the hypothesis that, in vivo, topo II preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.

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