Molecular Characterization of the t(15;17)(q22;21) in Epirubicin-Related Acute Promyelocytic Leukaemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 791-791
Author(s):  
Ashley Nicole Mays ◽  
Neil Osheroff ◽  
Carolyn Felix ◽  
Jo Ann Byl ◽  
Kandeepan Saravanamuttu ◽  
...  
Keyword(s):  
Long Range ◽  
Acute Promyelocytic Leukaemia ◽  
Topoisomerase Ii ◽  
The Other ◽  
Promyelocytic Leukaemia ◽  
Chromosome 15 ◽  
Accession Number ◽  
Long Range Pcr ◽  
Intron 2

Abstract Chromosomal translocations generating chimaeric oncoproteins play an important role in leukaemogenesis, but mechanisms underlying their formation are largely unclear. Substantial insights can be gained from the analysis of therapy-related acute myeloid leukaemias (t-AMLs), which are becoming an increasing healthcare problem as more patients survive their primary cancers. Exposure to agents targeting topoisomerase II (topoII) predisposes to the development of leukaemias with balanced translocations e.g. t(15;17), fusing PML and RARA genes, in therapy-related acute promyelocytic leukaemia (t-APL) which is particularly associated with prior treatment involving mitoxantrone or epirubicin. Using long-range PCR and sequencing to define genomic junction regions we found that in t-APL cases arising in breast cancer patients exposed to the former agent, chromosome 15 breakpoints clustered tightly in an 8 bp “hotspot” region in PML intron 6, which was shown by functional assay to be a preferred site of mitoxantrone-induced DNA topoII cleavage (Mistry et al, NEJM 2005). Subsequent analysis of an independent cohort of t-APL cases arising after mitoxantrone therapy for multiple sclerosis confirmed chromosome 15 breakpoint clustering in the “hotspot” and identified a recurrent breakpoint within RARA intron 2. This hotspot also was shown to be a preferential site of mitoxantrone-induced cleavage in vitro (Hasan et al, Blood 2008). However, the molecular basis of epirubicin-related APL remains uncertain. Therefore we used long-range PCR and sequence analysis to define translocation breakpoints in 6 patients who developed APL after treatment involving epirubicin-containing regimens for prior breast carcinoma. While mitoxantrone-related APL displayed a bias towards breakpoints occurring within PML intron 6 (bcr1), epirubicin-related breakpoints fell within intron 3 (n=2) or intron 6 (n=4) and were outside the mitoxantrone-related “hotspot” (located at position 1484 according to accession number S57791). Breakpoints within the RARA locus were distinct from those observed in mitoxantrone-related APL. Interestingly, the chromosome 15 or chromosome 17 breakpoints of 4 of the epirubicin-related t-APLs fell in close proximity (within 1–4bp) to one of the other cases. Two shared a breakpoint location in PML intron 6 that occurred at bases 1185 and 1186 (accession number S57791) and two fell within RARA intron 2 at base numbers 16193 and 16197 (accession number AJ297538). Given that PML intron 6 and RARA intron 2 are ~1kb and 17kb in length, respectively, such breakpoint clustering was unlikely to have occurred by chance and consistent with functional sites of topoII cleavage. In addition, in vitro DNA cleavage assays demonstrated that heat stable topoII cleavage complexes are formed at the exact location of the breakpoint identified in one of the other patients, at position 1969 in PML intron 6, which were enhanced by the presence of epirubicin. This study suggests that mitoxantrone and epirubicin exhibit preference differences in sites of DNA damage induced by topoisomerase II, which may underlie the propensity to develop specific molecularly-defined subtypes of t-AML according to the nature of the particular chemotherapeutic agent used.

scholarly journals Chromosome damage induced by DNA topoisomerase II inhibitors combined with g-radiation in vitro

1998 ◽  
Vol 21 (3) ◽  
pp. 407-417
Author(s):  
Maria Cristina P. Araújo ◽  
Francisca da Luz Dias ◽  
Andréa O. Cecchi ◽  
Lusânia M.G. Antunes ◽  
Catarina S. Takahashi
Keyword(s):  
Cho Cells ◽  
Topoisomerase Ii ◽  
Chinese Hamster ◽  
The Other ◽  
Additive Effects ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Topoisomerase Ii Inhibitors ◽  
Radiation Induced

Combined radiation and antineoplastic drug treatment have important applications in cancer therapy. In the present work, an evaluation was made of two known topoisomerase II inhibitors, doxorubicin (DXR) and mitoxantrone (MXN), with g-radiation. The effects of DXR or MXN on g-radiation-induced chromosome aberrations in Chinese hamster ovary (CHO) cells were analyzed. Two concentrations of each drug, 0.5 and 1.0 µg/ml DXR, and 0.02 and 0.04 µg/ml MXN, were applied in combination with two doses of g-radiation (20 and 40 cGy). A significant potentiating effect on chromosomal aberrations was observed in CHO cells exposed to 1.0 µg/ml DXR plus 40 cGy. In the other tests, the combination of g-radiation with DXR or MXN gave approximately additive effects. Reduced mitotic indices reflected higher toxicity of the drugs when combined with radiation.

scholarly journals In vitro activity of HSR-903, a new quinolone.

1997 ◽  
Vol 41 (6) ◽  
pp. 1326-1330 ◽  
Author(s):  
Y Takahashi ◽  
N Masuda ◽  
M Otsuki ◽  
M Miki ◽  
T Nishino
Keyword(s):  
Topoisomerase Ii ◽  
Inoculum Size ◽  
The Other ◽  
Vitro Activity ◽  
Penicillin G ◽  
In Vitro Activity ◽  
Postantibiotic Effect ◽  
The Family ◽  
Acidic Conditions

The in vitro activity of the new fluoroquinolone HSR-903 was compared with those of ciprofloxacin, lomefloxacin, sparfloxacin, and levofloxacin. HSR-903 inhibited 90% of methicillin-susceptible and -resistant Staphylococcus aureus (MRSA) clinical isolates at 0.78 and 1.56 microg/ml, respectively, and its activity against MRSA was 16-fold higher than those of sparfloxacin and levofloxacin and 64-fold higher than that of ciprofloxacin. The MICs at which 90% of the isolates are inhibited (MIC90s) of HSR-903 for Streptococcus pyogenes and penicillin G-susceptible and -resistant Streptococcus pneumoniae (PRSP) were 0.10, 0.05, and 0.05 microg/ml, respectively. Against PRSP, the activity of HSR-903 was 4-fold higher than that of sparfloxacin and 32- to 256-fold higher than those of the other quinolones. The MIC90 of HSR-903 for Enterococcus faecalis was 0.20 microg/ml, and HSR-903 was more active than the other quinolones against enterococci. The activity of HSR-903 against members of the family Enterobacteriaceae and Pseudomonas aeruginosa was roughly similar to that of ciprofloxacin and greater than those of the other quinolones. Against Haemophilus influenzae, Moraxella catarrhalis, and Helicobacter pylori, HSR-903 was the most potent of the quinolones tested. The activity of HSR-903 was not affected by the medium, the inoculum size, or the addition of serum, but decreased under acidic conditions, as did those of the other quinolones tested. HSR-903 exhibited rapid bactericidal action and had a good postantibiotic effect on S. aureus and P. aeruginosa. HSR-903 inhibited supercoiling by DNA gyrase from Escherichia coli, but it was much less active against human topoisomerase II.

A Paediatric Acute Promyelocytic Leukaemia Patient Harbouring a Cryptic PML-RARA Insertion due to a Complex Structural Chromosome 17 Rearrangement

2017 ◽  
Vol 153 (4) ◽  
pp. 181-189 ◽  
Author(s):  
Racha El-Hajj Ghaoui ◽  
Luke St Heaps ◽  
Dorothy Hung ◽  
Sumanth Nagabushan ◽  
Catherine Harris ◽  
...  
Keyword(s):  
Acute Promyelocytic Leukaemia ◽  
In Situ Hybridisation ◽  
Chromosome 17 ◽  
Promyelocytic Leukaemia ◽  
Chromosome 15 ◽  
Fluorescence In Situ Hybridisation ◽  
Rt Pcr ◽  
Snp Microarray ◽  
Complex Structural

Acute promyelocytic leukaemia with PML-RARA fusion is usually associated with the t(15;17)(q24.1;q21.1) translocation but may also arise from complex or cryptic rearrangements. The fusion usually resides on chromosome 15 but occasionally on others. We describe a cryptic PML-RARA fusion within a novel chromosome 17 rearrangement. We performed interphase fluorescence in situ hybridisation (FISH) using a dual-fusion PML-RARA probe, followed by reverse transcriptase-polymerase chain reaction (RT-PCR) for PML-RARA, karyotyping, and metaphase FISH using RARA break-apart, locus-specific, and subtelomere probes for chromosome 17. An 850K SNP microarray was also employed. Interphase and metaphase FISH showed atypical results involving a single PML-RARA fusion, no second fusion, but instead separate diminished PML and RARA signals. RT-PCR confirmed PML-RARA fusion; however, karyotyping detected only an altered chromosome 17. Metaphase FISH showed the single fusion and diminished 5′ RARA signals located unexpectedly in the subtelomeric short-arm and long-arm regions of the rearranged chromosome 17, respectively. SNP microarray revealed no copy number abnormality. This paediatric patient with PML-RARA fusion reflects a cryptic insertion that resides within a complex and novel chromosome 17 rearrangement. This rearrangement likely arose via 7 chromosome breaks with the insertion occurring first followed by sequential paracentric and then pericentric inversions.

scholarly journals Participation of ATP in the binding of a yeast replicative complex to DNA.

1987 ◽  
Vol 246 (1) ◽  
pp. 213-219 ◽  
Author(s):  
S M Jazwinski
Keyword(s):  
Topoisomerase Ii ◽  
Relative Sensitivity ◽  
The Other ◽  
Functional Assay ◽  
Dna Topoisomerase Ii ◽  
Dna Complexes ◽  
Dna Topoisomerase ◽  
Initial Interaction ◽  
Atp Analogues

The activity that replicates yeast DNA in vitro can be isolated from cells of the budding yeast Saccharomyces in a high-Mr (approximately 2 × 10(6] form. Several lines of evidence indicate that this fraction contains a multiprotein replicative complex. A functional assay has been developed for the analysis of the interaction of the replicating activity with DNA. Binding of the activity required Mg2+, but did not require the addition of ATP or the other ribo- or deoxynucleoside triphosphates. However, the ATP analogues adenosine 5′-[gamma-thio]triphosphate and adenosine 5′-[beta gamma-imido]triphosphate blocked the binding, suggesting that ATP participates in the interaction at some stage. The binding was template (origin)-specific in either the presence or the absence of ATP and the other nucleoside triphosphates; however, ATP stabilized the replicating activity. The preferential inhibition of binding that was observed in the presence of the DNA topoisomerase II inhibitor coumermycin suggests that the requirement for ATP may be at least partially accounted for by the involvement of this enzyme in the initial interaction of the replicating activity with DNA. Finally, the binding was rapid. In contrast, DNA synthesis displayed a lag when assayed directly without first allowing a period for the replicating activity to bind to the DNA. In addition, binding was ‘tight’, as judged by the resistance of the protein–DNA complexes to salt in comparison with the relative sensitivity of binding. The replicating activity was not readily displaced from the complexes by exogenous DNAs, either possessing or lacking yeast origins of replication. The results suggest that the interaction of the replicating activity with the DNA occurs in more than one stage.

The Ellipticine Derivative NSC 338258 Has Anti-Myeloma Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3379-3379
Author(s):  
Erming Tian ◽  
Owen Stephens ◽  
Susan Holbeck ◽  
Bart Barlogie ◽  
John Shaughnessy
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Topoisomerase Ii ◽  
Kinase Inhibitor ◽  
Myeloma Cell ◽  
The Other ◽  
Response Analysis ◽  
Time Points ◽  
Cell Kill

Abstract We recently discovered that amplification and overexpression of the CKS1B gene, which regulates the ubiquitination and proteasomal degradation of the cyclin-dependent kinase inhibitor p27Kip1, is linked to a poor prognosis in myeloma. We used the COMPARE algorithm (NCI, Developmental Therapeutics Program) to identify correlations between the expression of CKS1B in the NCI 60 cell line panel and the GI50 of nearly 50,000 anticancer compounds. This analysis revealed a strong correlation between CKS1B expression and the anticancer activity of Ellipticine and multiple derivatives of it. Ellipticine (5,11-Dimethyl-6H-Pyrido[4,3]Carbazole, MW=246.3), an alkaloid isolated from Apocyanaceae, is a topoisomerase II poison that induces topoisomerase II-dependent DNA cleavages. Previous studies have shown that the anti-neoplastic mechanism of Ellipticine is to form covalent DNA adducts mediated by human cytochromes P450. We obtained Ellipticine and twelve other CKS1B-correlated antineoplastic compounds from the NCI-DTP. In vitro analyses were carried out with 12 myeloma cell lines. Cell viability was detected using CellTiter-Glo Luminescent Assay (Promega, Co.). We found that one of the Ellipticine derivatives, NSC 338258 (EPED3) showed significant cell kill activity not observed with Ellipticine or the other compounds in all cell lines tested. In a dose response analysis of cell kill using 0.2, 0.02, and 0.002 uM EPED3, 12 out of 12 myeloma cell lines showed at least 50% reduction in cell viability within 24 hrs at 0.2 uM compared to no treatment controls. By 48 hours essentially all cell growth was inhibited. Only weak to moderate cell kill was noted at 0.02 and 0.002 uM at all time points tested. We next tested the activity of the topoisomerase II inhibitor VP-16-213 at the same concentrations and time periods and found only modest reduction of cell viability at day 5 at 0.2uM. Essentially no effect on cell viability was observed at 0.02 and 0.002 uM at any of the time points tested. We next tested whether EPED3 had a comparable anti-myeloma activity as Adriamycin at the same concentrations. Twelve myeloma cell lines were exposed to EPED3, VP-16-213, and Adriamycin at 0.2mM for a total of 5 days. Results of the mean relative luminescence showed that EPED3 promoted an immediate cell proliferation arrest within the first 24 hours of culture, with almost complete cell kill within 3 days. The other drugs were less effective with Adriamycin inducing a > 90% reduction only after 4 days and VP-16 a > 70% reduction at day 5. These data suggests that EPED3 may represent a potential new therapeutic for myeloma. Studies are currently underway to test the efficacy of EPED3 in our in-vivo SCID-Hu mouse model of primary myeloma and results of these studies will be presented.

scholarly journals Molecular analysis of t(15;17) genomic breakpoints in secondary acute promyelocytic leukemia arising after treatment of multiple sclerosis

Blood ◽  
2008 ◽  
Vol 112 (8) ◽  
pp. 3383-3390 ◽  
Author(s):  
Syed Khizer Hasan ◽  
Ashley N. Mays ◽  
Tiziana Ottone ◽  
Antonio Ledda ◽  
Giorgio La Nasa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Multiple Sclerosis ◽  
Acute Promyelocytic Leukemia ◽  
Dna Cleavage ◽  
Topoisomerase Ii ◽  
De Novo ◽  
Promyelocytic Leukemia ◽  
Chromosome 15 ◽  
Topoisomerase Iiα ◽  
Intron 2

Abstract Therapy-related acute promyelocytic leukemia (t-APL) with t(15;17) translocation is a well-recognized complication of cancer treatment with agents targeting topoisomerase II. However, cases are emerging after mitoxantrone therapy for multiple sclerosis (MS). Analysis of 12 cases of mitoxantrone-related t-APL in MS patients revealed an altered distribution of chromosome 15 breakpoints versus de novo APL, biased toward disruption within PML intron 6 (11 of 12, 92% vs 622 of 1022, 61%: P = .035). Despite this intron spanning approximately 1 kb, breakpoints in 5 mitoxantrone-treated patients fell within an 8-bp region (1482-9) corresponding to the “hotspot” previously reported in t-APL, complicating mitoxantrone-containing breast cancer therapy. Another shared breakpoint was identified within the approximately 17-kb RARA intron 2 involving 2 t-APL cases arising after mitoxantrone treatment for MS and breast cancer, respectively. Analysis of PML and RARA genomic breakpoints in functional assays in 4 cases, including the shared RARA intron 2 breakpoint at 14 446-49, confirmed each to be preferential sites of topoisomerase IIα-mediated DNA cleavage in the presence of mitoxantrone. This study further supports the presence of preferential sites of DNA damage induced by mitoxantrone in PML and RARA genes that may underlie the propensity to develop this subtype of leukemia after exposure to this agent.

SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation

1999 ◽  
Vol 112 (3) ◽  
pp. 381-393 ◽  
Author(s):  
E. Duprez ◽  
A.J. Saurin ◽  
J.M. Desterro ◽  
V. Lallemand-Breitenbach ◽  
K. Howe ◽  
...  
Keyword(s):  
Acute Promyelocytic Leukaemia ◽  
Consensus Sequence ◽  
Nuclear Bodies ◽  
Promyelocytic Leukaemia ◽  
Fusion Product ◽  
Nuclear Localisation ◽  
Target Proteins ◽  
Pml Nuclear Bodies

PML is a nuclear phosphoprotein that was first identified as part of a translocated chromosomal fusion product associated with acute promyelocytic leukaemia (APL). PML localises to distinct nuclear multi-protein complexes termed ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs), which are disrupted in APL and are the targets for immediate early viral proteins, although little is known about their function. In a yeast two-hybrid screen, we first identified a ubiquitin-like protein named PIC1 (now known as SUMO-1), which interacts and co-localises with PML in vivo. More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and IkappaBalpha and is proposed to play a role in either targeting modified proteins and/or inhibiting their degradation. The precise molecular role for the SUMO-1 modification of PML is unclear, and the specific lysine residues within PML that are targeted for modification and the PML sub-domains necessary for mediating the modification in vivo are unknown. Here we show that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain. Using site-specific mutagenesis, we have identified the primary PML-SUMO-1 modification site as being part of the nuclear localisation signal (Lys487 or Lys490). However SUMO-1 modification is not essential for PML nuclear localisation as only nuclear PML is modified. The sequence of the modification site fits into a consensus sequence for SUMO-1 modification and we have identified several other nuclear proteins which could also be targets for SUMO-1. We show that SUMO-1 modification appears to be dependant on the correct subcellular compartmentalisation of target proteins. We also find that the APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RARA. Our results provide significant insights into the role of SUMO-1 modification of PML in both normal cells and the APL disease state.

Inhibition of RNA Synthesis in vitro by Acridines - Relation between Structure and Activity

1998 ◽  
Vol 53 (5-6) ◽  
pp. 359-368 ◽  
Author(s):  
Mariola K. Piestrzeniewicz ◽  
Dorota Wilmańska ◽  
Kazimierz Studzian ◽  
Janusz Szemraj ◽  
Małgorzata Czyż ◽  
...  
Keyword(s):  
Biological Effect ◽  
Topoisomerase Ii ◽  
Rna Synthesis ◽  
Biological Effects ◽  
The Other ◽  
Inhibitory Potency ◽  
Enzyme Binding ◽  
Primary Mechanism ◽  
Structure And Activity

Abstract The effects of acridine derivatives (proflavine and 2,7-dialkyl derivatives, diacridines and triacridines, 9-aminoacridine carboxamides, and 9-anilinoacridine, amsacrine and its congeners) on overall RNA synthesis in vitro, on synthesis of initiating oligonucleotides and the binding of the enzyme to DNA were studied. The primary mechanism of action is related to inhibition of the enzyme binding to DNA. The acridines (intercalating or non-intercalating and bis-intercalating ligands) assayed here differ in the properties of their complexes with DNA. Correlation is generally observed between inhibition of RNA synthesis in vitro and cytotoxicity in cell cultures for di- and triacridines and 9-aminoacridine carboxamide derivatives. No relationship was found between the effect on RNA polymerase system and biological effects for amsacrine and its derivatives in contrast to the other series of acridines studied here. The aniline ring seems to decrease the inhibitory potency of a ligand. The discrepancy between the biological effect and RNA synthesis inhibition may be due to a different mechanism of cytotoxicity action of amsacrine which is a potent topoisomerase II poison.

scholarly journals Case Report: Prenatal Diagnosis for a Rett Syndrome Family Caused by a Novel MECP2 Deletion With Heteroduplexes of PCR Product

2021 ◽  
Vol 9 ◽  
Author(s):  
Honghong Zhang ◽  
Yixi Sun ◽  
Yuxia Zhu ◽  
Jiali Hong ◽  
Miaomiao Zheng
Keyword(s):  
Prenatal Diagnosis ◽  
Rett Syndrome ◽  
Long Range ◽  
Somatic Mosaicism ◽  
Heterozygous Deletion ◽  
Coding Region ◽  
Pcr Product ◽  
Exon 4 ◽  
Long Range Pcr

Rett syndrome is an X-linked dominant, postnatal neurological disorder. Approximately 80–90% of classic Rett syndrome patients harbor mutations in the coding region of MECP2. Somatic or germline MECP2 mosaicism is not rare, and paternal germline MECP2 mosaicism occurs in especially high proportions. Here, we report the case of a Chinese girl with Rett syndrome in whom a heterozygous deletion was found in exon 4 of MECP2 using multiplex ligation-dependent probe amplification. To obtain an accurate region of deletion, we narrowed down the deletion region using real-time quantitative PCR, and subsequent long-range PCR was performed to detect the deletion breakpoints. Surprisingly, three DNA bands from long-range PCR products were observed after gel electrophoresis. To exclude somatic mosaicism, we performed T-A cloning and DNA sequencing, the middle DNA band was proved to be a heteroduplex of the PCR product in vitro. Meanwhile, a prenatal diagnosis was performed for the pregnant mother of the patient. Our study showed that the patient was heterozygous for the deletion of 713-base pairs in exon 4 of MECP2 (MECP2: c.441_1153del713), resulting in a frameshift and premature termination of the 487 amino acid protein at the 154th codon. In summary, we reported a novel heterozygous deletion in the MECP2 gene with heteroduplexes of the PCR product in vitro, which can help in the genetic counseling and prenatal diagnosis of disorders of MECP2 defects.

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