A. Borkhardt

Ras-mediated activation of ERK by cisplatin induces cell death independently of p53 in osteosarcoma and neuroblastoma cell lines

Abstract. Activation of the mitogen-activated protein kinases ERK1/2 by the chemotherapeutic agent cisplatin has been shown to result in either survival or cell death. The downstream mediators of these opposing effects are unknown, as are the upstream signaling molecules. Activation of ERK is required for accumulation and phosphorylation of p53 following cisplatin treatment. We studied the role of ERK activation after cisplatin treatment under p53-negative and p53-positive conditions using a tetracycline-dependent expression vector in Saos-2 osteosarcoma cells. Dose-dependent activation of ERK first occurred 3–6xa0h after a 2-h cisplatin incubation and declined after 12–24xa0h in several tumor cell lines. Incubation of cell lines with the MEK1 inhibitors PD98059 or UO126 after, but not during, cisplatin treatment completely inhibited cisplatin-induced activation of ERK. The activation of ERK by cisplatin was inhibited by transient transfection with dominant-negative Ras-N17 in Saos-2 cells. Treatment of cells with PD98059 or UO126 after cisplatin incubation or inhibition of signaling through ERK by tetracycline-regulated expression of dominant-inhibitory ERK enhanced resistance to cisplatin in p53-negative osteosarcoma cells and reduced cisplatin-induced apoptosis. P53 was stabilized and phosphorylated in a MEK1-dependent manner after cisplatin incubation in Kelly neuroblastoma cells. Inhibition of signaling through ERK increased cell survival after cisplatin treatment in these cells as well. Expression of functional p53 did not change the proapoptotic effects of ERK activation in response to cisplatin in Saos-2 cells. Our results suggest that cisplatin-induced activation of ERK is mediated by Ras. ERK activation increased cisplatin-induced cell death independently of p53 in osteosarcoma and neuroblastoma cell lines.

Disinfectant contaminated with Klebsiella oxytoca as a source of sepsis in babies

We report an outbreak of sepsis from contaminated disinfectant in a neonatal and paediatric intensive-care unit. 28 infants were infected with Klebsiella oxytoca and basic measures to control the outbreak failed. The resistance of K. oxytoca against the disinfectant was probably mediated by capsule formation, visible as mucoid colonies.

Infant acute lymphoblastic leukemia - combined cytogenetic, immunophenotypical and molecular analysis of 77 cases.

We used karyotyping, fluorescence in situ hybridization (FISH), Southern blotting, and RT-PCR in order to analyze prospectively 77 infants (less than 1 year of age) with acute lymphoblastic leukemia for the occurrence of 11q23/MLL rearrangements and/or other cytogenetic abnormalities. Out of the 69 informative samples we found an 11q23/MLL rearrangement in 42 cases (61%). Regarding only pro-B ALL cases, the incidence of 11q23/MLL rearranged cases, however, reached more than 90% The infants were treated within the therapy studies ALL-BFM90, ALL-BFM95 and CoALL-05–92. For patients with an adequate follow-up of 4 years the event-free survival of the 11q23/MLL-positive and 11q23/MLL-negative group was 0.2 or 0.64, respectively (P = 0.024). The monoclonal antibody 7.1. (moab 7.1) does not react with normal hematopoetic precursors or mature blood cells but was shown to specifically react with leukemic cells bearing a rearrangement of chromosome 11q23 or the MLL gene, respectively. We, therefore, specifically addressed the question whether the reactivity of moab 7.1, as determined by flow cytometry, may substitute for molecular testing of an 11q23/MLL rearrangement in this cohort of infant ALLs. Reactivity of moab 7.1 indicated a 11q23/MLL rearrangement with a specificity of 100%. However, five of the 11q23/MLL-positive cases did not react with moab 7.1 indicating a sensitivity of 84% only. Three of these five moab 7.1-negative but 11q23/MLL-positive cases could be identified by their unique expression pattern of CD65s and/or CD15. Thus, 95% of all 11q23/MLL-positive ALL cases in infancy may be identified by flow cytometry based on their expression of CD15, CD65s and/or moab 7.1.

Polymorphisms of surfactant protein B encoding gene: modifiers of the course of neonatal respiratory distress syndrome?

Abstract. Surfactant protein B (SP-B) is a lipophilic protein and plays a major role in lung mechanics. Polymorphisms of surfactant protein A, another component of the surfactant system, have been previously described to be a risk factor for respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in preterms. The aim of this prospective study was to determine whether polymorphisms within intron 4 of the SP-B gene are related to the incidence, severity and complications of RDS in Caucasian newborns. In order to identify SP-B intron 4 polymorphisms, we analysed genomic DNA by means of polymerase chain reaction, fragment length and sequence analysis in 140 preterms and 58 healthy term neonates. The frequency of intron 4 variations did not differ between preterms and terms. A total of 111 preterms with the intron 4 wild type (group 1) and 29 preterms carrying the genetic variations (group 2) did not differ in gestational age, gender distribution and birth weight. Compared to group 1, the overall incidence of RDS (75.7% versus 93.1%, P<0.05), the frequency of severe RDS (28.4% versus 55.2%, P<0.01) and BPD (21.6% versus 48.3%, P<0.01) were all higher in group 2. The median duration of oxygen dependency (4 days versus 17 days, P<0.05) and the need for surfactant administration were also higher in group 2 than in group 1 (43.2% versus 72.4%, P<0.01). Duration of mechanical ventilation and rate of chronic lung disease at 36 weeks were comparable in both groups. Conclusion: we suggest that polymorphisms in intron 4 of the surfactant protein B gene independently modify the course of neonatal respiratory distress syndrome.

An ins(X;11)(q24;q23) fuses the MLL and the Septin 6/KIAA0128 gene in an infant with AML-M2

The MLL (HRX, ALL‐1 HTRX) gene at chromosome band 11q23 frequently is rearranged in acute lymphoblastic and myeloblastic leukemia. To date, more than 40 different 11q23 abnormalities have been described on the cytogenetic level, and at least 25 of the respective fusion partner genes are cloned. The vast majority of the respective reciprocal translocations generate a chimeric 5′‐MLL/partner‐3′ gene on the derivative 11q23. In this work, we report a unique ins(X;11)(q24;q23) in an infant with acute myeloid leukemia (AML‐M2) that fuses the human KIAA0128 gene at Xq24 with MLL. In contrast to the typical reciprocal MLL translocations, however, we provide evidence that the 5′‐MLL/KIAA0128‐3′ fusion resides on Xq24 rather than on 11q23. The KIAA0128 gene encodes the human Septin 6 protein, which contains an ATP‐GTP binding motif and three nuclear targeting sequences in its carboxy terminus. The maintenance of the reading frame of the 5′‐MLL/KIAA0128‐3′ mRNA fusion allows for the formation of a novel chimeric protein. Septin 6 is the third member of the Septins that is fused to the MLL protein; the other two are hCDCrel at 22q11 and MSF at 17q25.

Detection of a hepatitis B virus variant with a truncated X gene and enhancer II.

SummaryApproximately 15% of the viral genomes in the serum of a hepatitis B carrier were found to exhibit an 8 base-pair deletion within the enhancer II element, which would truncate the X protein by 26 amino acids at the carboxy-terminus.

Detection of chromosomal translocations in leukemia-lymphoma cells by polymerase chain reaction.

In recent years many chromosomal translocations involved in leukemia and lymphoma have been defined at the molecular level. In addition to advancing the understanding of pathological mechanisms underlying the transformation process, the cloning and sequencing of the genes altered by the translocations have provided new tools for diagnosis and monitoring of patients. In particular, the polymerase chain reaction (PCR) methodology yields rapid, sensitive and accurate diagnostic and prognostic information. As leukemias carrying certain translocations confer a higher risk of treatment failure, it is important to identify accurately all positive cases in order to give appropriate therapy. An important new initiative in the diagnostical setting and anti-leukemic therapy is the early detection of minimal residual disease (MRD). If MRD, implying an increased risk of relapse, is reliably detected during apparent clinical remission, alternative strategies could be applied early while the malignant cell burden is still minimal. The PCR assays are clearly more sensitive than other methods of MRD detection including morphology, immunophenotyping and cytogenetics; treatment failure is first detectable by PCR followed by cytogenetic relapse and finally clinical disease. PCR assays have been most often used in the MRD analysis of follicular lymphoma with t(14;18), chronic myeloid leukemia and acute lymphoblastic leukemia (ALL) with t(9;22), ALL with t(4;11), and acute myeloid leukemia (AML) with t(8;21) or t(15;17). PCR amplification is applicable to any other translocation provided the translocation is highly associated with the malignancy and the breakpoints are sufficiently clustered; a quickly increasing number of such specific molecular markers are now available for PCR assays. PCR still remains an experimental investigation for the detection of covert disease. However, the clinical relevance of MRD detection should be evaluated separately for each type of leukemia as significant prognostic differences between disease entities were found. This review describes the PCR assays available for the detection of leukemia cells with specific chromosomal translocations and summarizes the experience with the application of PCR techniques in monitoring patients during the course of the disease.

ERK signaling pathway is differentially involved in erythroid differentiation of K562 cells depending on time and the inducing agent

K562 cells can be induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, butyrate, cisplatin and ara‐C. Differential signaling through MAP kinases has been suggested to be involved in this differentiation process. We have investigated the involvement of ERK activation/inhibition in hemin‐, butyrate‐, cisplatin‐ and ara‐C‐induced erythroid differentiation using the K562 cell line. ERK activity decreased for 2–4 h after administration of either inducing agent. ERK was then activated by hemin and cisplatin, while ERK phosphorylation remained decreased during incubation with butyrate and ara‐C. There was no activation of JNK or p38. The MEK‐1 inhibitors UO126 or PD98059 induced erythroid differentiation in K562 cells and acted additively with butyrate. Inhibition of MEK‐1 reduced the hemoglobin accumulation by hemin and cisplatin; erythroid differentiation by ara‐C was unchanged. The results suggest that inhibition of signaling through ERK in K562 cells may be needed to enter the erythroid differentiation process, while after initiation both activation and inhibition of signaling through ERK enhance erythroid differentiation, which, however, is dependent on the inducing compound.

Frequency and DNA sequence of tal-1 rearrangement in children with T-cell acute lymphoblastic leukemia

SummaryUsing nested polymerase chain reaction (PCR) a gene rearrangement named tal-1 deletion was found in five of 56 leukemic bone marrow samples from children with T-cell acute lymphoblastic leukemia (ALL). The DNA sequences of the PCR fragments consisted of the known conserved germline sequences in addition to short DNA insertions at the breakpoint region, which were different in each patient. Moreover, one patient was examined at diagnosis and at relapse 11 months later, revealing identical DNA sequences at the rearrangement site. The recombination site of the tal rearrangement therefore may be used as a genetic marker for detecting minimal residual disease in about 10% of T-cell ALL in childhood.

Asymmetric multiplex‐polymerase chain reaction – a high throughput method for detection and sequencing genomic fusion sites in t(4;11)

Chromosomal translocations are a characteristic feature of leukaemia and other malignant diseases. As clonal markers, they can be applied to identify and quantify the number of malignant cells by polymerase chain reaction (PCR) methods. The translocation t(4;11) is present in >60% of infant leukaemia. In order to facilitate the sequencing of chromosomal breakpoints, we developed an optimized set of 30 PCR primers and a new approach, designated as asymmetric multiplex PCR (am‐PCR). Due to the high number of primers, small breakpoint‐spanning DNA fragments are obtained in one nested multiplex PCR reaction. All PCR products contain an identical binding site for the initiation of direct sequencing. By using am‐PCR, the translocation t(4;11) was examined in bone marrow and blood samples from children with acute leukaemia. Compared with previously described methods for the determination of genomic breakpoints, am‐PCR may be advantageous with regard to its simplicity and rapidity. Breakpoint‐spanning sequences were also evaluated with regard to their applicability as unique clonal markers to design primers and probes for minimal residual disease quantification by real‐time PCR. This approach can easily be adapted to other chromosomal translocations in malignant diseases for the detection and analysis of clone‐specific DNA markers.