Functional effects of endothelin and regulation of endothelin receptors in isolated human nonfailing and failing myocardium.

BACKGROUND: An activated endothelin (ET) system may be of pathophysiological relevance in human heart failure. We characterized the functional effects of ET-1, ET receptors, and ET-1 peptide concentration in left ventricular myocardium from 10 nonfailing hearts (NF) and 27 hearts in end-stage failure due to idiopathic dilative cardiomyopathy (DCM). METHODS AND RESULTS: Inotropic effects were characterized in isolated muscle strips (1 Hz; 37 degrees C). ET-1 0.0001 to 0.3 micromol/L significantly (P<0.05) increased twitch force by maximally 59+/-10% in NF and by 36+/-11% in DCM (P<0.05 versus NF). Preincubation with propranolol 1 micromol/L and prazosin 0.1 micromol/L did not affect the response to ET-1, but the mixed ET receptor antagonist bosentan and the ETA receptor antagonist BQ-123 shifted the concentration-response curves for ET-1 rightward. The ETB receptor agonist sarafotoxin S6c 0.001 to 0.3 micromol/L had no functional effects. The inotropic response to ET-1 was not associated with increased intracellular Ca2+ transients, as assessed in aequorin-loaded muscle strips. ET receptor density (Bmax; radioligand binding) was 62.5+/-12.5 fmol/mg protein in NF and 122. 4+/-24.3 fmol/mg protein in DCM (P<0.05 versus NF). The increase in Bmax in DCM resulted from an increase in ETA receptors without change in ETB receptors. ET-1 peptide concentration (radioimmunoassay) was higher in DCM than in NF (14 447+/-2232 versus 4541+/-1340 pg/mg protein, P<0.05). CONCLUSIONS: ET-1 exerts inotropic effects in human myocardium through ETA receptor-mediated increases in myofibrillar Ca2+ responsiveness. In DCM, functional effects of ET-1 are attenuated, but ETA receptor density and ET-1 peptide concentration are increased, indicating an activated local cardiac ET system and possibly a reduced postreceptor signaling efficiency.

Philadelphia chromosome in relapsed childhood acute lymphoblastic leukemia: a matched-pair analysis. Berlin-Frankfurt-Münster Study Group.

PURPOSE: The translocation t(9;22)(q34;q11), known as Philadelphia chromosome (Ph1) or its molecular equivalent the expression of BCR-ABL-mRNA, is one of the most striking and well-characterized cytogenetic abnormalities in leukemia. Although investigated for more than 30 years, it remains unclear whether the Ph1 is an independent risk factor for outcome of leukemia or not. METHODS: A matched-pair analysis was performed within a homogeneous group of patients, which consisted of children who presented with a first relapse of acute lymphoblastic leukemia (ALL) who were treated according to ALL relapse trials (ALL-REZ BFM) protocols. A total of 307 patients were eligible for this analysis: 30 positive and 277 negative for Ph1. Positive patients were matched exactly for time point of relapse (on [during] or off [after cessation of] front-line therapy), site, and immunophenotype, and as close as possible for duration of first remission, peripheral blast-cell count, WBC count, and year of relapse diagnosis. RESULTS: The probability of event-free survival is 0.46 at 5 years for negative and 0.11 for positive patients, respectively (P = .0006). Multivariate analysis showed risk ratios of 4.229 for relapse on therapy, 3.561 for Ph1 and/or expression of BCR-ABL- mRNA, 1.691 for high peripheral blast-cell count, and 0.232 for bone marrow transplantation. CONCLUSION: It was shown that the Ph1 is indeed an independent risk factor in childhood relapsed ALL. There are striking similarities between these patients and children at initial diagnosis, as well as adult patients. Therefore, it is highly suggestive that the Ph1 is also an independent risk factor under all of these circumstances.

Multiplex PCR for simultaneous detection of the most frequent T cell receptor-delta gene rearrangements in childhood ALL.

A rapid and simple multiplex polymerase chain reaction (PCR) is described that is capable of identifying the six most frequent rearrangements of the T cell receptor (TCR)-delta gene segments in childhood acute lymphoblastic leukemia (ALL). The PCR products amplified in a single reaction are of different size for each TCR-delta gene rearrangement. Therefore, they are readily and unambiguously distinguished after agarose gel electrophoresis and assigned to a specific V-D-J gene rearrangement. There is no need for labor-intensive and time-consuming Southern blot hybridization or nested PCR. To evaluate the multiplex assay we chose 45 DNA samples of childhood ALL analyzed beforehand for TCR-delta gene rearrangements by Southern blot and single PCR of which 30 showed TCR-delta gene rearrangements. The multiplex PCR results corresponded to the Southern blot and single PCR analyses. The described multiplex PCR enables the detection of clonal markers in about 50% of patients in order to monitor minimal residual disease (MRD) in prospective studies with a high turnover of samples.

Potential of LightCycler technology for quantification of minimal residual disease in childhood acute lymphoblastic leukemia.

A certain quantity of residual leukemic cells at several time points during chemotherapy of childhood acute lymphoblastic leukemia (ALL) was proved to predict outcome. Future childhood ALL treatment will take minimal residual disease (MRD) into consideration for stratification aiming at an individualization of chemotherapeutic regimens. Recently, the first quantitative real-time PCR assay for MRD detection was described using T cell receptor and immunoglobulin gene rearrangements as clonal markers. Quantitative real-time PCR was performed with TaqMan technology. Here, we present for the first time the potential of LightCycler real-time PCR technology to quantify MRD. We compare and assess different approaches for real-time PCR quantification of leukemic cells, based either on clone-specific primers and general fluorescence detection with SYBR Green, TaqMan probe or hybridization probes, or based on general PCR amplification and clone-specific detection with hybridization probes. MRD quantification with LightCycler real-time PCR technology is a sensitive, specific and incomparably rapid method that needs no post-PCR handling, hence eliminating contamination risk and saving time. Working towards the establishment of MRD quantification in routine diagnostics and towards treatment strategies based on these results, LightCycler quantitative PCR seems to be a promising new technique that makes results immediately available for treatment decisions.

Extensive alternative splicing of interleukin-7 in malignant hematopoietic cells: implication of distinct isoforms in modulating IL-7 activity.

Interleukin-7 (IL-7) plays a pivotal role in early stages of normal B and T cell development. In addition, IL-7 stimulates the proliferation of both antitumor reactive cells and a number of T and B cell malignancies, underlining its significance for leukemogenesis. However, its exact role in the process of pathologic maturation of lymphocytes and regulation of the immune response is not completely understood. As alternative splicing of pre-mRNA has been shown to be involved in the control of gene expression, and splicing-derived protein isoforms with antagonistic activity have been found, we assessed the mRNA-expression of IL-7 and its previously described alternative splice variant lacking exon 4, IL-7delta4, in leukemic cells from children with acute lymphoblastic leukemia (ALL). PCR of full-length IL-7 cDNA enabling the competitive amplification of both variants led to the amplification of diverse unexpected PCR products. The sequence data demonstrated the existence of three additional in-frame splice variants resulting from exon skipping of exon 3 or exon 5 or both in combination with exon 4. We named these IL-7delta3/4, IL-7delta4/5, and IL-7delta3/4/5. Furthermore, three out-of-frame splice variants were identified, IL-7(-56bpExon2), IL-7delta4(-56bpExon2), and IL-7delta3/4/5(-56bpExon2), in which, in addition to the aforementioned exon skipping, 56 bp of the 3' end of exon 2 are omitted. Our results led us to assume that splicing-derived IL-7 isoforms play a potential role in modulating IL-7-mediated biologic effects. Further studies are required to clarify the significance of the diverse IL-7 protein isoforms for the regulation of IL-7 function and the pathogenesis of leukemia.

Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR.

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.

Reduced expression of the metastasis suppressor gene KAI1 in advanced colon cancer and its metastases.

BACKGROUND: The expression of the KAI1 gene and its gene product were studied in metastatic and non-metastatic human colorectal cancer to evaluate its role in the metastatic process. METHODS: KAI1 mRNA and protein expression was examined in 36 primary colorectal carcinomas and 6 liver metastasis using Northern blot and Western blot analyses. Forty-six normal colonic tissue samples served as controls. The exact site of KAI1 expression was analyzed by in situ hybridization and by immunohistochemistry in primary tumors, in the corresponding normal tissues, in lymph node metastases and liver metastases. RESULTS: Densitometric analysis of Northern blots revealed overexpression of KAI1 mRNA in 87% of colonic cancer tissues in comparison with the corresponding normal colonic tissues. This increase was 9.1-fold in median (P < .001). KAI1 mRNA expression was strongly dependent on tumor stage. Colorectal cancer at stages II and III revealed significantly higher KAI1 mRNA levels than stage IV tumors (P < .03 and P < .015, respectively) or normal controls. In addition, liver metastases showed reduced KAI1 mRNA expression when compared with their corresponding primary tumor. In situ hybridization confirmed the stage-dependent expression results obtained by Northern blots, in which the KAI1 mRNA signal was exhibited almost exclusively in the epithelial cells. Lymph node and liver metastases were largely devoid of KAI1 mRNA. Western blot analysis showed a highly significant increase of KAI1 protein level in stage II cancers in comparison with the normal colon (P < .001) but also in comparison with the more advanced tumor stages III and IV (P < .03) and P < .02, respectively), when metastases were already present. In accordance, KAI1 immunostaining decreased successively with the advance of the tumor stage and was absent in lymph node and liver metastases. CONCLUSIONS: These data demonstrate that the KAI1 mRNA expression and the KAI1 protein level increase in an earlier tumor stage of colorectal cancer, decrease in advanced stages, and are lost in metastases. The loss of KAI1 might favor the ability of colorectal cancer cells to metastasize.

Absence of mutations in the CDKN2 binding site of CDK4 in childhood acute lymphoblastic leukemia.

The cell cycle regulatory circuit resulting in phosphorylation of the retinoblastoma protein (pRB) is frequently altered in human cancers. Several mechanisms of disruption are known in that pathway. In childhood acute lymphoblastic leukemia (ALL), the main disrupting mechanism is the homozygous deletion of the CDKN2 (cyclin dependent kinase inhibitor 2) genes: p16CDKN2a, p15CDKN2b, and p19ARF. Another pRB pathway disturbance is a previously described point mutation in the exon 2 of CDK4, a pRB phosphorylating enzyme, which abrogates binding of the latter to its inhibitors, p16CDKN2a and p15CDKN2b. Here we report the absence of point mutations in the CDKN2-binding site of CDK4 in 100 cases of childhood ALL, 2 cases of childhood chronic myeloid leukemia and 9 hematologic cell lines screened by PCR-SSCP (polymerase chain reaction single stranded conformational polymorphism gel electrophoresis), thereby minimizing the possibility of the existence of these specific CDK4 mutations in childhood ALL.

Plant DNA fingerprinting with radioactive and digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences.

The existence of hypervariable DNA sequences in nuclear genomes, and the use of appropriate "fingerprinting" probes to detect them, has gained widespread scientific interest, and also led to multiple applications in diverse areas. Two years ago, the new technique of "DNA fingerprinting" was also introduced into the analysis and characterization of plant genomes, initially by using human or M13 minisatellites as probes. In the present article, we demonstrate the applicability for plant DNA fingerprinting of oligonucleotide probes specific for simple repetitive DNA sequences. We show that various levels of intra- and interspecific polymorphisms can be detected; the information to be gained depends on the optimal combination of probe and species. Variety-specific patterns were obtained in several cases. Some probes revealed variability between individuals. Somatic variability was not observed. Different DNA isolation and purification procedures were tested in order to introduce a fast and easy-to-perform isolation method suitable for a large variety of plant species. Nonradioactive fingerprinting was performed using digoxigenated oligonucleotides as probes. Banding patterns obtained with radioactive and digoxigenin-based labeling techniques proved to be of similar quality.

Fingerprinting plant genomes with oligonucleotide probes specific for simple repetitive DNA sequences.

Oligonucleotides hybridizing to simple repetitive DNA patterns are highly informative as probes for DNA fingerprinting in all investigated animal species, including man. Here we demonstrate the applicability of this technique in higher plants. The oligonucleotide probes (GTG)5 and (GATA)4 were used to investigate the differences in DNA fingerprint patterns of the following angiosperm species: Triticum aestivum, Secale cereale, Hordeum vulgare, Beta vulgaris, Petunia hybrida, Brassica oleracea, and Nicotiana tabacum. Two species, Hordeum vulgare as a monocot and Beta vulgaris as a dicot, were analyzed in more detail. Their genomes differ considerably in both amount and organization of the simple repetitive sequences (GATA)n, (GACA)n, (GTG)n, and (CT)n due to the evolutionary distance of these two species. Furthermore, several lines and cultivars of Beta vulgaris and Hordeum vulgare can clearly be distinguished on the basis of their highly polymorphic patterns of these repetitive sequences.

Differentiation of species and strains among filamentous fungi by DNA fingerprinting.

We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)8, (GTG)5 and (GACA)4, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)4 produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.

Childhood all blasts retain phenotypic and genotypic characteristics upon long-term serial passage in NOD/SCID mice.

For children with an early bone marrow relapse or relapsed T-cell acute lymphoblastic leukemia (ALL), allogeneic bone marrow transplantation (BMT) is currently the only therapeutic option with a curative approach. Here, the graft versus leukemia (GvL) effect seems to play an important role for long-term immunological control of leukemia. If a bone marrow donor is not available, autologous stem cell transplantation after high-dose chemotherapy has been used as an alternative option. The objective of this work was the induction of tumor specific cytotoxic T-lymphocytes (CTL) against autologous leukemic cells in order to generate the missing GvL effect after autoBMT. The first step was the establishment of an optimized and reliable mouse model. The second step was the induction of a GvL effect in an allogeneic approach to serve as a basis for further GvL experiments in an autologous approach in this mouse model. Leukemic cells from 11 out of 16 different pediatric patients were successfully established in mice and in one case passaged over 19 generations without changes of genotype or phenotype. The antileukemic activity of allogeneic human MNC as a GvL reaction and an accompanying GvHD in the mouse model was shown. Xenotransplanted ALL can be considered a clinically relevant model mimicking the human conditions and as a useful preclinical tool for the evaluation of novel immuno- or genetherapeutic approaches.

Prognostic value of minimal residual disease in relapsed childhood acute lymphoblastic leukaemia.

Molecular monitoring by quantitative PCR techniques of residual leukaemia cells during the first phases of treatment can predict outcome in children with acute lymphoblastic leukaemia. We did a retrospective study of 30 children who had been treated according to the ALL-REZ BFM trials to assess whether amount of minimal residual disease during the first stages of treatment for relapsed acute lymphoblastic leukaemia could predict outcome. In children with minimal residual disease of less than 10(-3) at day 36, probability of event-free survival was 0.86 (95% CI 0.77-0.95), compared with 0 in children with minimal residual disease of 10(-3) or greater (p<0.001). Our results suggest that information about molecular response to treatment can be used to predict long-term outcome in relapsed childhood acute lymphoblastic leukaemia.

Clinical features and outcome of children with first marrow relapse of acute lymphoblastic leukemia expressing BCR-ABL fusion transcripts. BFM Relapse Study Group.

Although the Philadelphia chromosome (Ph1) has been identified as an adverse prognostic factor in acute lymphoblastic leukemia (ALL), little is known about the incidence and clinical course of relapsed Ph1-positive ALL in children. The incidence was determined by screening of 170 consecutive children with first bone marrow relapse of ALL using the reverse transcriptase-polymerase chain reaction (RT-PCR) and comparison, with cytogenetic analysis. Among these 170 children, 20 (12%) were found to be BCR-ABL-positive, representing a rate that is about three times higher than that reported for newly diagnosed ALL. Ten of the cases were identified by RT-PCR only. In none of the 21 patients with T-cell immunophenotypes could an expression of the BCR-ABL mRNA be detected. BCR-ABL positivity was associated with a significantly shorter duration of first remission (P = .0086) and higher white blood cell (P = .0157) and blast cell counts (P = .0304) at relapse diagnosis. All patients were treated according to the ALL-REZ BFM 87 and 90 relapse trials of the BFM Relapse Study Group. The intensive multiagent chemotherapy induced a second complete remission in only 60% of children with BCR-ABL-positive ALL compared with in 91% of those without BCR-ABL expression (P = .0023). The prognosis of BCR-ABL-positive ALL in children is poor, with a probability of event-free survival at 2 years of 8% versus 50% in those without BCR-ABL mRNA or cytogenetic analysis should become part of the routine diagnostic panel for children with newly diagnosed ALL and is fundamental for children presenting with an early bone marrow relapse.

TEL-AML1 fusion transcript in relapsed childhood acute lymphoblastic leukemia. The Berlin-Frankfurt-Münster Study Group.

The cryptic translocation t(12;21)(p13;q22) has been recently recognized as the most common genetic rearrangement in B-lineage childhood acute lymphoblastic leukemia (ALL). The resulting fusion transcript, termed TEL-AML1, has been associated with an excellent prognosis at initial ALL diagnosis. Hence, we postulated that the incidence of TEL-AML1 fusion should be lower in patients with ALL relapse. To address this assumption and to investigate the prognostic significance of TEL-AML1 expression in relapsed childhood ALL, bone marrow samples of 146 children were analyzed by reverse-transcriptase (RT)-polymerase chain reaction (PCR). All children were treated according to Berlin-Frankfurt-Münster (BFM) ALL relapse trial protocols (ALL-REZ BFM 90-96). Their clinical features and outcome were compared with those of 262 patients who could not be tested due to lack of bone marrow samples. Thirty-two of 146 children with relapsed ALL were TEL-AML1-positive. Four of the negative patients had T-lineage and nine Philadelphia chromosome (Ph)-positive leukemia. Thus, the incidence of TEL-AML1 in relapsed Ph1-negative, B-cell precursor ALL is 32 of 133 (24%). The 32 TEL-AML1-positive and 101 negative patients differed significantly with respect to duration of last remission (42.5 v 27 months; P = . 0001) and age at initial diagnosis (53.5 v 74 months; P = .0269). At a median follow-up time of 21.5 months, children positive for TEL-AML1 had a significantly (P = .0011) higher probability of event-free survival (EFS; 0.79 v 0.33). The predominant majority of patients had been treated for initial ALL according to German multicenter BFM (108 of 133) or Cooperative ALL study group (CoALL) (19 of 133) frontline protocols. The comparison of tested and not-tested (N = 262) patients showed no significant difference. TEL-AML1 positivity predicted a favorable short-term outcome; long-term results are unknown. Screening for TEL-AML1 should become routine at relapse diagnosis and might be used for therapy stratification in future trials.

Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation.

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.