Expression of the p210BCR-ABL oncoprotein drives centrosomal hypertrophy and clonal evolution in human U937 cells.

Centrosomes play fundamental roles in mitotic spindle organization, chromosome segregation and maintenance of genetic stability. Recently, we have shown that centrosome aberrations occur early in chronic myeloid leukemia (CML) and are induced by imatinib in normal fibroblasts in vitro. To investigate the influence of BCR-ABL on centrosomes, we performed long-term in vitro experiments employing the conditionally p210BCR-ABL-expressing (tetracycline-inducible promoter) human monocytic cell line U937p210BCR-ABL/c6 as a model of CML chronic phase. Centrosome hypertrophy was detectable after 4 weeks of transgene expression onset, increasing up to a rate of 25.7% aberrant cells within 13 weeks of propagation. This concurred with clonal expansion of aneuploid cells displaying a hyperdiploid phenotype with 57 chromosomes. Partial reversibility of centrosome aberrations (26-8%) was achieved under prolonged propagation (14 weeks) after abortion of induction and bcr-abl silencing using small interfering RNA. Therapeutic doses of imatinib did not revert the aberrant phenotype, but counteracted the observed reverting effect of bcr-abl gene expression switch off. Suggesting a mechanistic model that features distinct abl-related tyrosine kinase activity levels as essential determinants of centrosomal integrity, this is the first report mechanistically linking p210BCR-ABL oncoprotein activity to centrosomal hypertrophy.

Gene expression signature of primary imatinib-resistant chronic myeloid leukemia patients.

Although the selective tyrosine kinase inhibitor imatinib is successfully used in the treatment of chronic myeloid leukemia (CML), inherent mechanisms confer primary resistance to leukemic patients. In order to search for potentially useful genes in predicting cytogenetic response, a retrospective gene expression study was performed. Leukocyte RNA isolated before imatinib from interferon-alpha-pretreated chronic phase CML patients (n=34) with or without major cytogenetic remission (< or =35% Philadelphia (Ph)+ metaphases) during the first year of treatment was comparatively analyzed using Affymetrix U133A chips. Using support vector machines for gene classification, an outcome-specific gene expression signature consisting of 128 genes was identified. Comparative expression data of specific genes point to changes in apoptosis (e.g. casp9, tumor necrosis factor receptor-associated protein 1, hras), DNA repair (msh3, ddb2), oxidative stress protection (glutathione synthetase, paraoxonase 2, vanin 1) and centrosomes (inhibitor of differentiation-1) within primary resistant patients. Independent statistical approaches and quantitative real-time reverse transcriptase-polymerase chain reaction studies support the clinical relevance of gene profiling. In conclusion, this study establishes a candidate predictor of imatinib resistance in interferon-alpha-pretreated CML patients to be subjected to future investigation in a larger independent patient cohort. The resulting expression signature point to involvement of BCR-ABL-independent mechanisms of resistance.

Gene expression profiling of CD34+ cells identifies a molecular signature of chronic myeloid leukemia blast crisis.

Despite recent success in the treatment of early-stage disease, blastic phase (BP) of chronic myeloid leukemia (CML) that is characterized by rapid expansion of therapy-refractory and differentiation-arrested blasts, remains a therapeutic challenge. The development of resistance upon continuous administration of imatinib mesylate is associated with poor prognosis pointing to the need for alternative therapeutic strategies and a better understanding of the molecular mechanisms underlying disease progression. To identify transcriptional signatures that may explain pathological characteristics and aggressive behavior of BP blasts, we performed comparative gene expression profiling on CD34+ Ph+ cells purified from patients with untreated newly diagnosed chronic phase CML (CP, n=11) and from patients in BP (n=9) using Affymetrix oligonucleotide arrays. Supervised microarray data analysis revealed 114 differentially expressed genes (P<10(-4)), 34 genes displaying more than two-fold transcriptional changes when comparing CP and BP groups. While 24 of these genes were downregulated, 10 genes, especially suppressor of cytokine signalling 2 (SOCS2), CAMPATH-1 antigen (CD52), and four human leukocyte antigen-related genes were strongly overexpressed in BP. Expression of selected genes was validated by real-time-polymerase chain reaction and flow cytometry. Our data suggest the existence of a common gene expression profile of CML-BP and provide new insight into the molecular phenotype of blasts associated with disease progression and high malignancy.

Assessment of imatinib as first-line treatment of chronic myeloid leukemia: 10-year survival results of the randomized CML study IV and impact of non-CML determinants.

Chronic myeloid leukemia (CML)-study IV was designed to explore whether treatment with imatinib (IM) at 400 mg/day (n=400) could be optimized by doubling the dose (n=420), adding interferon (IFN) (n=430) or cytarabine (n=158) or using IM after IFN-failure (n=128). From July 2002 to March 2012, 1551 newly diagnosed patients in chronic phase were randomized into a 5-arm study. The study was powered to detect a survival difference of 5% at 5 years. After a median observation time of 9.5 years, 10-year overall survival was 82%, 10-year progression-free survival was 80% and 10-year relative survival was 92%. Survival between IM400 mg and any experimental arm was not different. In a multivariate analysis, risk group, major-route chromosomal aberrations, comorbidities, smoking and treatment center (academic vs other) influenced survival significantly, but not any form of treatment optimization. Patients reaching the molecular response milestones at 3, 6 and 12 months had a significant survival advantage. For responders, monotherapy with IM400 mg provides a close to normal life expectancy independent of the time to response. Survival is more determined by patients' and disease factors than by initial treatment selection. Although improvements are also needed for refractory disease, more life-time can currently be gained by carefully addressing non-CML determinants of survival.

Induction of centrosome and chromosome aberrations by imatinib in vitro.

Imatinib (STI571, Gleevec/Glivec) is a potent selective tyrosine kinase inhibitor and is used successfully in the treatment of chronic myeloid leukemia (CML). While karyotype alterations, in addition to the Philadelphia chromosome, are a common phenomenon of progressing CML, the observation of BCR-ABL-negative leukemic clones with distinct aberrant karyotypes under an imatinib regimen is not yet understood. Here we test the hypothesis that such tumor clones may be induced de novo from normal cells by imatinib. In vitro experiments with varying drug concentrations (5-20 microM) were performed on normal human dermal fibroblasts (NHDF), Chinese hamster embryonal and Indian muntjak fibroblasts. After 3 weeks of treatment, analysis of cell cultures by centrosome immunostaining and conventional cytogenetics revealed that imatinib induced centrosome and chromosome aberrations in all cultures in a significant dose-dependent and species-independent manner. Moreover, the results of NHDF long-term culture experiments demonstrated that aberrant phenotypes, emerging under imatinib treatment for 12 weeks, were not reversible after prolonged propagation omitting the drug. These observations suggest a causative role of imatinib in the origin of centrosome and karyotype aberrations (genetic instability) and thus may explain the emergence of clonal chromosomal abnormalities in BCR-ABL-negative progenitor cells under imatinib therapy.

Centrosome aberrations in chronic myeloid leukemia correlate with stage of disease and chromosomal instability.

Centrosome abnormalities are hallmarks of various cancers and have been implicated in chromosome missegregation, chromosomal instability, and aneuploidy. Since genetic instability is a common feature in chronic myeloid leukemia (CML), we sought to investigate whether centrosome aberrations occur and correlate with disease stage and cytogenetic findings in CML. We examined 34 CML samples including CD 34+Ph+cells of 18 newly diagnosed patients (chronic phase (CP)) and 16 blast crisis (BC) specimens by using a centrosome-specific antibody to pericentrin. All CP and BC samples displayed centrosome alterations as compared with corresponding CD 34+control cells. Centrosome abnormalities were detected in 29.1+/-5.9% of CP blasts and in 54.3+/-4.8% of BC blasts, but in only 2.4+/-1.1% of controls (P<0.0001). Additional karyotypic alterations to the t(9;22) translocation were found in only 1/18 CML-CP patients. In contrast, 11/16 (73%) CML-BC patients displayed additional karyotype alterations in 48.7% of analyzed cells, correlating with an abnormal centrosome status (P=0.0005). Our results indicate that centrosome defects are a common and early detectable feature in CML that may contribute to acquisition of chromosomal aberrations and aneuploidy. They may be considered as the driving force of disease progression and could serve as future prognostic markers.

Abyss1: a novel L2-like non-LTR retroelement of the snakelocks anemone (Anemonia sulcata).

Non-LTR retrotransposons are a diverse and taxonomically widely dispersed group of retroelements that can be divided into at least 14 distinguishable clades. Basal metazoans have not been examined in great detail for their retrotransposon content. In order to screen for the presence of reverse transcriptase (RT) related sequences in Cnidaria and Ctenophora, basal phyla of metazoans, PCR with highly degenerate oligonucleotides was performed and an RT-like sequence was identified from the sea anemone species Anemonia sulcata. Further screening identified a related element in another anemone species Actinia equina. Significant homology to non-LTR retrotransposon RTs was observed, particularly to L2-like elements of fish such as Maui. The sequence was not detected among other cnidarians and we have designated the A. sulcata and A. equina elements Abyss1 and Abyss2 respectively. Phylogenetic analysis of Abyss1 compared with members of 14 known non-LTR retroelement clades suggests that the sequence represents a novel L2 element.

Therapeutic progress and comparative aspects in chronic myelogenous leukemia (CML): interferon alpha vs. hydroxyurea vs. busulfan and expression of MMTV-related endogenous retroviral sequences in CML. German CML Study Group.

In summary, it can be expected that the availability of unrelated donors will increase the number of CML patients that can be treated curatively with allogeneic BMT. Hydroxyurea has replaced busulfan as first line treatment in CML since it prolongs survival. Ongoing randomized studies comparing IFN-based treatment regimens with standard chemotherapy or IFN-monotherapy probably will answer the question whether IFN can cure a small percentage of CML patients and whether this small percentage can be increased by additional chemotherapy. The present attempts to improve prognostic scores and to apply them to early treatment decisions will allow treatment adaptation more individually. The implications of endogenous retroviral sequences expressed in CML cells are not known now, but may be far reaching.

Double induction strategy including high dose cytarabine in combination with all-trans retinoic acid: effects in patients with newly diagnosed acute promyelocytic leukemia. German AML Cooperative Group.

A prospective multicenter study was performed to investigate the clinical and molecular results of intensified double induction therapy including high-dose cytarabine (ara-C) in combination with ATRA in newly diagnosed acute promyelocytic leukemia (APL), followed by consolidation and 3 years maintenance therapy. Fifty-one patients, diagnosed and monitored from December 1994 to June 1999, were evaluated. The median age was 43 (16-60) years. The morphologic diagnosis was M3 in 40 (78%) and M3v in 11 (22%) patients. In 15 (30%) patients the initial white blood cell counts were > or =5 x 10(9)/l. The cytogenetic or molecular proof of the translocation t(15;17) was a mandatory prerequisite for eligibility. The diagnosis was confirmed by karyotyping in 46 and by RT-PCR of the PML/RARalpha transcript in 45 cases. The rate of complete hematological remission was 92% and the early death rate 8%. Monitoring of minimal residual disease by RT-PCR of PML/RARalpha (sensitivity 10(-4)) showed negativity in 29 of 32 (91%) evaluable cases after induction, in 23 of 25 (92%) after consolidation, and in 27 of 30 (90%) during maintenance, after a median time of 2, 4 and of 18 months after diagnosis, respectively. After a median follow-up of 27 months, the estimated actuarial 2 years overall and event-free survival were both 88% (79, 97), and the 2 years relapse-free survival 96% (90, 100). The high antileukemic efficacy of this treatment strategy is demonstrated by a rapid and extensive reduction of the malignant clone and by a low relapse rate. The results suggest that the intensity of the induction chemotherapy combined with ATRA is one of the factors which may have a critical influence on the outcome of APL. A randomized trial should assess the value of an induction therapy including ATRA and high-dose ara-C in comparison to standard-dose ara-C.

HERV-IP-T47D, a novel type C-related human endogenous retroviral sequence derived from T47D particles.

A new type C retrovirus-related endogenous pol sequence (ERV-FTD) found to be occasionally copackaged in retrovirus-like particles released by the human mammary carcinoma cell line T47D was used to screen a human genomic library (Seifarth W, Skladny H, Krieg-Schneider F, Reichert A, Hehlmann R, and Leib-Mösch C: J Virol 1995;69:6408-6416). The DNA sequence of one full-length clone now reveals a human endogenous proviral sequence (HERV) of 4190 bp in length comprising a 5' LTR (489 bp) and regions with 37 and 74% overall amino acid homology to RTVL-Ia gag and pol genes, respectively. About 35 related elements were found to be distributed on all human chromosomes except 16, 17, and Y. Sequence comparisons with Mo-MuLV and various type C-related HERVs suggest that despite a proline primer-binding site this novel HERV element, now named HERV-IP-T47D, can be assigned to one family together with known HERV-I elements. Phylogenetic analyses of 5 proviral and 25 solitary LTR sequences confirmed the existence of two distinct but closely related subgroups of the HERV-IP superfamily in the primate genome. In contrast to most known HERV-families, the evolutionary age of HERV-IP elements dates back prior to the divergence of New and Old World monkeys. Despite their old age, members of the HERV-IP family are still transcriptionally active and were found to be highly expressed in specific human tissues such as liver and kidney.

Rapid identification of all known retroviral reverse transcriptase sequences with a novel versatile detection assay.

We have developed a highly sensitive, universal assay that allows detection as well as identification of all known retroviral reverse transcriptase (RT)-related nucleic acids in a biological sample by a single two-step experiment. The assay combines polymerase chain reaction (PCR) and reverse dot-blot hybridization (RDBH), using an array of immobilized synthetic retrovirus-specific oligonucleotides and two sets of mixed oligo primers (MOPs). These primers were derived from highly conserved motifs found in all known reverse transcriptase genes. The PCR/RDBH assay was used for qualitative analyses of human endogenous retrovirus (HERV) transcription in peripheral blood mononuclear cells (PBMCs) and in particles released by the human mammary carcinoma-derived cell line T47D. Sensitivity was further demonstrated by detection of down to 10 copies of pig endogenous retrovirus (PERV) DNA in human cDNA samples. Therefore, this assay is particularly useful for the identification of retroviral sequences in xenografts as well as in recipients of xenografted tissues and organs. Moreover, it is a valuable tool to detect retroviral transcripts and particles in cell cultures used for production of therapeutic polypeptides. The assay is further suitable for monitoring vector preparation used in human gene therapy to exclude transfer of copackaged endogenous retroviruses into target cells.

Congress Report: XIX symposium of the International Association for Comparative Research on Leukemia and Related Diseases, Mannheim/Heidelberg, Germany, July 13 - 18, 1997.

The XIX Symposium of the International Association for Comparative Research on Leukemia and Related Diseases (IACRLRD, President: Prof. Dr. R. Hehlmann) was held in Mannheim and Heidelberg, Germany, July 13 - 18, 1997. Comparative research in cancer was systematically established in the 1950s. Similarities in morphology, biology and pathology between animal and human leukemias and related diseases and the viral origin of a variety of animal leukemias and related diseases (lymphomas, sarcomas, breast tumors etc.) led to the concept that comparative research should promote the understanding of human leukemias and related diseases. In 1960 the World Health Organization inaugurated the establishment of a World Committee for Comparative Leukemia Research. The first symposium took place in Hannover, Germany, in 1963. After the fifth symposium in Padova, Italy, in 1971 the International Association for Comparative Research on Leukemia and Related Diseases (IACRLRD) was founded to complement the World Committee and to expand the international effort. The history of the symposium shows the evolution from a meeting on animal leukemia viruses into one dealing with viral and genetic aspects of human and animal leukemia and related diseases. The scientific evolution of the Abelson murine leukemia virus with its abl oncogene in the 1970s to what currently appears as the most reliable marker for human chronic myeloid leukemia is merely one example. Comparative research has reached a new dimension with the the recent advances in sequencing of the genomes of a variety of species and of humans. Many genes identified in the human genome and relevant for disease can be found in the genomes of animal species and even in the genomes of bacteria and of yeast. This reminds us that not just human and animal biology but also pathology must be regarded as a continuum of evolution and that much can be learned from comparing the genetic information of different species. Comparative genome research will allow conclusions to be drawn from principles recognized in animal species which are relevant to human diseases. It is likely that the application of comparative research to genome analysis will provide basic new insights in molecular medicine into the function of living beings for both animal species and humans. The current revolution in genomics is the latest phase in a rich history of medical progress related to the comparative approach. Meetings and organizations that have grown out of IACRLRD, include, at least to some extent: the Meeting of the International Human Retrovirology Association, the Gallo Lab Meeting , the Feline Retrovirus Meeting, the Cold Spring Habor Retrovirus Meeting, international and regional AIDS meetings, and many others. The XIX symposium in Mannheim included five memorial lectures, seven plenary sessions, 18 parallel sessions, two round table discussions and a public forum. In addition, six associated satellite symposia were held. The general meeting, attended by participants from 27 countries, integrated thematically contributions of genetic, cellular, and viral factors toward the development of leukemia and lymphoma and sought unifying concepts in leukemogenesis.

Expression of transketolase-like gene 1 (TKTL1) depends on disease phase in patients with chronic myeloid leukaemia (CML).

PURPOSE: Overexpression of transketolase-like gene 1 (TKTL1) on RNA and protein level has been linked to tumour progression, metastasis and unfavourable patient outcome in many solid tumours. Chronic myeloid leukaemia (CML) cells show metabolic characteristics resembling deviations observed in TKTL1 overexpressing solid tumour cells. We therefore sought to evaluate TKTL1 gene expression in different phases of CML. METHODS: A total of 120 peripheral blood samples from 69 patients in various phases of CML and 21 healthy individuals were investigated. TKTL1 expression levels were determined by real-time quantitative polymerase chain reaction using LightCycler technology and normalised against beta-glucuronidase expression. RESULTS: A significantly lower TKTL1 expression was found in chronic phase (CP) CML patients compared to healthy controls. Lowest expression levels were observed in patients during blast crisis (BC). Baseline TKTL1 expression in CP patients did not have value in prognostication of subsequent favourable or dismal outcome. Further, more mature granulocytes showed significantly higher TKTL1 expression compared to immature CD34+ and CD34-/CD33+ cells both in healthy controls and in CML patients. CONCLUSION: TKTL1 expression levels appear to decline in the course of CML with lowest levels during BC. A potential reason is a shift of TKTL1-high-expressing mature granulocytes towards TKTL1-low-expressing immature cells and blasts.

An evidence-based prehospital guideline for external hemorrhage control: American College of Surgeons Committee on Trauma

This report describes the development of an evidence-based guideline for external hemorrhage control in the prehospital setting. This project included a systematic review of the literature regarding the use of tourniquets and hemostatic agents for management of life-threatening extremity and junctional hemorrhage. Using the GRADE methodology to define the key clinical questions, an expert panel then reviewed the results of the literature review, established the quality of the evidence and made recommendations for EMS care. A clinical care guideline is proposed for adoption by EMS systems. Key words: tourniquet; hemostatic agents; external hemorrhage.

Evolution and biological significance of human retroelements.

Retroelements comprise a substantial portion of the human genome. Their large number and ubiquitous distribution has led scientists to speculate about their evolutionary origin and their biological functions. Human endogenous retroviruses and their retrotransposon relatives represent a reservoir of possibly pathogenic retroviral genes that may be activated spontaneously or by environmental conditions. They can act as insertion mutagens and activate or inactivate cellular genes, or may be involved in chromosome aberrations by recombination of related elements on different chromosomal locations. Retroviral gene products themselves may also be pathogenic and, for example, could be implicated in the development of tumors and autoimmune diseases. On the other hand, endogenous retroviral elements and nonviral retroposons are thought to have played an important role in shaping the genomes of vertebrates by intracellular transposition events and by generating hot spots of recombination. In the course of time, some of these elements have acquired cellular functions, such as, for instance, in the regulation of gene expression. Therefore, the role of human endogenous retroviruses and retroposons in biological processes is currently a subject of great interest.

Identification of the genes coding for the second-largest subunits of RNA polymerases I and III of Drosophila melanogaster.

We have isolated cDNA and genomic clones of Drosophila melanogaster by cross-hybridization with a 658 bp fragment of the yeast gene coding for the second-largest subunit of RNA polymerase III (RET1). Determination of the sequence by comparison of genomic and cDNA regions reveals an ORF of 3405 nucleotides which is interrupted in the genomic sequence by an intron of 48 bp. The deduced polypeptide consists of 1135 amino acids with a calculated molecular weight of 128 kDa. The protein sequence shows the same conserved regions of homology as those observed for all the second-largest subunits of RNA polymerases cloned so far. The gene (DmRP128) obviously codes for a second-largest subunit of an RNA polymerase which is different from DmRP140 and DmRP135. We have purified three distinct RNA polymerase activities from D. melanogaster. By using specific RNA polymerase inhibitors in enzyme assays and by comparing their subunit composition we were able to distinguish between RNA polymerase I, II, and III. RNA polymerase preparations of D. melanogaster were blotted and the second-largest subunits were identified with antibodies raised against polypeptides expressed from DmRP128 and DmRP135. Anti-DmRP135 antibodies react strongly with the second-largest subunit of RNA polymerase I but do not react with the respective subunits of RNA polymerase II and III. The second-largest subunit of RNA polymerase III is only recognized by anti-DmRP128. Previously, we have claimed that DmRP135 codes for the second-largest subunit of RNA polymerase III.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrovirus-like particles released from the human breast cancer cell line T47-D display type B- and C-related endogenous retroviral sequences.

The human mammary carcinoma cell line T47-D releases retrovirus-like particles of type B morphology in a steroid-dependent manner (I. Keydar, T. Ohno, R. Nayak, R. Sweet, F. Simoni, F. Weiss, S. Karby, R. Mesa-Tejada, and S. Spiegelman, Proc. Natl. Acad. Sci. USA 81:4188-4192, 1984). Furthermore, reverse transcriptase (RT) activity is found to be associated with particle preparations. Using a set of degenerate primers derived from a conserved region of retroviral pol genes, we repeatedly amplified three different retroviral sequences (MLN, FRD, and FTD) from purified T47-D particles in several RT-PCR experiments. Screening of a human genomic library and Southern blot analysis revealed that these sequences are of endogenous origin. ERV-MLN represents a multicopy family of human endogenous retroviral elements (HERVs) with two closely related copies and up to 20 more distantly related members. In contrast, ERV-FRD and ERV-FTD comprise only one copy and five to seven related elements per haploid human genome. DNA sequence analysis of the proviral pol region of ERV-MLN revealed an uninterrupted stretch of 241 amino acids that shows 65% identity with the RT of the type B-related HERV designated HERV-K10. ERV-FRD and ERV-FTD are defective type C-related HERVs. The pol gene of ERV-FRD displays a nucleotide homology of 54% to the gibbon ape leukemia virus, and the pol gene of ERV-FTD is about 67% homologous to members of the RTVL-I family of HERVs. Our results thus indicate that the retroviral particles released by the breast cancer cell line T47-D are probably generated by complementation of several endogenous proviruses and can package retroviral transcripts of different origins.

Primary structure and functional aspects of the gene coding for the second-largest subunit of RNA polymerase III of Drosophila.

We have cloned and sequenced the gene coding for the second-largest subunit of RNA polymerase III of Drosophila melanogaster (DmRP135). The gene, interrupted by two introns of 62 and 59 bp, respectively, codes for an mRNA of 3.6 kb. As for other housekeeping genes transcription initiates at several sites (between positions -98 and -76) none of which is preceded by a clear TATA sequence. The deduced polypeptide consists of 1129 amino acids with an aggregate molecular weight of 128 kDa. The protein sequence features the same regions of similarity as observed for the corresponding subunits of RNA polymerase II of Drosophila and yeast and the Escherichia coli beta subunit. As in the second-largest subunit of RNA polymerase II there is a zinc-binding motif which is absent in the beta subunit of E. coli. Antibodies directed against a fusion protein expressing 164 amino acids of the DmRP135 polypeptide cross-react with the second-largest subunit of RNA polymerase III of yeast and generate a distinct banding pattern on Drosophila polytene chromosomes distinguishable from that obtained with anti-RNA polymerase II antibodies.