High level EGFR amplification in a newly established glioblastoma cell line 170-MG-BA.

Glioblastoma multiforme is a highly invasive and incurable primary brain tumor. The most frequent genetic alteration therein is amplification of the epidermal growth factor receptor (EGFR) gene, the target of current clinical trials. However, EGFR amplification is poorly represented in glioblastoma cell lines. From the 30 cultures attempted herein, we were able to establish two glioblastoma permanent cell lines. The remaining cultures showed limited life span and underwent senescence between passage numbers (PN) 8 to 15. Our newly established glioblastoma cell lines, designated 170-MG-BA and 538-MG-BA, both originated between PN 3 and 5 when areas of smaller, more rapidly proliferating cells appeared. Both cell lines showed similar rates of growth, moderate morphological differences, cytoskeletal heterogeneity and multiple chromosome rearrangements. Analysis by molecular cytogenetics and comparative genomic hybridization (aCGH) revealed two copies of a stable marker chromosome in 170-MG-BA cells effecting focal amplification at 7q11 of the EGFR locus. Comparative RqPCR analysis confirmed that EGFR was uniquely highly expressed in 170-MG-BA cells. Combined targeted expression analysis and aCGH data excluded the recurrent EGFRvIII activating mutation. In contrast, EGFR expression in 538-MG-BA cells which lacked genomic EGFR amplification was not raised. Immunofluorescent staining showed high EGFR protein expression only in the 170-MG-BA cells. Cytogenetic, genomic and transcriptional analyses then confirmed high-level genomic amplification and transcriptional upregulation of wild type EGFR in 170-MG-BA; the first conventional cell line model for investigating the biology and targeted therapy of this key alteration in glioblastoma. Both cell lines are freely available from the DSMZ cell repository.

Use of zinc deposited in deciduous teeth as a retrospective measurement of dietary zinc exposure during early development.

Purpose: We proposed that zinc (Zn) deposition in deciduous teeth would be a timed record of exposure to this essential micronutrient over very early life. We tested this hypothesis by gathering information on the maternal and child's diet during pregnancy and early infancy and measuring mineral deposition in the dentine at points during deciduous tooth development. Methods: We developed a short food frequency questionnaire (S-FFQ) to record consumption of food containing Zn during pregnancy and over the first year of life of the child in an Indonesian population. Zn, Sr and Ca were measured by laser ablation ICP-MS in a series of points across the developmental timeline in deciduous teeth extracted from 18 children undergoing the process as part of dental treatment whose mothers completed the SFFQ. Mothers and children were classified into either high Zn or low Zn groups according to calculated daily Zn intake. Results: The Zn/Sr ratio in dentine deposited over late pregnancy and 0-3 months post-partum was higher (p < 0.001, 2-way ANOVA; p < 0.05 by Holm-Sidak post hoc test) in the teeth of children of mothers classified as high Zn consumers (n = 10) than in children of mothers classified as low Zn consumers (n = 8). Conclusion: The S-FFQ was validated internally as adequately accurate to measure zinc intake retrospectively during pregnancy and post-partum (∼7 years prior) by virtue of the correlation with measurements of zinc in deciduous teeth. The ratio of Zn/Sr in deciduous teeth appears to be a biomarker of exposure to zinc nutrition during early development and offers promise for use as a record of prior exposure along a timeline for research studies and, potentially, to identify individuals at heightened risk of detrimental impacts of poor early life zinc nutrition on health in later life and to implement preventative interventions.

Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin.

We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

Transient expression of the tartrate-resistant acid phosphatase (TRAP) gene in hamster cells: a pilot study.

Tartrate-resistant acid phosphatase (TRAP) became known as the characteristic, albeit not specific, marker enzyme for hairy cell leukemia (HCL). It is also expressed in other types of leukemic cells and in a variety of normal hematopoietic cells under both physiological and artificial conditions. The role of this distinctive enzyme is still unknown. TRAP hydrolyzes several chemical compounds, but its physiological substrate remains to be elucidated. Here, the insertion of a human TRAP cDNA into mammalian expression vector pSBC-2 yielded a construct that encoded enzymatically active TRAP in transfected baby hamster kidney cells (BHK-21). TRAP was expressed transiently, as shown by Northern blot analysis, polyacrylamide gel electrophoresis, isoelectric focusing and cytochemical staining. BHK-21 cells over-expressing the enzyme had a growth rate that was approximately 50% of that observed in control cells. A stable expression could not be achieved. Recent evidence suggested that TRAP might function as a protein tyrosine phosphatase. The effect of TRAP on protein tyrosine phosphorylation was examined by immunoblotting with an anti-phosphotyrosine monoclonal antibody (MoAb). The level of tyrosine phosphorylation was clearly lower in transfected BHK-21-TRAP cells than in the control cultures. Phorbol ester-mediated induction of protein tyrosine phosphorylation could overcome the status of reduced phosphorylation in BHK-21-TRAP cells. Furthermore, upon treatment with 12-O-tetra-decanoylphorphol-13-acetate tyrosine phosphorylation reached similar levels to stimulated control cells, indicating the existence of a functional endogenous phosphorylation network. Thus, TRAP might function as an antagonistic counterpart of cellular protein tyrosine kinases and could be involved in the control of cellular activation, proliferation, and differentiation.

Cloning and characterization of the human tartrate-resistant acid phosphatase (TRAP) gene.

The expression and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, have been analyzed extensively in the past. In some diseases, like hairy cell leukemia and Gaucher's disease, cytochemically detected TRAP expression is used as a disease-associated marker. In this paper we describe the isolation of a genomic cosmid clone of the human TRAP gene. Restriction mapping revealed a 22-kb insert and the complete genomic structure of the TRAP gene. A 6-kb HindIII-fragment harboring the entire TRAP gene was subcloned and the 5'-flanking region of 3026 bp was sequenced. Analysis of the sequence data showed the presence of potential transcription factor binding sites. Two transcriptional start sites were identified in the untranslated exon 1 at positions -349 and -347 bp relative to the translational start codon. Linked to a luciferase-encoding reporter gene the 5'-flanking region was sufficient to direct transcription in the heterologous cell line BHK-21. Treatment of the transfected cells with different modulators of the intracellular iron content showed that regulation of TRAP expression is dependent on iron. In summary, these data imply a possible functional role of the TRAP gene product either in the storage or the transport of iron.

False human hematopoietic cell lines: cross-contaminations and misinterpretations.

The risk of adventitious contamination and subsequent overgrowth of cell lines by unrelated cells is a potential and often recurring problem where cells are grown and studied. This problem of intraspecies and interspecies cross-contamination among human cell lines has been recognized for over 25 years; incidences of cell cross-contamination between 17 and 35% have been reported. The most useful methods to detect human cell cross-contamination are DNA fingerprinting and cytogenetic analysis, each complementing the other. Using this combination, we found that in total 14.8% of the human hematopoietic cell lines received either from the original investigator (n = 117 cell lines) or from secondary sources (n = 72 cell lines) were cross-contaminated with another hematopoietic cell line and were thus false cell cultures. Another problem relates to the fact that not every cell line established from a patient with a hematopoietic malignancy is a malignant cell line; unintended immortalization of non-malignant B cells by 'passenger' Epstein-Barr virus (EBV) leads to the establishment of B-lymphoblastoid cell lines (termed EBV+ B-LCLs), an event which is much more frequent than the establishment of a 'true' leukemia-lymphoma-myeloma cell line. These EBV+B-LCLs are most often (albeit not always) unrelated to the malignant clone. The misinterpretation of such EBV+ B-LCLs as true malignant hematopoietic cell lines (particularly in research areas investigating B cell-derived neoplasms such as myeloma) and the indiscriminate use of these cell lines may render some of the results of such studies irrelevant to the pathobiology of the disease concerned. However, a combination of markers commonly allows for an accurate determination of the nature of EBV+ B-LCLs: immunoprofile, cellular morphology, EBV status, and karyotype. In summary, the continuous need for vigilant quality and identity control procedures is emphasized by the high incidences of cross-contaminated cell lines. Most laboratories using cells cultured in vitro maintain multiple cell lines. Such cell lines should be monitored regularly for their identity and specific characteristics in order to prevent invalidation of research work due to incidents of cell line cross-contamination or misinterpretation.

Pathobiology of NPM-ALK and variant fusion genes in anaplastic large cell lymphoma and other lymphomas.

Despite its clinical and histological heterogeneity, anaplastic large cell lymphoma (ALCL) is now a well-recognized clinicopathological entity accounting for 2% of all adult non-Hodgkin's lymphomas (NHL) and about 13% of pediatric NHL. Immunophenotypically, ALCL are of T cell (predominantly) or Null cell type; by definition, cases expressing B cell antigens are officially not included in this entity. The translocation (2;5)(p23;q35) is a recurring abnormality in ALCL; 46% of the ALCL patients bear this signature translocation. This translocation creates a fusion gene composed of nucleophosmin (NPM) and a novel receptor tyrosine kinase gene, named anaplastic lymphoma kinase (ALK). The NPM-ALK chimeric gene encodes a constitutively activated tyrosine kinase that has been shown to be a potent oncogene. The exact pathogenetic mechanisms leading to lymphomagenesis remain elusive; however, the synopsis of evidence obtained to date provides an outline of likely scenarios. Several t(2;5) variants have been described; in some instances, the breakpoints have been cloned and the genes forming a new fusion gene with ALK have been identified: ATIC-ALK, TFG-ALK and TPM3-ALK. Cloning the translocation breakpoint and identifying the ALK and NPM genes provided tools for screening material from patients with ALCL using various approaches at the chromosome, DNA, RNA, or protein level: positive signals in the reverse transcriptase-polymerase chain reaction (RT-PCR) and the immunostaining with anti-ALK monoclonal antibodies (McAb) serve as the most convenient tests for detection of the t(2;5) NPM-ALK since the fusion gene and ALK protein expression do not occur in normal or reactive lymphoid tissue. The wide range of NPM-ALK positivity reported in different series appears to be dependent on the inclusion and selection criteria of the ALCL cases studied. Overall, however, 43% of ALCL cases were NPM-ALK+ (83% of pediatric ALCL vs 31% of adult ALCL). Occasional non-ALCL B cell lymphomas (4%) with diffuse large cell and immunoblastic histology and Hodgkin's disease cases (3%) were NPM-ALK-, but these data are questionable. The aggregate results indicate that, in contrast to primary nodal (systemic) ALCL, the t(2;5) may be present in only 10-20% of primary cutaneous ALCL and rarely, if at all, in lymphomatoid papulosis, a potential precursor lesion; however, these 10-20% positive cases were not confirmed by anti-ALK McAb immunostaining and may represent an overestimate. Positivity for NPM-ALK is associated to various degrees with the following parameters: 44% and 45% of ALCL cases with T cell and Null cell immunophenotype, respectively, are positive, whereas only 8% of cases with a B cell immunoprofile are positive; the mean age of positive patients is significantly younger than that of negative patients; positive cases carry a better overall prognosis (but not in all studies). Recently, the homogenous category of ALK lymphoma ('ALKoma') has emerged as a distinct pathological entity within the heterogenous group of ALCL. The fact that patients with ALK lymphomas experience significantly better overall survival than ALK- ALCL demonstrates further that analysis of ALK expression has important prognostic implications. The term ALK lymphoma signifies a switch in the use of the diagnostic criteria: cases are selected on the basis of a genetic abnormality (the ALK rearrangement), instead of the review of morphological or immunophenotypical features which are clearly more prone to disagreement and controversy. Since its initial description in 1985 ALCL has become one of the best characterized lymphoma entities.

False leukemia-lymphoma cell lines: an update on over 500 cell lines.

Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

Identity of original and late passage Dami megakaryocytes with HEL erythroleukemia cells shown by combined cytogenetics and DNA fingerprinting.

During routine authentication checks, we noticed untoward karyotypic similarities between late-passage stocks of the Dami megakaryocyte and HEL erythroleukemia cell lines. Genetic identity of Dami with HEL was demonstrated by DNA fingerprinting with a (gtg)5 multilocus probe and confirmed for earlier passages of Dami deposited by its originators with the ATCC. As initial passage stocks of Dami are no longer available for comparison, we investigated whether cross-contamination by HEL was more likely to have occurred during Dami's establishment or subsequently, by karyotyping currently available stocks of both cell lines for comparison with that originally reported for Dami. We found that the karyotype of current stocks of Dami overwhelmingly resembles that described in the original report, having retained at least 16/18 structural chromosome rearrangements as described therein, cf. 12/20 shared by current stocks of Dami and HEL. HEL's antecedence is shown by the retention of normal homologs of chromosomes, 6, 18, and 21--all rearranged in Dami. These results confirm the identity of current and early passage stocks of Dami and their common origin by cross-contamination with HEL which had occurred by July 1987, a year prior to publication. Thus, most, if not all, studies using Dami are likely to have employed HEL instead and may require reappraisal.

Preservation of functional and regulatory domains of expressed bcl-2 genes in non-Hodgkin's lymphoma.

The most common translocation in human lymphoma, t(14;18)(q32;q21), recombines the bcl-2 gene with the immunoglobulin (Ig) heavy-chain locus leading to the production of high levels of chimeric RNAs and the resulting 26 kDa bcl-2 protein. The oncogenic role of the bcl-2 gene has been shown by the suppression of a variety of programmed cell deaths (apoptosis). Bcl-2 is able to interact with other members of the bcl-2 family through at least one of its conserved dimerization domains. Although overproduction of the wild-type protein appears sufficient for conferring a selective growth or a survival advantage to hematopoietic cells, the mode of activation of the proto-oncogene remains to be elucidated. In a first step, we examined and quantitated the expression of the bcl-2 gene in primary biopsies of non-Hodgkin's lymphomas (NHL) as well as in cell lines derived from NHLs. The results show that bcl-2 expression is found in a variety of hematopoietic lineages, but is most strongly associated with the B cell lineage. Within the B cell lineage, the expression levels vary depending on the differentiation as well as on the t(14;18) rearranged status. The quantitative measurements show high steady-state mRNA levels in early and in t(14;18) arranged B cells, whereas bcl-2 expression decreases with further B cell maturation and differentiation. In a second step we analyzed the bcl-2 mRNA for secondary genetic alterations, which may alter regulatory regions rendering it more tumorigenic. For this purpose, we chose a combined RT-PCR/SSCP method in order to screen out mutations of alleles which are not expressed. Different migration patterns of SSCP products were found only in two cell lines and subsequent sequencing revealed that the functional domains are not affected. Our data suggest that the dimerization properties of this protein are preserved in tumor cells and that modifications of the bcl-2 gene by the somatic hypermutation mechanism are not involved and do not influence the pathobiology of NHL.

The (2;5)(p23;q35) translocation in cell lines derived from malignant lymphomas: absence of t(2;5) in Hodgkin-analogous cell lines.

The CD30+ anaplastic large cell lymphoma (ALCL) represents a new lymphoma entity thought to be related to Hodgkin'S disease (HD), but displaying also its own unique features. Cytogenetic studies of ALCL have demonstrated the presence of a (2;5)(p23;q35) translocation in a substantial number of these cases. Recently, the t(2;5) has been cloned and described to represent fusion of the NPM gene with the ALK gene on chromosome 5. To better define the spectrum of lymphomas containing this abnormality we have analyzed 50 continuous human cell lines established from various types of non-Hodgkin's lymphoma, ALCL and HD. In a first step, the expression of the NPM-ALK fusion gene was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). In a second step, the t(2;5)-carrying cells were tested for the translation of functional chimeric mRNA into a fusion protein by immuno-staining of single cells with a polyclonal antibody. The NPM-ALK fusion transcript and the p80 protein were detected in eight of nine ALCL cell lines. We were unable to find PCR evidence for the t(2;5) in any of the non-ALCL cell lines including other CD30+ cell lines. As all seven bona fide HD cell lines were NPM-ALK-negative, these results do not support the notion that the t(2;5) represents a chromosomal aberration common to both ALCL and HD.

Expression of FLT3 receptor and FLT3-ligand in human leukemia-lymphoma cell lines.

The FLT3 gene encodes a receptor tyrosine kinase that is closely related to two well-known receptors, KIT and FMS, that regulate with their respective ligands, stem cell factor (SCF) and macrophage colony-stimulating factor (M-CSF), proliferation and differentiation of hematopoietic cells. The ligand for FLT3, FL, is active in both soluble and membrane-bound forms. We examined expression of FL and FLT3 mRNA in a panel of some 110 continuous human leukemia-lymphoma cell lines from all major hematopoietic cell lineages by Northern blot analysis. FLT3 mRNA is expressed primarily in pre-B cell lines, myeloid and monocytic cell lines whereas FL mRNA was detected in most cell lines from all cell lineages. Analysis of FLT3 receptor protein expression examined with a specific anti-FLT3 monoclonal antibody and flow cytometry in 17 cell lines confirmed the results obtained at the mRNA level. Forty of 110 cell lines displayed both receptor and ligand mRNA suggesting a possible autocrine or intracrine stimulation. In normal hematopoietic cells expression of FLT3 was reported to be associated with CD34 positivity, a cell surface marker of immature and precursor cells. No correlation between FLT3 and CD34 expression was found in the cell lines analyzed. These studies served to illustrate further the importance of the FL-FLT3 ligand-receptor system in the regulation of hematopoietic cells.

Tumor necrosis factor-alpha-induced proliferation requires synthesis of granulocyte-macrophage colony-stimulating factor.

OBJECTIVE: Tumor necrosis factor- alpha (TNF-alpha) induces a variety of cellular responses, some of them being at least seemingly contradictory. Thus, we set out to find differences in the modes of proliferative and apoptotic responses to TNF- alpha. MATERIALS AND METHODS: We screened a panel of acute myeloid leukemia-derived cell lines for TNF- alpha-responsiveness. In two lines (OCI-AML-1, OCI-AML-11), TNF- alpha acted as an apoptotic agent; in others (HU-3, M-07e, TF-1), it had the opposite effect, preventing apoptosis and inducing proliferation. Direct and indirect signaling mechanisms, including NF-kappaB activation and cytokine synthesis, were analyzed. RESULTS: All cell lines tested expressed TNF- alpha receptors I and II and responded to TNF- alpha by upregulation of intercellular adhesion molecule-1. In contrast to granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF- alpha did not activate the MAP kinase and p70S6 kinase pathways. Nevertheless, inhibitors of these pathways clearly reduced the TNF-alpha-induced cell growth, indicating that TNF- alpha-proliferative cells produced a growth factor that induced proliferation upon stimulation of the above pathways. Anti-GM-CSF antibodies inhibited the TNF-alpha-induced growth, suggesting the presence of an autocrine loop for cell proliferation mediated by GM-CSF. Supporting this notion, TNF-alpha-induced upregulation of GM-CSF mRNA levels and protein secretion in the TNF-alpha-proliferative, but not in the TNF-alpha-apoptotic cell lines. CONCLUSION: These data identify GM-CSF synthesis as an early and essential step in TNF- alpha-induced proliferation. We show for the first time that TNF-alpha-treated cell lines producing no or only minimal amounts of GM-CSF demonstrate an apoptotic phenotype, while cell lines with high GM-CSF expression rates can escape from growth arrest or even apoptosis. In this context, we discuss arguments pointing at NF-kappaB as regulator of GM-CSF synthesis and thus indirectly as regulator for the escape of TNF-alpha-induced apoptosis.

Dicistronic transcription units for gene expression in mammalian cells.

Dicistronic vectors utilizing the internal ribosomal entry site sequence of poliovirus as the intercistronic region were constructed for gene expression in mammalian cells. We have developed two monocistronic expression vectors which facilitate the creation of dicistronic expression plasmids. The dicistronic expression plasmids encode transcription units which allow the coordinated translation of the two genes. Using internal luciferase and secreted alkaline phosphatase, we show the correlated expression of both reporter genes and expression levels comparable to those achieved by the respective monocistronic expression vectors.

A new hybrid promoter directs transcription at identical start points in mammalian cells and in vitro.

A hybrid promoter which generates large amounts of mRNAs with transcription start points (tsp) differing in one nt, both in mammalian cells and in vitro, was constructed by integrating the promoter of bacteriophage T7 in between the TATA box and the tsp of the retroviral myeloproliferative sarcoma virus (MPSV) long terminal repeat (LTR). This promoter was designed for sequence and secondary structure studies of 5' untranslated regions (UTR) with respect to mRNA stability and translatability.

A multifunctional vector family for gene expression in mammalian cells.

A family of different promoters and vector backbones for multiple use in mammalian cells in vivo and in vitro experiments has been created. The elements allow high-level constitutive, as well as adjustable expression with mono- and dicistronic transcription units and several in vitro manipulations of the inserted ORFs. The vector components are designed to be combined in a simple cloning step.

Expression of the growth arrest-specific gene 6 (GAS6) in leukemia and lymphoma cell lines.

The initial identification of GAS6 as a protein expressed in response to growth arrest suggested that it might function as a negative regulator of cell proliferation. Since the transforming activity of the GAS6 receptor (AXL/UFO) was documented, GAS6 might stimulate rather than inhibit proliferation. In order to detect aberrant expression of GAS6 we examined gene expression in 46 cell lines of precursor B-, B- and T-cell origin as well as from Hodgkin's disease and cell lines established from various myeloproliferative disorders. In our study, the expression of GAS6 reveals a constitutive transcriptional activation in 8/46 cases of proliferating cell lines. The GAS6 mRNA expression could be shown in 4/22 cell lines of the lymphoid arm and in 4/17 of the myeloid lineages of the hematopoietic system. No transcripts could be detected in the CD30+ Hodgkin and anaplastic large cell lymphomas (0/7). Interestingly, the steady state mRNA levels showed neglectable GAS6 expression in precursor B and B-cell lines (1/9), but could be detected in terminally differentiated plasma cell lines (4/4). The predominantly GAS6-expressing cell lines of non-lymphoid origin have been established from acute myeloid leukemias of the M4 subtype (3/4). In order to demonstrate evidence for an autocrine regulation of growth in permanent hematopoietic cell lines, we measured the GAS6 expression in cell lines with strong positivity for the AXL/UFO receptor mRNA. Constitutive basal levels of GAS6 mRNA and protein expression could be only detected in 3/23 AXL/UFO expressing cell lines. Although a general mechanism seems most unlikely, further studies are necessary to demonstrate the involvement of GAS6 in single cases of disordered growth or chemotaxis/adhesion of leukemia and lymphomas.

The human tartrate-resistant acid phosphatase (TRAP): involvement of the hemin responsive elements (HRE) in transcriptional regulation.

The biochemical properties and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, are well known. In contrast, little is known about the physiology and genic structure of this unique enzyme. In some diseases, like hairy cell leukemia, Gaucher's disease and osteoclastoma, cytochemically detected TRAP expression is used as a disease-associated marker. In order to begin to elucidate the regulation of this gene we generated different deletion constructs of the TRAP 5'-flanking region, placed them upstream of the luciferase reporter gene and assayed them for their ability to direct luciferase expression in human 293 cells. Treatment of these cells with the iron-modulating reagents transferrin and hemin causes opposite effects on the TRAP promoter activity. Two regulatory GAGGC tandem repeat sequences (the hemin responsive elements, HRE) within the 5'-flanking region of the human TRAP gene were identified. Studies with specific HRE-deletion constructs of the human TRAP 5'-flanking region upstream of the luciferase reporter gene document the functionality of these HRE-sequences which are apparently responsible for mediating transcriptional inhibition upon exposure to hemin. In addition to the previously published functional characterization of the murine TRAP HRE motifs, these results provide the first description of a new iron/hemin-responsive transcriptional regulation in the human TRAP gene.

U-2932: two clones in one cell line, a tool for the study of clonal evolution.

Genetic heterogeneity is common in tumors, explicable by the development of subclones with distinct genetic and epigenetic alterations. We describe an in vitro model for cancer heterogeneity, comprising the diffuse large B-cell lymphoma cell line U-2932 which expresses two sets of cell surface markers representing twin populations flow-sorted by CD20 vs CD38 expression. U-2932 populations were traced to subclones of the original tumor with clone-specific immunoglobulin IgVH4-39 hypermutation patterns. BCL6 was overexpressed in one subpopulation (R1), MYC in the other (R2), both clones overexpressed BCL2. According to the combined results of immunoglobulin hypermutation and cytogenetic analysis, R1 and R2 derive from a mother clone with genomic BCL2 amplification, which acquired secondary rearrangements leading to the overexpression of BCL6 (R1) or MYC (R2). Some 200 genes were differentially expressed in R1/R2 microarrays including transcriptional targets of the aberrantly expressed oncogenes. Other genes were regulated by epigenetic means as shown by DNA methylation analysis. Ectopic expression of BCL6 in R2 variously modulated new candidate target genes, confirming dual silencing and activating functions. In summary, stable retention of genetically distinct subclones in U-2932 models tumor heterogeneity in vitro permitting functional analysis of oncogenes against a syngenic background.

Online imaging of initial DNA damages at the PTB microbeam.

In an inter-disciplinary collaboration of Physikalisch-Technische Bundesanstalt (PTB), German Collection of Microorganisms and Cell Cultures (DSMZ) and Heinrich-Heine University, live-cell imaging has been established at the charged-particle microbeam facility of PTB. Candidate genes participating in DNA strand-break repair pathways such as PARP-1, MRE11, MSH2, MDC1 and p53BP1 have been modified to generate fluorescent fusion proteins. Using multi-cistronic expression vectors, stable genomic integration was achieved in HT-1080 fibroblasts. The aim of this study is to characterise and use these highly reliable cell lines for studying initial steps of DNA damage responses and kinetics of repair after microbeam irradiation with high- and low-linear energy transfer (LET) particles in living cells at physiological conditions.