Mycoplasma hyopneumoniae resides intracellularly within porcine epithelial cells.

Enzootic pneumonia incurs major economic losses to pork production globally. The primary pathogen and causative agent, Mycoplasma hyopneumoniae, colonises ciliated epithelium and disrupts mucociliary function predisposing the upper respiratory tract to secondary pathogens. Alleviation of disease is reliant on antibiotics, vaccination, and sound animal husbandry, but none are effective at eliminating M. hyopneumoniae from large production systems. Sustainable pork production systems strive to lower reliance on antibiotics but lack of a detailed understanding of the pathobiology of M. hyopneumoniae has curtailed efforts to develop effective mitigation strategies. M. hyopneumoniae is considered an extracellular pathogen. Here we show that M. hyopneumoniae associates with integrin ß1 on the surface of epithelial cells via interactions with surface-bound fibronectin and initiates signalling events that stimulate pathogen uptake into clathrin-coated vesicles (CCVs) and caveosomes. These early events allow M. hyopneumoniae to exploit an intracellular lifestyle by commandeering the endosomal pathway. Specifically, we show: (i) using a modified gentamicin protection assay that approximately 8% of M. hyopneumoniae cells reside intracellularly; (ii) integrin ß1 expression specifically co-localises with the deposition of fibronectin precisely where M. hyopneumoniae cells assemble extracellularly; (iii) anti-integrin ß1 antibodies block entry of M. hyopneumoniae into porcine cells; and (iv) M. hyopneumoniae survives phagolysosomal fusion, and resides within recycling endosomes that are trafficked to the cell membrane. Our data creates a paradigm shift by challenging the long-held view that M. hyopneumoniae is a strict extracellular pathogen and calls for in vivo studies to determine if M. hyopneumoniae can traffic to extrapulmonary sites in commercially-reared pigs.

[Investigation on the 3 D geometric accuracy and on the image quality (MTF, SNR and NPS) of volume tomography units (CT, CBCT and DVT)]. / Untersuchung zur geometrischen 3-D-Genauigkeit und zur Bildqualität (MTF, SRV und W) von Volumentomografie-Einrichtungen (CT, CBCT und DVT).

PURPOSE: The study aims at investigating how far image quality (MTF and NPS) differs in between CT, CBCT and DVT units and how far the geometrical 3 D accuracy and the HU calibration differ in respect to surgical or radio therapeutic planning. MATERIALS AND METHODS: X ray image stacks have been made using a new designed test device which contains structures for measuring MTF, NPS, the 3 D accuracy and the Hounsfield calibration (jaw or skull program). The image stacks of the transversal images were analyzed with a dedicated computer program. RESULTS: The MTF values are correlated with the physical resolution (CT and DVT) and are influenced by the used Kernel (CT). The NPS values are limited to an intra system comparison due to the insufficient HU accuracy. The 3 D accuracy is comparable in between the system types. CONCLUSIONS: The values of image quality are not yet correlated with dose values: NPS. Investigations to an appropriate dosimetry are ongoing to establish the ratio between dose and image quality (ALARA principle). No fundamental difference between the systems can be stated in respect radio therapeutic planning: improper HU calibration accuracy in CBCT and DVT units. The geometric 3 D accuracy of high performance DVT systems is greater than that of CT Systems.

Elimination of mycoplasma from leukemia-lymphoma cell lines using antibiotics.

Mycoplasmal contamination of cell culture systems continues to present major problems for basic research and for manufacturing of bioproducts. Previous work suggested that certain antibiotics have strong anti-mycoplasma properties and raised the prospect that the technically rather simple antibiotic treatment may be an appropriate means for mycoplasma eradication. We have developed and validated an effective strategy to eliminate mycoplasma from chronically infected cell cultures using antibiotics which have shown strong activity against these contaminants. Here, we describe our experience with the treatment of 123 consecutive mycoplasma-positive leukemia-lymphoma cell lines, comparing five different antibiotic regimens (in total 433 treatments). We optimized the antibiotic dose schedules and the duration of treatments. The various antibiotic treatments which were employed in parallel had a high efficacy, as 71% to 86% of the infected cultures were cleansed. Treatment failure may result from the resistance of the mycoplasmas to antibiotic therapy and the inability of the eukaryotic cells to survive the cytotoxic effects of the antibiotics. Resistance to mycoplasma eradication was observed in 3% to 20% of the cultures. Loss of the cell culture caused by cytotoxicity was seen in 3% to 11% of the treatments. With regard to the overall outcome, 96% of the cell lines were rendered mycoplasma-free with at least one of the antibiotic treatments and were permanently cured. In conclusion, antibiotic treatment represents the most practical and efficient option to cleanse mycoplasma-positive cell lines.

Detection of mycoplasma in leukemia-lymphoma cell lines using polymerase chain reaction.

The specific, sensitive and reliable detection of mycoplasma contamination in cell cultures is an important part of mycoplasma control. We have sought to develop and validate a method for mycoplasma detection which is sensitive and accurate, but also practical in the sense of time spent, costs, and applicability in the standard laboratory; finally, the method should be suitable for screening large numbers of test specimens. To that end, we adapted a previously developed polymerase chain reaction (PCR) method for daily routine application. This single-step PCR uses a mixture of primers annealing to gene sequences coding for evolutionarily conserved 16S rRNA of different mycoplasma species, including the ones most commonly found in cell cultures. An internal control was introduced to exclude any false-negative tests resulting from technical PCR problems. This mycoplasma detection by PCR has been validated prospectively on 201 consecutive leukemia-lymphoma cell lines received at the institute over a 3-year period and on 118 initially positive cell lines after anti-mycoplasma treatment with antibiotics. The sensitivity (detection of true positives) of this PCR detection assay was 96% and the specificity (detection of true negatives) was also 96%, with positive and negative predictive values (probability of correct result) of 86% and 99%, respectively. PCR defined the mycoplasma status with 96% accuracy (detection of true positives and true negatives). Besides the high sensitivity and specificity, further attractive features of the PCR approach are the ease and speed with which large numbers of specimens can be tested. PCR mycoplasma analysis provides a readily available, quick and reliable test system with which to manage the important issue of mycoplasma contamination of cell lines.

Prevention of mycoplasma contamination in leukemia-lymphoma cell lines.

The contamination of cell lines with mycoplasmas is certainly one of the major problems occurring in cultured cells. Analyzing more than 460 human leukemia-lymphoma (LL) cell lines, we found that 28% of the cultures were mycoplasma-positive. Mycoplasmas can produce extensive changes, growth arrest and cell death in the infected cultures. While mycoplasma-infected cell lines can be truly cleansed from the contaminants, all the efforts would be in vain when the cells return to a mycoplasma-infested environment or are handled with unsuitable culture practices. Hence, the main focus of mycoplasma control should be on preventing cell culture contamination. Mycoplasmas can be introduced through several routes including culture reagents and laboratory personnel. Cross-contamination from infected cell cultures within one laboratory continues to be the major source for the spread of mycoplasma. Specific technical protocols and cell culturing guidelines may be followed in order to minimize the risk of mycoplasma contamination of cell lines. This "good culture practice" is of utmost importance as faulty cell culture techniques appear to be also the main reason for the high incidence of cross-contaminated LL cell lines which according to our experience using DNA fingerprinting of some 500 LL cell lines is about 15%.

p53 alterations in human leukemia-lymphoma cell lines: in vitroartifact or prerequisite for cell immortalization?

Alteration of the p53 gene is one of the most frequent events in human tumorigenesis. The inactivation of p53 tumor suppressor function can be caused by chromosome deletion, gene deletion, or mainly by point mutations. p53 mutations occur moderately often in hematopoietic malignancies. A significantly higher frequency of p53 alterations in cell lines vs primary samples has been observed for all types of malignant hematopoietic cell lines. It has been postulated that p53 gene abnormalities arise in cell lines during in vitro establishment of the culture or prolonged culture; but it is also conceivable that those cases that carry p53 mutations may be more suitable for in vitro establishment as permanent cell lines. We analyzed data on the p53 gene status in a panel of matched primary hematopoietic tumor cells and the respective cell lines derived from this original material. In 85% (53/62) of the pairs of matched primary cells and cell lines, the in vivo and in vitro data were identical (both with p53 wild-type or both with the same p53 mutation). In some instances, serial clinical samples (eg at presentation and relapse) and serial sister cell lines were available. These cases showed that a clinical sample at presentation often had a p53 wild-type configuration whereas the derived cell line and a relapse specimen carried an identical p53 point mutation. These findings suggest that a minor clone, at first undetectable by standard analysis, represents a reservoir for the outgrowth of resistant cells in vivo and also a pool of cells with a growth advantage in vitro, providing a significantly higher chance of immortalization in culture. This was further supported by studies employing mutant allele-specific gene amplifications, a technique which is significantly more sensitive (100- to 1000-fold) than the commonly applied SSCP assay with a sensitivity threshold of about 10% mutated cells within a pool of wild-type cells. Taken together, this analysis confirms the usefulness of human hematopoietic cell lines as in vitro model systems for the study of the biology of hematopoietic malignancies. It further underlines the notion that p53 gene alterations confer a survival advantage to, at least some, malignant cells in vitro and presumably also in vivo; however, it is highly unlikely that a p53 mutation alone would suffice for the immortalization of a cell line in vitro or tumor development in vivo. Leukemia (2000) 14, 198-206.

Biology of monocyte-specific esterase.

The monocyte-specific (carboxyl)esterase (MSE) is a marker enzyme which is well-known to hematologists as its detection is part of the traditional cytochemical stainings of leukemia cells. There are a variety of synonyms for MSE among hematologists and biochemists. Biochemically, MSE is well-characterized, but should be discerned from other esterases with similar or identical substrate specificities and other features. Intensive analysis of normal and malignant hematopoietic cells and leukemia-lymphoma cell lines using isoelectric focusing established the specificity of this enzyme for monocytes and related cells, hence its designation as monocyte-specific esterase. Cloning of the gene led to its molecular characterization and provided new opportunities to examine MSE expression also at the RNA level which confirmed the monocyte/macrophage specificity. The availability of the gene sequences of various serine esterases and lipases which also hydrolyze ester bonds allowed for the identification of identical isolates from different tissues and the construction of an unrooted dendrogram based on sequence homologies of 22 enzymes. The detailed regulation of the gene and the functional role of MSE have remained largely unknown as of yet. However, DNA binding sites for various transcription factors have already been detected. Some evidence suggests involvement in physiological detoxification processes and in the immune defense against tumor cells. A more thorough understanding of the in vivo function of this truly unique enzyme should be helped by characterizing the signals and signal transduction mechanisms which lead to MSE expression.

ABL-BCR expression in BCR-ABL-positive human leukemia cell lines.

Expression of normal ABL and BCR and of reciprocal fusion genes BCR-ABL and ABL-BCR was examined in a panel of 53 BCR-ABL-positive cell lines by RT-PCR to determine the influence of the various transcripts on leukemogenesis. Seventeen out of 18 lymphoid cell lines expressed ABL1a and/or ABL1b, whereas only 16 out of 35 myeloid cell lines expressed one or both normal ABL transcripts. Normal BCR was expressed in seven lymphoid cell lines; all cell lines from the m-bcr group (n = 9) were BCR-negative. Among the myeloid cell lines, 77% expressed the BCR gene. The M-bcr and m-bcr translocations were equally distributed among cell lines with lymphoid phenotype. The m-bcr translocation was not found in myeloid cell lines. b3-a2 constitutes the predominant form of fusion gene in myeloid cell lines with an incidence of about 68%. One myeloid cell line exhibited the mu-bcr variant. An ABL-BCR transcript of the 1a splice variant was not detected in any of the cell lines. ABL1b-BCR was expressed in all varieties of cell types and translocation forms: 56 and 66% in the lymphoid and myeloid cell lines, respectively; similar distributions were found for the fusion gene types: 67% among e1-a2, 73% among b2-a2, and 61% among b3-a2 translocations. Except for the lack of expression of normal BCR in m-bcr cell lines and of ABL1a-BCR expression in all cell lines, no consistent correlation of expression or lack of expression of BCR and ABL or of ABL-BCR reciprocal fusion genes could be found with cell lineages and translocation types. Further work is required to determine the exact role of the reciprocal fusion gene transcripts on the pathophysiological mechanisms of leukemogenesis.

Expression of receptor tyrosine kinase HTK (hepatoma transmembrane kinase) and HTK ligand by human leukemia-lymphoma cell lines.

HTK (hepatoma transmembrane kinase) is a receptor tyrosine kinase belonging to the EPH subfamily of tyrosine kinases. Binding of its ligand (HTKL) results in tyrosine phosphorylation of HTK. In the present study, we analyzed the possible involvement of this ligand-receptor signalling system in hematopoiesis by examining the expression of both HTK and HTKL in a large and comprehensive panel of 70 continuous human leukemia-lymphoma cell lines. HTK and HTKL mRNA expression were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). HTK mRNA was detected in 68/70 cell lines; 58/70 cell lines were positive for HTKL mRNA expression; consequently, co-expression of both receptor and ligand was demonstrated in the majority of cell lines. Collectively, the wide-spread expression suggests a role for this ligand-receptor pair in hematopoietic development and/or function. Investigation of the details of signal transduction pathway that is activated by the HTK tyrosine kinase will help to define the exact biological function of the HTK-HTKL system.

Leukemia cell lines: in vitro models for the study of Philadelphia chromosome-positive leukemia.

The Philadelphia (Ph) chromosome, the main product of the (9;22)(q34;q11) translocation, is the cytogenetic hallmark of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder of the hematopoietic stem cell; the Ph chromosome is also found in a sizeable portion of acute lymphoblastic leukemia (ALL) patients and in a small number of acute myeloid leukemia (AML) cases. At the molecular level, the t(9;22) leads to the fusion of the BCR gene (on chromosome 22) to the ABL gene (translocated from chromosome 9); this fusion gene BCR-ABL with its elevated tyrosine kinase activity must to be central to the pathogenesis of these disorders. Three different breakpoint cluster regions are discerned within the BCR gene on chromosome 22: M-bcr, m-bcr, and mu-bcr. Ph + leukemia cell lines are important tools in this research area. More than 20 ALL-and more than 40 CML-derived Ph + leukemia cell lines have been described. Furthermore, three Ph + B-lymphoblastoid cell lines, established from patients with Ph + ALL or CML, are available. Molecular analysis has documented BCR-ABL fusion genes in three apparently Ph chromosome-negative cell lines, all three derived from CML. Nearly all Ph + ALL cell lines have the m-bcr e1-a2 fusion gene (only two ALL cell lines have a b3-a2 fusion) whereas all CML cell lines, but one carry the M-bcr b2-a2, b3-a2 or both hybrids. The mu-bcr e19-a2 has been detected in one CML cell line. Four cell lines display a three-way translocation involving chromosomes 9, 22 and a third chromosome. Additional Ph chromosomes (up to five) have been found in four Ph + ALL cell lines and in 18 CML cell lines; though in some cell lines the extra Ph chromosome(s) might be caused by the polyploidy (tri- and tetraploidy) of the cells. Another modus to acquire additional copies of the BCR-ABL fusion gene is the formation of tandem repeats of the BCR-ABL hybrid as seen in CML cell line K-562. Both mechanisms, selective multiplication of the der(22) chromosome and tandem replication of the fusion gene BCR-ABL, presumably lead to enhanced levels of the fusion protein and its tyrosine kinase activity (genetic dosage effect). The availability of a panel of Ph + cell lines as highly informative leukemia models offers the unique opportunity to analyze the pathobiology of these malignancies and the role of the Ph chromosome in leukemogenesis.

Detection of mycoplasma contaminations in cell cultures by PCR analysis.

Mycoplasma contamination is still one of the main problems in using cell cultures in biological and medical research and in the production of bioactive substances, because mycoplasma can alter nearly all parameters and products of the cell. They can persist undetected in the culture if no special detection methods are applied. In recent years, the PCR technology has become a commonly used method to analyze genomic DNA and the expression of genes, with both high specificity and sensitivity. This technique can be effectively employed for the detection and even the identification of mycoplasma contaminations in cell cultures applying primers complementary to the 16S rDNA region. Although this technique, once established, is characterized by simplicity and speed, PCR is still a complex process and its sensitivity and specificity can be influenced by a number of different parameters, e.g. inhibiting compounds originating from the preparation process of the DNA, RNA or cDNA, contamination of the solutions with PCR products, and the selection of a primer pair which does not cover all the mycoplasma species occurring in cell cultures. Thus, adequate controls have to be included to obtain reliable results. The present review examines the use of different primers of the 16S rDNA region including their specificity, the sensitivity applying various DNA or RNA preparation procedures, and the methods to detect finally the amplicons. In conclusion, basic nucleic acid preparation and PCR product detection methods offer a simple, fast and reliable technique for the examination of mycoplasma contaminations in cell cultures, provided that the indispensable control assays are implemented.

Expression patterns of the JEM-1 gene in normal and tumor cells: ubiquity contrasting with a faint, but retinoid-induced, mRNA expression in promyelocytic NB4 cells.

The JEM-1 gene, recently identified in acute promyelocytic leukemia (APL) cells, codes for a novel nuclear factor (Duprez et al Oncogene 1997; 14: 1563-1570). JEM-1 is kept silent in the APL cell line NB4, but up-regulated (3 kb transcript) during cell maturation. Here, we show that retinoic acid (RA)-induced JEM-1 expression is biphasic (peaks at 6 h and 48 h) and associated with the later stages of maturation. Retinoids, which cooperates with cAMP to induce maturation, also cooperates with cAMP to up-regulate JEM-1, either in maturation-responsive NB4 cells or in NB4-R1 resistant subclones. APL patients showed a low, yet variable, level of JEM-1 mRNA in bone marrow. RA treatment induced an increase in the level of JEM-1 mRNA, as detected by a semi-quantitative PCR. This increase can result from both gene up-regulation or replacement of leukemia cells by differentiated ones. Analysis of JEM-1 expression patterns in normal and tumor cells revealed that JEM-1 expression was ubiquitous. Cell lines derived from monocytic and erythroid leukemias, expressed low and high amounts of JEM-1 mRNA, respectively. Using a JEM cDNA probe, distinct profiles of expression and different transcript sizes (4 kb, 3 kb and 2 kb) were also identified in tumour and normal non-hematopoietic tissues, while interestingly only the 3kb transcript was up-regulated in NB4 cells. This work identifies JEM-1 as a novel ubiquitous gene whose expression is low in APL cells, but can be restored by RA treatment, concomitant with cell maturation.

HHV-8 infection is specific for cell lines derived from primary effusion (body cavity-based) lymphomas.

Human herpes virus 8 (HHV-8; or KSHV, Kaposi's sarcoma-associated herpes virus) is a gamma herpes virus with sequence homology to Epstein-Barr virus (EBV). It was first isolated from Kaposi's sarcoma tumor cells and subsequently from tumor cells and peripheral blood mononuclear cells from patients with primary effusion lymphomas (PEL; or body cavity-based lymphomas). PEL has been recognized as an individual nosologic entity based on its distinctive biological-pathological features and its consistent infection with HHV-8 (commonly, but not always co-infected with EBV), occurring predominantly in human immunodeficiency virus (HIV)-infected patients but occasionally also in HIV-negative cases. Whether HHV-8 sequences can be found also in non-hematopoietic tumor cells other than Kaposi's sarcoma and in malignant hematopoietic malignancies other than PEL, has been the focus of the present studies. We examined the presence of HHV-8 sequences by polymerase chain reaction (PCR) using (1) a panel of 133 human cell lines established from a large variety of solid tumors; (2) a spectrum of 114 hematopoietic cell lines derived from the different cell lineages including 50 B cell leukemia/lymphoma-derived cell lines and seven cell lines established from patients with PEL. Besides the seven PEL cell lines, 46 cell lines that were derived from malignant pleural effusion or ascitic fluid material (25 non-hematopoietic and 21 hematopoietic cell lines) were examined. Except for the seven PEL cell lines that were strongly HHV-8+ in the PCR, all solid tumor cell lines and all hematopoietic cell lines scored consistently negative for the presence of HHV-8 sequences. These results confirm the absolute specificity of HHV-8 infection (within the hematopoietic malignancies) for PEL. PEL cell lines represent useful tools for the analysis of the biology of this neoplasm and of the pathogenetic role of the virus in the disease development.

Lymphoma cell lines: in vitro models for the study of HHV-8+ primary effusion lymphomas (body cavity-based lymphomas).

Primary effusion lymphoma (PEL; also known as body cavity-based lymphoma) is recognized as a new and unique lymphoma entity occurring predominantly, but not exclusively in human immunodeficiency virus (HIV)-seropositive patients with acquired immunodeficiency syndrome (AIDS). PEL grows exclusively in body cavities as serous lymphomatous effusion without evidence of mass disease or dissemination. Their most unique feature is infection with the newly discovered human herpesvirus-8 (HHV-8; also known as Kaposi's sarcoma-associated herpesvirus), often accompanied by co-infection with Epstein-Barr virus (EBV). A number of continuous lymphoma cell lines have been established from the malignant pleural effusion, ascitic fluid and peripheral blood of patients with AIDS- and non-AIDS-associated PEL. While all cell lines are HHV-8+, about half of them also contain EBV sequences. Stimulation of the cell lines causes switch from latent to lytic HHV-8 infection. The cells are generally negative for T and B cell immunomarkers (except for CD138 suggesting a pre- or terminal plasma cell stage) and positive for some activation and adhesion markers; they are genotypically B cells with their immunoglobulin genes rearranged. Complex, hyperdiploid karyotypes with multiple structural abnormalities are seen in the cell lines examined. No alterations of known proto-oncogenes are detected in PEL, with the exception of BCL-6 mutations occurring in a large percentage of cases. Heterotransplantation of the cell lines into immunodeficient mice leads to the development of lymphomatous effusion and marked angiogenesis. As HHV-8 contains DNA sequences of several protein homologues, the cell lines express various cytokines, cytokine receptors, chemokines, cell cycle and anti-apoptosis modulators which are upregulated upon stimulation. Indeed, some cell lines produce high levels of (human) interleukin-6 and interleukin-10. Taken together, these cell lines represent very important model systems for the elucidation of the pathobiology of PEL; furthermore, the cell lines are extremely useful scientific tools providing a resource to pursue studies of HHV-8-mediated pathogenic mechanisms.

Growth-inhibitory effects of transforming growth factor-beta 1 on myeloid leukemia cell lines.

Transforming growth factor-beta1 is a pleiotropic cytokine involved in a variety of biological processes in both transformed and normal cells, including regulation of cellular proliferation and differentiation; its predominant action on hematopoietic cells is to inhibit cell growth. We used growth factor-dependent cell lines to assess TGF-beta1 effects on human myeloid leukemia cell growth. While four lines were completely or predominantly resistant, TGF-beta1 inhibited effectively, albeit to various extents, the growth of 12 other cell lines. This effect was dose dependent and specific, because a neutralizing anti-TGF-beta1 antibody prevented TGF-beta1-induced growth suppression. In the present system, basic fibroblast growth factor, known as an antagonist of TGF-beta1 counteracting its inhibitory effects, did not abrogate the suppressive effects of TGF-beta1. Other growth-stimulatory cytokines negated the TGF-beta1-induced inhibition in several cell lines, again to various extents. When proliferation was enhanced by growth-promoting cytokines (e.g. granulocyte-macrophage colony-stimulating factor, GM-CSF, stem cell factor, SCF, or PIXY-321), some previously TGF-beta1-sensitive cell lines acquired cellular resistance toward TGF-beta1-mediated growth suppression, whereas four other cell lines remained susceptible to TGF-beta1 growth inhibition despite possible counteraction by other cytokines. Thus, three growth response patterns to TGF-beta1 were seen: (1) constitutive resistance; (2) factor-dependent relative resistance; and (3) sensitivity to growth inhibition indifferent to counteracting cytokines. In the latter case, TGF-beta1 did not downregulate expression of one specific growth factor receptor. These studies indicate that human myeloid leukemia cells, represented here by leukemia cell lines as model systems, exhibit heterogeneous growth responses to TGF-beta1; its inhibitory effects can be modulated or completely alleviated by positive antagonistic cytokines. The availability of TGF-beta1-susceptible and -refractory cell lines allows for detailed investigations on the mechanisms of these regulatory pathways, the nature of TGF-beta1-resistance, and the possible contribution of acquired TGF-beta1-resistance to disease progression.

Expression of hepatocyte growth factor and its receptor c-met in human leukemia-lymphoma cell lines.

Recent studies have shown that hepatocyte growth factor (HGF) is a regulatory protein for the proliferation and differentiation of hematopoietic progenitors. The proto-oncogene c-met encodes a tyrosine kinase receptor that binds HGF. To obtain information about their possible involvement in the pathogenesis of hematopoietic tumors, we have examined the expression of HGF and c-met in a large panel of leukemia-lymphoma cell lines encompassing all major hematopoietic cell lineages. HGF and c-met mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blotting. The panel of 92 cell lines analyzed comprised seven B-cell precursor, ten B-cell, six plasma cell, 13 T-cell, four natural killer (NK) cell, 16 myelocytic, 12 monocytic, 13 erythroid-megakaryocytic and 11 Hodgkin-anaplastic large cell lymphoma (ALCL) lines. In total 64 (70%) were RT-PCR-positive for HGF and 43 (47%) for c-met. The highest percentages of expression were found for HGF in the plasma cell (100%), NK (100%) and myeloid (75-92%) cell line categories, whereas c-met was found predominantly in plasma cell (100%) and Hodgkin-ALCL (91%) cell lines. The concomitant expression of HGF and c-met in plasma cell lines (100%) and Hodgkin-ALCL (73%) cell lines should be noted. The high HGF expression in myelocytic-monocytic cell lines (75 and 92%) contrasts with the low c-met expression (18 and 8%) in these cell lineages. In 50 cell lines, mRNA expression of these two genes was also examined at the Northern blot level: 12/50 (24%) and 4/48 (8%) were positive for HGF and c-met mRNA expression, respectively. Of note, three of the four c-met + lines belonged to the category Hodgkin-ALCL; the Hodgkin cell line SUP-HD-1 showed both HGF and c-met mRNA bands suggesting the possibility of an autocrine loop. In conclusion, we detected HGF expression in various types of leukemia-lymphoma cell lines, particularly in plasma cell and myeloid malignancies; c-met expression was found in plasma cell and Hodgkin-ALCL cell lines. Further detailed analysis of the role of this ligand-receptor pair in the pathogenesis of hematopoietic neoplasms is indicated; to this end the HGF + and c-met + cell lines described here represent exquisite model systems.

Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23).

We describe two new human leukemia cell lines, MOLM-13 and MOLM-14, established from the peripheral blood of a patient at relapse of acute monocytic leukemia, FAB M5a, which had evolved from myelodysplastic syndrome (MDS). Both cell lines express monocyte-specific esterase (MSE) and MLL-AF9 fusion mRNA. Gene fusion is associated with a minute chromosomal insertion, ins(11;9)(q23;p22p23). MOLM-13 and MOLM-14 are the first cell lines with, and represent the third reported case of, MLL gene rearrangement arising via chromosomal insertion. Both cell lines carry trisomy 8 which was also present during the MDS phase, as well as the most frequent trisomies associated with t(9;11), ie, +6, +13, +19 variously present in different subclones. Despite having these features in common, differences in antigen expression were noted between the two cell lines: that of MOLM-13 being CD34+, CD13-, CD14-, CD15+, CD33+; whereas MOLM-14 was CD4+, CD13+, CD14+, CD15+, CD33+. Differentiation to macrophage-like morphology could be induced in both cell lines after stimulation with INF-gamma alone, or in combination with TNF-alpha, which treatment also induced or upregulated, expression of certain myelomonocyte-associated antigens, including CD13, CD14, CD15, CD64, CD65 and CD87. Together, these data confirm that both cell lines are likely to be novel in vitro models for studying monocytic differentiation and leukemogenesis.