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Second International Symposium on the Molecular Biology of Hematopoiesis University of InnsbrucWNew York Medical College Innsbruck, Austria, July 14-18, 1991 Honorary President E. Donna11 Thomas, Nobel Laureate (USA)

Honorary Members Eugene Goldwasser (USA) Paul Marks (USA) Albert0 Marrnont (Italy)

Donald Metcalf (Australia) Leo Sachs (Israel) Mehdi Tavassoli (USA)

Chairmen Herbert Braunsteiner (Austria) Richard D. Levere (USA)

Organizing Committee Nader G. Abraham (USA) Gunther Konwalinka (Austria)

Local Organizers Richard Bilgeri Francoise Geisen

Gunther Konwalinka Andreas Petzer Margot Haun

Symposium Secretary Udo Zilian

International Advisory Committee J.W. Adamson (USA) G.C. Bagby (USA) E.3. Benz (USA) H. Beug (Austria) S.C. Clark (USA) M. Dierich (Austria) B. Forget (USA)

D. Holzer (FRG) W. Ostertag (FRG) I. Pragnell (UK) R.K. Shadduck (USA) F. Takaku (Japan) E.G.Wright (UK) N.S.Young (USA)

Abstracts Abstracts received by June 7, 1991 are listed in alphabetical order by first author’s name within each of the following sections:

I. Gene Regulation in Hematopoiesis II. Regulation of Hematopoiesis: Growth Factors - Receptors III. Stroma and Hematopoiesis

IV. Viruses - Hematopoiesis - AIDS V. Retroviral-Mediated Gene Transfer

Acknowledgments The Organizing Committee gratefully acknowledges the following organizations and companies who supported this meeting: Bundeskanzleramt fur Gesundheitswesen Bundesministerium fur Wissenschaft und Forschung Amt der Tiroler Landesregierung Magistrat der Stadt Innsbruck Aesca Amgen Bayer Biomedica Bio-Trade Boehringer BYk Cetus Cilag Cyanamid Ebewe Genetics Institute

Gerot Glaxo Heilmittelwerke Immunex Institute Henri Beaufour Kirin Laevosan Miles ortho Pfizer Sandoz

Preface

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International Journal of Cell Cloning 9:1 (1991) The Second International Conference on the Molecular Biology of Hematopoiesis is dedicated to the Nobel Laureate Dr. E. Donna11 Thomas, and to the contributions of Dr. E. Goldwasser, Dr. €! Marks. Dr. A . Mannont, Dr. D. Metcalf, Dr. L. Sachs and Dr. M. Tavassoli. This symposium is the second international symposium on Molecular Biology of Hematopoiesis held in collaboration between the University of Innsbruck and New York Medical College. However, it is the seventh conference held in this series. A great deal has been learned in the past few years about bone marrow failure, gene expressiodgene therapy and growth factors from the point of view of basic science and clinical trials. In addition to erythmpoietin and colony-stimulating factors, several other growth factors have passed phase 11 clinical trials and their applications have been shown to have great therapeutic potential. It seemed to us that the time was ripe for a conference to bring together investigators from around the world to discuss different aspects of these and other areas related to normal and abnormal hematopoiesis. The goals of this conference were to permit scientists to review and integrate existing knowledge, exchange ideas, provide a forum for new information and stimulate new approaches in each other’s laboratories at what appears to be a critical time in the development of this field. Certain growth factors can act on progenitors in conjunction with other cytokines. Exciting data are being generated in linking growth factors to proto-oncogenes, and the role of non-hormone growth factors such as heme and arachidonic acid metabolites in cell proliferation and differentiation. More data will continue to accrue, in particular, with respect to the cell surface receptors, the mechanisms of shut-off of local production of these factors and possible clinical side effects of their continued administration. Retrovirus gene transfer techniques and genetic manipulation of murine and human cells have added new information on the regulatory steps involved in cell development and genetic disorders. Transfection of eukaryotic cells, specifically bone marrow cells, and subsequently in vivo transplantation further suggest that hemopoietic reconstitution is possible. The third international meeting in this series will be held in either Europe or the U.S.

N.G. Abraham G. Konwalinka

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Section I. Gene Regulation in Hematopoiesis Quantitation of Human Erythroid-Specific Porphobilinogen Deaminase mRNA by the Polymerase Chain Reaction N.G. Abraham, B. Grandchamp, R.D. Levere Department of Medicine, New York Medical College, Valhalla, NY and Faculte de Medecine Xavier Bichat, Paris, France Porphobilinogendeaminase (PBG-D), the third enzyme in the heme synthetic pathway, possesses two isoforms encoded by distinct mRNAs that are the result of transcription of a single gene from two promoters through differential splicing. During erythroid differentiation, only the expression of the erythroid-specific isoform (E-PBGD) was increased. A system was developed to evaluate genetic expression of E-PBGD in samples limited in cell number and/or mRNA copy. Total RNA from human cells was reverse-transcribed and amplified by the polymerase chain reaction in the same tube with an internal standard that is an in vitro transcript of a cDNA differing from its sample counterpart by a few restriction sites and 24 bp (10%)in the target region. The primers spanned through regions where sample and standard templates were identical in sequence. Amplified templates were resolved by restriction enzyme digestion and gel electrophoresis and quantified by densitometer tracing of corresponding bands on autoradiograms. When an appropriate amount of internal standard is present in the reaction mixture, the ratio of amplified sample versus standard template is proportional to the amount of sample RNA and it is therefore possible to calculate the number of specific mRNA molecules in the original sample.

Hemin Stimulation of Hematopoiesis in Long-Term Bone Marrow Culture N.G. Abraham, J. D. Lutton. J. L. Chertkov, R.D. Levere New York Medical College, Valhalla, NY, USA The effect of various concentrations of exogenous hemin on cellularity and hemopoietic clonal potential of cells maintained in murine long-term bone marrow cultures (LTBMC) was studied. Hemin, at concentrations of 1 and 10 pM added weekly to LTBMC, produced a significant increase in cellularity for up to 8 weeks in culture. Lower concentrations of hemin (0.1 pM) were more effective for sustained cellularity in older cultures (10 - 12 weeks). Prior exposure of the adherent cell layer to high concentrations of hemin (10 pM) had a beneficial effect on the support of newly seeded cultures, however, the effect of lower hemin concentrations (0.1 - l pM) on stromal cell layer formation was not significant. Supplementation of hemin for the first week in culture increased cumulative cell production as well as the number of CFU-gm and longevity of hematopoiesis in LTBMC was significantly increased with 0.1 pM hemin. In contrast with data obtained in short-term cultures, hemin in this system primarily affected the myeloid line of differentiation, but not the early erythroid progenitors (BFU-e). Hemin, at a concentration of 0.1 pM,increased CFU-s at several fold higher than that of the control. Results suggest that hemin may produce mobilization of hematopoietic cells and committed precursors from adherent cells into suspension. Further, supplementationwith hemin in LTBMC significantly increased the myeloid progenitor compartment and longevity of culture without altering the erythroid compartment.

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Mechanisms of Activation of ABL

P B. Allen, L. M.

Wiedemann

Leukaemia Research Fund Centre at the Institute for Cancer Research, Fulham Road, London SW3 6JB, UK Studies aimed towards a better understanding of the origin and development of disease in human chronic myeloid leukemia (CML) are dominated by attempts to elucidate the contribution of a deregulated ABL tyrosine kinase. This is the principal genetic lesion s&n associated with CML, resulting from a reciprocal translocation which juxtaposes BCR gene sequences with those of ABL, culminating in the translation of a ECR/ABL fusion protein (~210).CML will develop into an aggressive leukemia involving multiple hematopoietic lineages, including the lymphoid compartment. The BCR/ABL oncogene is also detected in a minority of de novo acute lymphoblastic leukemia with the chromosomal breaks occurringin some mes at an alternativelocation within the BCR gene generating a fusion protein (~190)with a lesser contribution from ECR sequences. The focus of current investigation is to definethe role of BCR residues upon modulation of ABL kinase activity, as well as attemptingto characterizeother putative regulatory regions within the ABL protein. A series of in-frame deletion mutants have been constructed in the first exon of BCR which is common to both p1W and p210. These proteins have been successfully expressed in factor dependent hematopoietic cell lines and all variants exhibit an elevated in vitro tyrosine kinase activity characteristic of the activatedABL oncogene. The critical question with respect to malignancy is which of these mutants behave as an oncogene in vivo. The factor dependent nature of the cells transfected can be used as a guideline in this respect. The parental BCR/ABL oncogenewill relieve factor dependence in these cells and this in turn has been shown to correlate with their tumorigenicity in vivo. Other studies are directed tawards expression of regulatory domains of ABL out of the context of the catalytic domain in an attempt to titrate out putative negative andlor positive regulatory functions that may act upon AEL within the normal signaling pathway.

Inhibition of Heme Synthesis and Modulation of Globm Chains Synthesis, 'Ransferrin Receptors Number and Ferritin Content in Differentiating Friend Erythroleukemia Cells A . Buttistini", E.M. Coccia'. G. Marziali", D. Bulgarinib. S. ScaLzob, G. Fioruccid, G. Romeod, E. Affabrif, U. Testab, G.& Rossi", C.Peschleb 'Dept. of Mrology, Istituto Superiore di Sanid; bept. of Hematology and Oncology, Istituto Superioredi Sanit& 'Institute of Microbiolcgy, Univ. of Messina; 'Institute of Biomedical Technology, CNR. Rome, Italy Friend leukemic cells (FLC) are erythroid precursors blocked in their differentiationpath- at the proerythroblastic stage. Treatment of FLC with dmethylsulfoxide (ME,SO) and other inducers leads to a sequence of differentiativeevents, which mimic normal differentiation of erythroid cells. The effect of succinylacetone(SA), a highly specific inhibitor of ALAdehydratase and heme synthesis,on hemoglobin (Hb) production, transferrinreceptor (TfR) and femtin expression was analyzed in differentiating FLC. This compound exerted a pronounced inhibitory effect not only on heme synthesis, but also on all the other above mentioned parameters. In particular, SA induced: (a) a reduction of the level of a-globin mRNA; (b) a decreased number of exposed TfR molecules, without modification of their affinity for the ligand; (c) a reduced level of TfR RNA, without significant change of TfR gene transcription rate; and (d) a lower ferritin content. The addition of exogenous hemin to differentiating FLC exerted opposite effects. and particularly induced an increase of both the number of TfRs and ferritin content. These findings suggest that in erythropoieticdifferentiationoptimal heme synthesis is required to coordinately modulate (i) globin chains synthesis, (ii) iron uptake and its iotracellular pathway, i.e.. the TfWferritin system.

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The Use of Fluorescent In Situ Hybridization in Flow Cytometry and Confocal Microscopy to Detect Gene Expression and EBV RNA Tracks in Hemopoietic Cells Jan G.J.Bauman, Jan Bayer, Jeanne B. Lawrence Departmentsof Cell Biology, ITRI-TNO, Rijswijk, Holland and UMMC, Worcester, MA, USA We have developed a FISH method for hemopoietic cells in suspension to detect specific RNA species. Detection is by flow cytometry to quantitatethe signal' or by confocal microscopyzto localize the RNA inside the cells. An example is detection by flow cytometry of 8-globin mRNA levels in bone marrow of normal and anemic mice and fetal liver cells? It was possibleto quantitate the amount of P-globin mRNA expressed per individual cell and to count the number ofa-globin expressingcells. These numbers correlated well with the amount of mRNA isolated from the cells. Detection of specific RNA in 3D by confocal microscopy was recently applied to the Namalwa Lymphoma line. Namalwa cells contain two integrated copies of the EBV genome? The RNA transcribed from these viral genome is present in a curvilinear track inside the nucleus? The shape and position of the EBV RNA tracks inside the cell nucleus has been determined by double staining using immuno-fluorescencedetection of the nuclear pore complexes in combination with FISH detection of the viral RNA. The tracks were always found running towards and very close to the nuclear pores. The examplesgiven illustrate the power of FISH analysis of specific RNA in hemopoietic cells. 1

2 3 4 5

Bauman and Bentvelzen, Cytometry 9, 1988, 517-524. Bauman, Bayer and van Dekken, J. Microscopy 157, 1990, 73-81. Bayer and Bauman, Cytometry 11, 1990, U2-143. Lawrence, Villnave and Singer, Cell 52, 1988, 51-61. Lawrence, Singer and Marselle, Cell 57, 1989, 493-502.

An NFxB-Like Protein is Involved in the Transcriptional Regulation of the cgua Gene in Human Myelo-Monocytic Leukemia Cells but Not in Normal Monocytef M.A.Brach, E Herrmann, D.W fbfe Dept. of Internal Medicine 1, University of Freiburg Medical Center, Freiburg, Germany, and Laboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Boston, MA We have identified a novel DNA binding protein in human myelo-mo&c leukemia cells THP-1, HL-60, and KG-1responsiblefor basal transcriptionalactivity of the c-jun gene. This protein recognizes a sequence in the c-jun promoter which is different from the AP-1 site previously reported to mediate autoinduction of the cjun gene. Binding of this protein is enhanced upon stimulation with inducers of monocytic differentiationsuch as TPA, TNF-a, and the DNA synthesis inhibitor ARA-C. As determined by DNAse I protection assay, the element recognizes an ll bp palindromic sequence in the c-jun promoter which has similarity to the NFxB binding site. In addition, the protein shares several other features with the NFxB transcription factor in that i) binding activity can be induced by treatment of the human myeloid cells with cyclohurimide; ii) the binding activity of the specific sequence in the cjun promoter is enhanced in the presence of GTP; iii) binding activity can be generated from cytosolic fractions of unstimulated cells by a combined treatment with desoxycholicacid and formamide, suggesting that this protein is released from an inhibitory protein in the cytoplasm by dissociating agents. A 54 bp region of the c-jun promoter containing this element confers basal transcriptional activity as well as the induction of the c-jun gene in these cells after treatment with TPA, TNF-a, or ARA-C. Deletion of this fragment abolishesbasal transcriptionand inducibility. Analysis with normal human monocytes, however, reveals, that this protein is not expressed and not inducible with either substance tested. The molecular size of the protein differs from that of the known members of the NFxB family, suggesting that this transcription factor is a novel DNA binding protein, which regulates transcription of the c-jun gene in human myeloid leukemia cells but not in normal monocytes.

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Immediate-Early Genes: PAF Activates Transcription of c-fos and c-jun f! Braquet', N.G.Bazanb 'hstitut Henri Beaufour, LePlessis Robinson, France; bLSU Eye Center and Neuroscience Center, New Orleans, LA, USA Platelet-activating factor (PAF, l-O-alkyl-2-acetyl-sn-glycem-3-phosphwhol~e) is a biologically active phospholipid mediator that plays a role as a cell modulator or messenger in inflammation, immune responses, ischemia-reperfusion, and in tissue injury. In addition, PAF transcriptionally activates the immediate-early genes c-fos and c-jun in neuroblastoma cells, human T-lymphocytes, B-cells, and retinoblastoma cells. The rapid expression of immediate-earlygenes comprises the nuclear response to intracellular signals such as growth factors, neuronal depolarization, and agents which induce cellular differentiation. The protooncogenesfos andjun are the best characterized of these genes, are known to encode transcriptional regulatory proteins and to be responsive to second messengers, and are associated with events collectively known as cellular activation. The mediator PAF,which is formed during tissue injury, ischemia, and inflammatory and immune reactions, is capable of activating the expression of the fos/jun/AP-l transcriptional signaling system (J. Neurosci. Res. 24558; 1989) in cultured human lymphocytes and neuroblastoma cells. PAF antagonists (e.g. BN52021) inhibit immediate-earlygene expressionin cultured cells. The PAF antagonism results argue for the involvement of the recently disclosed intracellular PAF-binding sites (J. Biol. Chem. 2609140; 1990) and the couplingof stimulatory events (ischemia, neurotransmitters)to immediate-earlygene expression. PAF, in its role as an intracellular signaling molecule, may be centrally involved in eliciting the expression of genes whose products determine the long-term responses to injury, inflammation, and events of cellular differentiation.

Regulation of Vascular Integrity by PAF,Cytokines and Adhesion Molecules: Implications in Inflammatory Diseases P: Bmquet, D. Hosford, J. M. Mencia-Huerta, M. Paubert-Braquet" IHB, 17 avenue Descartes, 92350 Le Plessis Robinson, France and BIO-INOVA, 48-52 rue de la Gare, 78370 Plaisir, France In addition to shock, sepsis, and thermal injury, several pathologies including asthma, ischemia, and graft rejection are characterized by an underlying pathology consisting of endothelial injury, excessive blood cell infiltration and vascular leakage. These alterations contributeto hemodynamicdisturbances, edema formation and most notably in shock, organ failure. Interactionsbetween endothelial cells (EC) and circulating cells (CC) are regulated by various mediators which influence the expression of cell surface markers and adhesion molecules. These molecules, particularly leukocyte cell adhesion molecules (Leu-CAM) and VLA 4, appear to play a central role in EC-CC crosstalk. We have shown that PAF primes the synthesis and release of various cytokines such as interleukin 1 and 6 (IL-I, IL-6) and tumor necrosis factor (TNF). In addition PAF enhances the IL-4 induced expression of the low affinity receptor for IgE (FceIIICD23) on rnonocytes. We examined the role of PAF in expression of a and common chains of Leu-CAM's (CD lla, CD llb, CD llc, CD 18), and CD 2/LFA3 (CD 58) on neutrophils and monocytes, by comparing effects of the autacoid with those of IL-4. Both PAF and IL-4induced a significant increase in CD llb, CD llc and CD 18. In contrast, no influenceof either PAF or IL-4 on CD lla expressionwas observed. Anti L-4antibodies inhibited the L-4-induced expression of CD llb and CD llc, while the PAF antagonistsBN 52021 and BN 50730 inhibited the PAF-induced appearance of CD llb, CD llc and CD 18, a similar effect being observed on CD 2/LFA3 expression. This indication that PAF is implicated in EC-CC adhesion processes is further supported by data showing that antagonistsof the mediator can reduce the adhesion of leukocytes to TNF- and IL-1-treated EC by up to 35-4046. Indeed, PAF antagonists have already proved therapeuticallyeffective in animal models of diverse inflammatoryconditions, particularly shock and bum, where in addition to other activities, they may modulate expression of adhesion molecules. Encouraging p r e l i a r y results have been obtained in clinical studies on BN 52021 in burn injured patients.

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Co-ordinate Expression of the Product of the Proto-Oncogene c-kil and CD34 on Normal Bone Marrow Cells and Leukemic AML Blasts H.-J.Buhring", C.A. Muller", FW Buschb a Section for Transplantation Immunology and Immunohematology, Second Department of Internal Medicine, Medical University Clinic of Tubingen, 7400 Tiibingen, Germany, and bSecondDepartment of Internal Medicine, Medical University Clinic, 7400 Tiibingen, Germany Six monoclonal antibodies recognizing Pl45c-kit, the product of the proto-oncogene c-kit, and three antibodies specific for CD34 (43A1, ICH-3, QBEND/10 were used to study the correlated expression of these antigens on human bone marrow mononuclear cells (BMMNC) and on leukemic blasts from patients with AML. One of the P145c-kit reactive antibodieswas raised by immunization with the leukemic blasts of M, type AML, three were obtained by immunization with a transfectant expressing PM5c-kit (NM-3T3/hckit),and two antibodies were kindly provided by A. Ullrich, Munich, Germany. By two-color fluorescence analysis it could be shown that about 50% of CD34-positive BMMNC co-express P145c-kit. Vice versa, 50-8546 of P145c-kit-positive cells express also CD34. Double-labeling experiments on 14 AML blasts positive for both CD34 and P145c-kit antigens revealed a highly heterogeneousexpression of these antigens. W14 blast samples showed double-positive subpopulations, 10/14 expressed also CD34+P145c-kif and only 3/14 CD34-PI45c-kif populations. The blasts from one patient contained two P145c-kit-positive populations: a double-positive population with a relatively low expression of PW5c-kit and one with the exclusive expression of P145c-kit at a high level.

Retinoic Acid in Acute Promyelocytic Leukemia: From Cellular to Molecular Biology C. Chomienne, H. de Thd, M. Comic, A. Dejean. L. Degos Lab. Biol. Cell. Hemat. Dpt. Med. Nuc., Dpt. Hematol. Adulte, Hopital Saint Louis, U 163 Inserm, Institut Pasteur, Paris, France Retinoic acid, active metabolite of vitamin A specifically induces fresh human promyelocytic leukemic cells (AML3) to differentiate in vitro to mature functional granulocytes which lose their self-renewal potency and spontaneously die. These results were successfully transposed in vim: AML3 patients treated with all-trans RA 45 mg/sqm/d alone achieved complete remission. Two distinct classes of proteins directly interact with RA: nuclear receptors (RAR) and specific cytoplasmic proteins (CRABP). We have shown that the retinoic acid receptor (RAR) (Y gene located on chromosome 17 is rearranged through the t(l5;17) translocation observed in these cells, gives abnormal transcripts, and results from the fusion of the truncated RAR (Y gene to a newly identified gene myl localized on chromosome 15. This genomic alteration may be implicated in the leukemogenesis of this disease. The mechanism of RA efficacy in AML3 patients remains however an enigma. We propose a model in which the normal proteins (RAR (Y and CRABP) may play a crucial role in the efficacy of RA therapy in these patients.

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The Role of Erythmid 5-hinolevdinate Synthase in the Regulation of Hemoglobin Heme Biosyathesis 7imthy C. Car.Michael J. Bawden, C. Raman0 Bhasker, Sylvia S. Bofik~tnley, Lukar C. Kuhn, Brian K. May University of Adelaide, Adelaide, Australia; University of Oklahoma Health Sciences Center, Oklahoma City, USA; Swiss Instituteof ExperimentalCancer Research, Epalinges, Switzerland Following the induction of erythroid differentiation, the biosynthesis of heme duriag the subsequent proliferation and maturation of erythroid cell is closely co-ordinated with the supply of iron for optimal hemoglobin production. That this co-ordination is mediated through erythroid 5-aminolevulinatesynthase (ALAS) is beimg explored. After isolating cDNA and genomic clones of erythroid ALAS, its gene was characterized to consist of ll exons spanning 22 kb within band pll.2 of the X chromosome. lbo mRNA transcripts were also found, one being devoid of the exon 4 sequence. 30th mRNAs were discovered to contain a stem-loop element in their 5 ' unvanslated region resembling the iron responsive element (IRE) found in fedtin and transfeninreceptor mRNAs. The ALAS IRE was shown to mediate iron-dependentregulation of ALAS translationthrough a mechanism similar to that elucidated for ferritin. Analysis of the binding constantsof the IRE-bindingprotein for the 3 IREs revealed the following order of affinity: femtin IRE > transfenin receptor IREs > erythroid ALAS IRE. These findings indicate that erythroid ALAS is probably the prinicipal modulator of ironlprotoporphyrin homeostasis in the developing red cell.

PCR-Bnsed Method for Measurement of Ejm und Arachidonatew Hydroxylase mRNAs in Rat Nephron Segments J.L. da Silw, K. Omata, M.L. Schwrartvnan, N.G. Abmham Departments of Medicine and Pharmacology, New York Medical College, Valhalla, NY The cellular localization of Epo mRNA in mammalian kidney is controversial, pertidly due to the limited sensitivity and accuracy of in situ hybridization and the limited amounts of isolated puntied specific renal cells. We therefore developed a highly sensitive PCR method to measure the expression of a specific mRNA in microdissected nephron segments of the rat kidney. The nephron of Wistar-Kyoto rats was microdissected into 8 distinct segments, the purity of which was determined by morphology and enzyme markers. Total RNA from the segments was reverse-transcribedand amplified by PCR. The PCR products were analyzed on agamse gels, blot to nylon membraneand hybridized with a specific Epo pmbe. Total RNA fromanemicmouse kidney and normal rat kidney amplified using primers for Epo exhibitedstrong bands correspondingto Epo mRNA, whereas the RNA amplified from the 8 different tubular segments showed no signal. Hybridizationwith a specific Epo probe confmed the above observations; i.e.. a strong signal of Epo mRNA in the mouse and rat kidney with no signal in the microdissectedtubules. For comparison, wc amplifiedtotal RNA from microdissected tubules using primers for AA w hydroxylase. The specific band for AA w hydroxylase was detected in 2 out of the 8 segments, as well as in total rat kidney. Our results indicate that Epo mRNA is not expressed in tubular structures along the nephron of the normal rat kidney.

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Detection of Circulating Lymphoma Cells Carrying the t(14;lS) Translocation by PCR G. Ddlken, E. Kbhlund, Li Nowicki. E. Schalipp. J. Rnke, U! Lunge, R. Mertehann Dept. Hematol. Oncol., Med. University, Hugstetter Str. 55, D-7800 Freiburg, Germany The t(14;18) (q32;q21) translocation involves reciprocal translocation of the J region of the immunoglobulinheavy chain gene and the BCL-2 gene. It occurs in about 90% of follicular lymphoma. About 60% of the breakpointson chromosome 18 are located within the so-called major breakpoint region (mbr) of BCL-2 exon 2, about 30% within the minor cluster region (mcr). We have used the PCR technique to preferentially amplify in separate reactions the two different BCL-2/J hybrid sequences using a consensus primer for all six human J-gene segmentsand two different BCL-2 primers. The specificity of the amplified products has been confirmed by two step PCR with nested primers, hybridizationwith specific oligonucleotidesand finally direct sequencing. The high sensitivity of this technique made it possible to detect minimal circulating lymphoma cells in the peripheral blood of patients with follicular lymphoma being in clinical remission for 1-10 years: 9/26 patients (35%) were positive by PCR, two patients had a clinical relapse after 6 and l2 months. 3/19 patients with high grade malignant lymphoma (16%)had t(l4;18) positive circulating tumor cells in their peripheral blood. A prospective study is underway to define the risk of relapse with regard to positive findings by PCR during clinical remission.

Induction of Erythropoietin Gene Expression by Hypoxia: Analysis by Competitive M y merase Chain Reaction Elia Duh, H. Fmnklin Bunn Hematology Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, M A Erythropoietin (Epo) production by the fetal liver and the adult kidney is stimulatedby reduced oxygen delivery to these tissues. The human hepatoma cell line Hep3B is able to produce biologically active Epo in response to hypoxia with a >50-fold increase in Epo messenger RNA levels. This induction is due both to increased transcription and enhanced mRNA stability. We have developed a system for studying the Epo gene in its native context by transient transfection into Hep3B cells of a plasmid containing a mutated full length genomic copy of Epo. The mutation consists of four basepair changes which ablate an AccI site and create a new HindIII site in the fifth exon. These changes allow the expression of transfected Epo to be distinguished from that of endogenousEpo.Transfected Epo RNA levels are assayed by competitive polymerase chain reaction (PCR) analysis, b l v h g reverse transcription of Hep3B RNA under nonnoxic and hypoxic conditions foliowed by PCR amplification using primers spanning the Epo fifth coding exon. Endogenous and tmdwted Epo expressionis then independently quantified using the AccI and H i n d m restriction enzymes. We are using this system to study the effects of deletions in the 5 ' and 3 ' untranslated regions of Epo. We have shown that a copy of Epo containing 400 base pairs 5 ' to the transcription start site and 800 base pairs 3 ' to the translationstop site is sufficientfor most of the hypoxic induction of Epo. We are making further deletional analyses, including those sequences which show marked homology between mouse and man. Epo mRNA production is completelyablated by deletion of a conserved 180 base pair sequence downstream of the translation stop site. This novel experimental approach should allow identification of the cis-acting regulatory elements of the Epo gene.

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hlymerase Chain Reaction (PCR)-Based Methods of Diagnosing Hemoglobin Disorders Stephen H. Embury San Francisco General Hospital and the University of California, San Francisco, CA,USA Globin-gene mutations that cause quantitative or qualitative disorders of hemoglobin production, known respectively as thalassemias or hemoglobinopathies, result in the generation of abnormal red blood cells. Complex combinations of mutant genes provide a challenge for clinical, population, and prenatal diagnosis which often necessitates diagnosis at the DNA level. The development of a method for enzymatically amplifying DNA in vitro called the polymerase chain reaction (PCR) has provided many advantages for diagnosing hemoglobin mutationsdirectly. The amazing sensitivity derived from exponential amplification of selected DNA sequences and exquisite accuracy provided by WatsonCrick base pairing provides the basis for a whole new spectrum of diagnostic approaches. PCR-based tests to be applied to routine clinical diagnosis should be simple, rapid, reliable, radioisotope-free, and adaptable to automation. The liquid hybridization/oligonucleotiderestriction method originally described proved unacceptable for routine use because of its technical complexity and reliance on radioisotopes. Methods employing direct visualization of restriction fragments, allele-specific dot blotting, allele-specific amplification, denaturing gradient gel electrophoresis, and direct sequence analysis also have technical and theoretical drawbacks. Among the methods that have been applied to diagnosing globin-gene mutations, two have particular potential for routine clinical application. The capacity to screen for several mutations with a single hybridization makes reverse dot blotting a promising method for routine diagnosis and population screefig. The ability to distinguish normal frommutant sequences without the use of radioisotopes or electrophoresis makes allele-specific fluorescence PCR (the color complementation assay) adaptable to automation. Each of these PCR-based methods will be discussed, and their specific advantages, disadvantages, and pitfalls will be reviewed.

Precursor/Product Ratio For mRNAs Encoded by c-myc, Calcyclin, S14 and rRNA Gene in Human Normal and Leukemic Blast Cells S. Fermri, E. Tagliajico. R. Manfredini. A . G r a d e , E. Rossi. U Torelli Experimental Haematology Center, II Medical Clinic, University of Modena, 4UOO Modena, Italy

In order to improve our knowledge on the importance of the several steps involved in the regulation of gene expressionin different functional states and particularly in quiescent, proliferating, differentiating and leukemic cells, we have evaluated the abundance of the precursor RNA molecules and of the processed products of four different genes, namely c-myc, calcyclin, S14 ribosomal protein and the rRNA gene. The study has been performed by the RT-PCR technique for the detection of precursor RNA molecules and by Northern blot analysis for the detection of mature messenger RNA. The synthetic oligodeoxynucleotides we used in the study are specific for the different genes and we selected the intronlexon junction regions for the RT-PCR amplification reaction. A 33 mer aODN is specific for the human internal ribosomal transcribed spacer ITS2 which is able, by Northern blot analysis, to detect the 45 S and 32 S precursor rRNA molecules. The study was performed in human quiescent and PHA stimulated lymphocytes, in myeloid HL-60 cell line before and after induction of terminal differentiation with retinoic acid and in four different leukemic blast cell populations. The whole of the results we have obtained point to the importance of the processing of primary transcript among the mechanisms of regulation of gene expression. In fact the experiments we have performed indicate clearly that the post-transcriptional modulation of the primary transcript plays an important role in basic cellular processes such as proliferation and differentiation. As far as the leukemic cells are Concerned, all our data point to the fact that the ratio between the precursor and mature mRNA is often different as far as the genes studied are concerned. Supported by a grant from A.I.R.C.

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Expression Pattern and Functional Characterisation of the Erythroid Transcription Factor GATA-1 K.D. Fischer, C. Heberlein, C. Stocking. J. Nowock Heinrich-Pette Institut fur Experimentelle Virologie und Immunologie, Hamburg, Germany We have investigated the expression and function of the transcription factor GATA-1 which recognizes a sequence motif that is frequently found in regulatory elements of genes specific for erythroid cells. Analyzing the expression of GATA-1 by quantitative PCR and protein-DNA binding assays in cell lines representing various states of differentiation we found low but significant expression in embryonal stem cells and hematopoietic multipotential progenitors. High expression was observed in cells committed to the erythroid pathway. In cells representing later stages of GM- and M- lineage GATA-1 expression was repressed. Results from in vitro transcription assays and from transfection experiments have shown that GATA-1 has a low activation potential on its own. This suggests that interaction with other transcription factors is required to establish high level expression. GATA motifs are regularly seen in close proximity to sequences known to bind Spl or Spl like proteins, pointing to a functional relevance. As a model system we used the promoter of the human y-globin gene, which contains two recognition sites, each for GATA-1 and Spl. Drosophila SL-2 cells which are devoid of Spl and GATA-I, served as a heterologous expression system. In cotransfection experiments, Spl activated strongly the y-globin promoter. An effect of GATA-1 was only observed in the presence of Spl. Short oligonucleotides containing each one pair of GATA-1 and Spl recognition sites were cloned 5 ‘ to a rudimentary promoter which allowed the analysis of different combinations from a natural setting. These constructs reacted only weakly with GATA-1 and more efficiently with Spl. Coexpression of both transcription factors resulted in a synergistic response. The activation potential differed with the sequence context of the various pairings reflecting different binding affinities and topological effects.

Formation and Biological Actions of “Epoxygenase”-Derived Eicosanoids FA. Fitzpatrick Dept. of PharmacologyC236, Univ. of Colorado Health SciencesCenter, Denver, CO 80262, USA The term “epoxygenase” pathway refers to transformations of arachidonic acid catalyzed by cytochrome P-450 mixed-function oxidases. This terminology designates chemical features of certain enzymatic products and it distinguishes the enzymology involved in their formation from that of other pathways of eicosanoid biosynthesis. There are two main types of “epoxygenase” product: i) cis-epoxyeicosatrienoic acids (EETs), ii) hydroxyeicosatetraenoic acids (HETEs). Both types have attracted attention because of their effects on several cellular processes. Evidence has also accumulated indicating that the EETs occur naturally, and have mechanisms of action which differ significantly from other eicosanoids. This review aims to summarize information on the biosynthesis, metabolism, occurrence and activities of these novel metabolites of arachidonic acid. Emphasis will be placed on: i) comparative features of cyclooxygenase, lipoxygenase and “epoxygenase”-dependent metabolism of arachidonic acid, ii) the actions of EFTS on blood cells, typified by platelets and monocytes, iii) the occurrence of EETs as constituents within phospholipids of cell membranes.

Molecular Biology of Hematopoiesis

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Immunophenotype and Genotype Analysis in CLL Patients from Northern Italy G. Gandini"'4 E. Bianchini', E W b A. ,Latorraca', G.L. Castoldi', L. del Senno" 'Centro di Studi Biochimici delle Wtologie del Genoma; bIstituto di Ematologia e deU'Emostasi, Universith degli Studi di Ferrara, Fermra, Italy The expression of surface immunoglobulins in lymphocytes of a small group of CLL patients from the region of the Po river delta has been analyzedand compared with the rearrangementpatterns of Ig heavy and light chain genes. Patients have been diagnosed as CLL by morphological, histological and immunophenotypicalstudies, and stages according to the Rai system. In the 143 CLL examined, K+ patients were more frequent than 1+ (60:40),with a ratio smaller than that reported in other CLL populations. However, this varied within the different stages because in stage II there was a prevalence of I+ (46:54),and in stage IV there w89 a strong prevalence of K+ (7733).This is suggesting of a different behavior of lymphocytesexpressing different light chains. A mild clinical course in I + patients may be proposed or, alternatively, there could be a low survival of these patients who might have been died earlier and hence their in stage IV.Therefore, Ig gene analysis in 25 of these CLL patients was performed in order to identify possible characteristics in our population. Results confirmed the monoclonalityof leukemic cells, but failed to show any rearrangedpattern typical of each stage. Fourteen patients have been analyzed in t m repeat determinations in a period of one year. Only in the ten who received chemotherapy an increase of the intensity of the Ig germline band was observed, indicating the reduction of lymphocytosisby the therapy. In conclusion, though Ig gene rearrangement could not distinguish the CLL stage, our data confirm the usefulness of Ig genes analysis as a tool in the evaluation of the efficiency of the therapy, independently of clinical stage.

An Autocrine Role for Erythropoietin at the Multipotent Cell Stage of Hernopoiesia Eugene GuldHlasser, Olivier Hennine, Nega Bern, Nancy Pech Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL

Normal bone marrow cells contain the mRNA for eryfhropoietin (Epo) detectable by the polymerase chain reaction. Since this finding is not compatible with the well established endocrine role of Epo (formation in kidney, transportin the circulationand target in hemopoietictissue) we WBmined the effect of anti-sense oligonucleotidesbased on the sequences of 5 ' regions of exons I and II of the Epo gene; with the sense oligonucleotidesused as controls. The oligonuclaotideswere incubated with bone manow cells and the culturrs scond for erythroid bursts, mixederythroid: non-erythroid@:non-E) colonies and mixed non-erythroid colonies (non-E). The sense oligonucleotides had no effects; the anti-sense oligonucleotidescaused a decrease in mixed E:non-E, an increase in non-E and no change in bursts. The same results were found with marrow depleted of adherent cells, of late cycling cells and of lymphocytes. In addition anti-sense oligonucleotide based on the Epo receptor had the same effects. We conclude that, in early hemopoiesis, Epo and its receptor act in an internal autocrine mechanism.

Gene Regulation in Hematupoiesis

314

Interference of Glucocorticoids with Monocytic Differentiation

M. Goppelt-Siruebe, R. Has, 1: Spencker, 1: Hoff Institute of Molecular Pharmacology, Medical School Hannover, Hannover, Germany The human monocytic cell line U937 was used as a model system to investigate the effects of glucocorticoids on monocytic differentiation. Upon incubation with the phorbolester PMA for 48 to 72 h, the immature U937 cells ceased to proliferate and became morphologically and functionally macrophage-like cells. Preincubationof the cells with glucocorticoidshad no effect on the PMA-induced translocation and activation of protein kinase C. Glucocorticoidsdid not interferewith the growth arrest of the U937 cells. but markedly interfered with parameters associated with differentiation such as cellular adhesion and expression of adhesion molecules, NBT reduction, release of lysozyme and induction of arachidonic acid metabolism. Upon differentiation with PMA the expression of the protooncogenes c-myc and c-myb was downregulated within the first 24 h. Glucocorticoids, which by themselves slightly reduce the c-myc and c-myb levels, only marginally attenuatedthe downregulationof these oncogenes by PMA. These data might indicate 1. that c-myc and c-myb are involved in the regulation of cell proliferation rather than cell differentiationin U937 cells, or 2. that the signal transduction cascades of PMA and glucocorticoids interfere at a later stage.

NF-xB: An Intracellular Signal Transduction Apparatus Composed of a Family of RelRelated Enhancer-Binding Proteins Warner C. Greene Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA The xB enhancer represents a potent cis-acting regulatory sequence present in multiple cellular and viral genes, including those encoding the cytokine IL-2, the IL-2 receptor alpha subunit, and HN-1. The induced expression of these transcription units during human T cell activation reflects, in part, the combined action of a family of inducible enhancer binding proteins (designated p50, p55, p75, and p85) that exhibit intrinsic sequence-specific DNA binding activitiescharacteristicof the pleiotropic 50 kD nuclear factor NF-xB. Notably, these cellular proteins appear sequested in the cytoplasm of unstimulated human T cells as two distinct pairs (p55/p75 and p50/p85) and are converted into active nuclear forms after phorbol ester treatment by the dissociation of one or more IxB-like inhibitors. While p55 and p75 are expressed in the nucleus within minutes following mitogenic stimulation, the nuclear expression of p50 and p85 is delayed for several hours, suggesting that the activation and nuclear translocation of these early and late factorsoccurs with distinctly biphasic kinetics. Biochemical and molecular cloning studies demonstratethat these four inducible proteins are encoded by three distinct genes that share significant N-terminal structural homology with the v-re1oncogene product Specifically, the p50 and p55 proteins appear and the Drosophila pattern formation gene to represent C-terminally truncated forms of human NF-xBIKBFl, while p85 is encoded by the human c-rei (hc-re/) proto-oncogene. In contrast, the human p75 polypeptide is closely related to, and may represent a post-translationally modified form of the 65 kD regulatory subunit of NF-xB that has been shown to mediate the functional association of NF-xB with its cytoplasmic inhibitor. Both p75 (early) and p85 (late) can independentlyassociate with the 50 kD DNA binding subunit of NF-xB to form stable heterodimers that can engage the xB enhancer. Together these data provide evidence for at least two distinct NF-xB transcription factor complexes, formed by combinationalassociations between these four Rel-relatedproteins, whose temporally regulated nuclear expressionmay underlie differing functional effects on the multiple T cell activation genes now recognized to be under NF-xB control.

w.

Molecular Biology of Hematopoiesis

315

“In Situ” Quantitation of Nuclear c-myc Expression in Human Hematopoiesis R. Greil’? B. Fasching’, A . Weger? I? Loidl’ Departments of ‘Internal Medicine, *Pathology, and 3Microbiology at the University of IMSbruck, Austria The biological role of nuclear oncogenes is dosage-dependent. Thus, heterogeneity of c-myc protein expression may reflect differences in biological behavior of immunologic+lly and morphologically uniform normal and neoplastic hematopoietic cells. Since in situ analysis may thus improve our understanding of tumor biology, immunocytochemical staining of c-myc was performed and semiquantitatively evaluated. Four categories were used and correlated with percentage of positive nuclear area (A), staining intensity (I) and the parameter A X I as assessed by computer-assisted image analysis (p = .MOl). Applying a standardized staining protocol, significantly different c-myc amounts were found in well-defined control cell types (HL-60 cells, PHA-blasts, myeloma cells, resting lymphocytes; p = ,0095).This result was highly reproducible (day/day variance 0.5 U)stimulated proliferation for 21 days, and a sizeable fraction of the non-dividing mature normoblasts were arrested in the Gz phase. Erythroid maturation required only a short exposure to Epo; following 3-5 days, r e m d of Epo resulted in accelerated maturation. Thus, Epo functions mainly as a growth Eactor for the erythroid precursors, while the kinetics of cell maturation, after being triggered by Epo, can proceed autonomously even faster.

Two Distinct Epitope Regions of Human Erythropoietin DepLwd by Neuealizing Monoclonal Antibodies

M.R. Fibi, E! Hino-Obertreis. H . 2 Harthus Seven hybridoma cell lines were established from a mouse immunized with recombinant human erythropoietin (rhuEpo). Antibodies secreted by these cell lines are neutralizing the biological function of human erythropoietin in an erythroid precursor cell proliferation assay. Binding inhibition studies revealed two sets of antibodies composed of four and three neutralizing antibodies, respectively. Complete cross-inhibition was observed, if one antibody of each set was labelled with peroxidase and competed for the binding to rhuEpo with a n t i w e s of the same set. In contrast, no cross-inhibitionwas observed when the competitionwas carried out with antibodies of the other set. Thus, rhuEp0 neutralizingmouse monoclonal antibodies bind to two distinct epitope regions of the hormone.

Molecular Biology of Hematopoiesis

355

Cytotoxic and GTP-Depleting Ef€ects of Tiamfurin on HL-60 Cells: Synergism with G r n n u l e M a c r o p h a g e Colony-Sthulathg Factor (GM-CSF') M. FnQer, I: Szekeres. K. Gharehbaghi. Z Felzmann, K. Pillwein. H. Goldenberg, l? Chiba Department of Medical Chemistry, University of Vienna, Austria Tiazofurin, an effective agent in the treatment of refractory chronic granulocytic leukemia in blast crisis (C.Rber, Adv. Em. Regul. 29, l989), selectively depletes GTP-pools by inhibiting de n o w synthesis via the enzyme IMPdehydrogenase. This enzyme activity is increased during the S-phase of the cell cycle. The GTP-Icwering effect of tiamfurin is enhanced in elutriated S-phase enriched cell fractions, parallel to an increased enzyme activity found in these fractions. The hematopoietic growth factor GM-CSF stimulatesproliferationof HLaO cells and increases IMP-dehydrogenase activity. Cells treated with GM-CSF respond to tiazofurin with a synergistically enhanced depletion of GTP-levels compared to controls. The cytotoxicity of tiamfurin, assayed as a decrease in tetrazolium reductase activity, is also increased in GM-CSF-treated cells. We concludethat tiamfurin provides a possibility for combinationchemotherapywith hematopoietic growth factors.

In Vitro and In Vivo Regulation of Hematopoiesis by Combination Therapy with Interleukin-3 and GM-CSF A. Gumer

Johann Wolfgang Goethe University, Frankfurt/M., Germany Hematopoiesis is regulated by an interacting network of cytokines. Some of the recombinant human cytokines, including IL-3 and GM-CSF have undergone phase IA clinical trials. IL-3has been given to patients with prolonged bone marrow Mureafkr chemdradiotherapyor bone marrow transplantation, myelodysplastic syndromes (MDS), and severe aplastic anemia (AA). In most cases of secondary bone marrow failure, patients responded to IL-3 with increases in peripheral platelet and leukocyte counts while the erythroid response was less pronounced. Whereas patients with MDS exhibited partial responses of leukocyte and platelet counts, only minimaland transient improvements were observed in patients with AA. Sequential combination of IL-3 and GM-CSF in a phase-I trial resulted in an increased leukocyte response while the platelet response to IL-3could be preserved. Assessmentof bone marrow and peripheral blood pmgenitor cells of patients treated with either IL-3 or IL31GM-CSF demonstratedan increased cell cycle rate of progenitorcells. An expansion of circulating progenitor cells, however, was only observed after ILJ/GM-CSF combination therapy and not after treatment with IL-3 alone. Further clinical trials will have to demonstrate the efficacy of treatment with combinations of cytokines.

Regulation of Hematopoiesis: Growth FactorslReceptors

356

Randomized Phase I1 Study with GM-CSF and Low-DoseAraC in Patients with "High Risk" Myelodysplastic Syndromes 0 H.H. Gerhartz.R. Ma-, A. Delmer, H.Zwierzina, I: de Wine,A. Jacobs, G. Vim', D. Eire, R Sonneveld, 8. Lobar, A.K H&mnd, I! Fenaur. M.H a p t , A. *ss, L. Debucher, B COifFer, U! Sizoo. R. Wilkmze, M. Ribeiro, S. S u c k G. Solbu. A. Stern, R. Zittoun EOKK Leukemia Group (Intr. by S. lhra), Med. Dept. ID, Klinikum Grofihadern, 8 Munich 70, FRG Symptomaticpatients with MDS and 10-3096 blast in the bone marrow were treated with LD-AraC (2 x 10mg/m2,S.C. day 1-14) and GM-CSF (fully glycosylated, Sandoz/Schering-Plough,2 x 150pg proteinld s.c.) given either subsequently (day 15 - 21) or simultaneously (day 8 - 14 and 1 week rest). Evaluation was done after 3 courses (9 weeks); responding patients could be continued for 2 further cycles. Between start (10/88) and closing ofthe study (05/90) 145 patients were entered, however 29 cases are inaaluable because they are too early (n = 24) or treatment was stopped prematurely (n = 5). Moreover, 34 patients were excluded for not meeting the inclusion criteria. Thus 82 cases were evaluable: 45 W E B and 37 RAEBt. mean age 64 years (range 17 - 80). Complete remission was achieved in 14 cases (17%), 11 had a good response (U%)and 12 a partial response (15%), stable disease was found in 21 cases (26%).There were 12 cases of toxic death (l5%),progression was noted in 8 patients (10%)and death due to disease in 3 (4%).No differencesexisted between the two treatment arms with respect to response. Major adverse events during treatment were hemorrhage (25%). infections (23%)and fever with GM-CSF (21%).Presently 47 patients are still alive (mean survival 10.5 months, 14.5 months in responders). GM-CSF did not induce leukemia nor contributed to hemorrhage induced by AraC, but allowed to achieve an overall response rate of 46% which is high and relatively durable as compared to other treatments in this disease.

Growth Factor Receptors Involvement in Experimental Leukemias S. Gisselbrecht, I. Vigon, L. &nit, S. Fichelson, I! Tambourin, E W d i n g , M. Souyri INSERM U152, ICGM, H6pital Cochin, 75014 Paris, France

Our group has been working on experimental leukemias induced by the Friend complex. We have previously reported the frequent activation of the M-CSF receptor in mouse myeloblastic leukemias by proviral insertion of F-MuLV 5 ' to the M-CSF receptor gene. We also recently observed LTR insertions 5 ' to the Epo receptor gene in mouse. SFFVp induced erythroleukemias.In both systems, the receptors are averexpressed and able to bind their physiological ligands. Moreover, the myeloproliferativeleukemia v i m (MPLV)isolated from a mouse inoculated with F-MuLV has transduced a truncated but not mutated form of a new member of the cytokine receptor superfamily (v-mpl). Using various viral constructs, we characterized the MPLV domains necessary for in vim pathogenesis. In addition, recent data on $e structure and expressionof the c-mpl protooncogene will be presented.

Molecular Biology of Hernatopoiesis

357

Characterization of an Inhibitor of Stem Cell Proliferation C.J. Gmham, E. K. Parkinson, I.B. Pmgnell Beatson Institute for Cancer Research, Garscube Estate,SwitchbackRoad, Bearsden, Glasgow G61 184 UK The hemopoietic stem cell (as detected by the CFU-s assay) appears to be under both positive and negative proliferative control. We have recently purified an inhibitor of hemopoietic stem cell proliferationwhich is identical to a previously described inflammatory, cytokine Macrophage Inflammatory Protein 1 alpha. This protein (stem cell inhibitor, SCI)consists of a peptide with a molecular weight of 8 kDa which forms large non-covalent aggregatesof varying size (c. 100 kDa in physiological buffers). The peptide is a member of a cytokine superfamily defined on the basis of a limited sequence homology and the presence of four positionally conserved cysteine residues. The inhibitory effects of this protein appear to be specific for the stem cell compartment within the hemopoietic system, having no apparent inhibitory effects on the more mature progenitor compartment. We have cloned both the murine and human SCI genes, neither of which are species specific in their actions. In the CFU-a in vitm stem cell assay, SCI is half maximally active at 100 - 200 pM. Initial results indicate that the inhibitoryrole of this factor may not be confinedtothe hemapoietic system and results demonstratinginhibition of clonogenicepidermal cells (human) will be presented.

Class Il Histocompatibility Antigens In the Regulation of Early Hematopoiesis H.7:Greinir, S. & r t C h e Z , R. Stmb Fred Hutchinson Cancer Research Center, Seattle, WA, USA

In a previous study using a canine model, we reportedthat certain anti-class Il monoclonal antibodies (MAB) prevented long-term engraftment of autologous manrnv cellsif M A B s were administend i.v. during the first 4 days after 9.2 Gy total body inadiation. Injectionof0.6 mg/kg/d MAB H81.9821, reactive with an cpitope formed by the a and B subunits of HLA-DR, resulted in an initial engraftment which persisted for 15 days, but lategraft failurewas consistentlyobserved. Another MAB, BlF6, reactive with only the fl subunit of HLA-DR + DP, had no adverse effect on engrahent, even though both MABs detect antigens on repopulating cells as determined from complement lysis experiments. Preliminary experimentshave excluded severalmechanisms, includingremoval of MABcoated cells by the RES and canine complement-mediatedor antibodydependent cytotoxic effects on repopulating cells. In 2 dogs, a second infusion of autologous marrow cells depleted of “accessory cells” by L-leucyl L-leucine methylesterresulted in long-term repplation, indicatingthat the Suppomve function of the marrow microenvironmentwas not permanently impaired by MAB injections. In agar cultures, MAB H81.98.21 inhibited stem cell factor dependent colony formation of early canine hematopietic progenitor cells. MAB H81.98.21 specifically inhibited the growth of b u m high proliferativeptential colony-formingcells (HPP-CFC), but had no effect on more mature progenitor cells. In contrast, MAB BIF6 did not impair HPP-CFC growth. These results support the notion that certain class Il epitopes are involved in proliferative signal pathways of long-term repopulating marrow cells.

Regulation of Hematopoiesis:

Growth Factors/Receptors

358

Differential Sensitivity of Human Normal and Leukemiccells to the lktriipeptideMDm. Relevance to Marrow Ptotection During Chemotherapy M. Guigon. D.Bonnet, EM. Lemoine, R. Cisaim, A. Najman Department of Hematology, Facult&de M&cine St. Antoine, Paris, France The tetrapeptide acetyl-N-ser-asplys-pro (AcSDKF'), isolated from fetal calf marrow and now chemically synthetized, inhibits the entry into DNA synthesis of murine CFU-s and protects mice against the toxicity of high doses of chemotherapy. In view of its clinical use as a marmw protector, we have investigated its effects on human normal and leukemic cells. When incubated for 24 hours with normal marrow or card blood mononuclearcells, the synthetictebapeptide (Ipen-Biotech, France) inhibits the growth of granulo-macrophagicand erythroid progenitors by about 40% at nanomolar concentrationand reduces significantlytheir percentage in DNA synthesis. The tetrapeptide was then assayed on HL-60 cells and on fresh leukemic cells from patients with acute myeloid leukemia (5 AML) and chronicmyeloid leukemia (10 CML). Whatever the doses used, AcSDKP does not modify the proliferation of HL-60 cells and AML cells even enhanced by interleukin-3 or GM-CSF. Furthermore, no change in the number and the percentage in S phase of both clonogenic HL-60 cells and CML progenitors is observed. Our data clearly demonstratethat AcSDKP is ineffectiveon leukemic cells and therefore by acting selectivelyon normal progenitorsrepresents a potent therapeuticalagent for the protection of normal bone marrow progenitors during chemotherapy.

High Dose Erythropoietin (Epo) as Treatmentof MyeiodysplasticSyndromes @IDS) and Wroxysmal Nocturnal Hernoglobmuria 0 C. Stebler Gysi. A. lichelli, A. Gmtwohl, T Hoffmonn, M. Uhr, R Speck Division of Hematology, University Hospital, Basel, Switzerland Treatment of anemia in patients (pts) with MDS and PNH is unsatisfactory. Their endogenous

Epo secretion is normal or increased and erythropoiesisineffective. Epo is now an established treatment for pts with anemia due to chronic renal failure. The role in pts with MDS is stiU controversial and until now only a small number of pts have received Epo. We treated 2 pts with PNH (secondary to severe aplastic anemia)and 5 MDS pts with recombinant human Epo at a dose of 500 IIJlkg X

3lweek S.C. for at least 18 weeks. The longest follow up is 48 months in pt 1. No side effects were observed and the results were as follows: retix 10911 ~~~10911 beforelafter treatment 1 PNH 8.9112.5 40160 3.914.1 2 PNH 8.51 14.7 1101110 3.0/3.4 3 MDS 10.419.2 12110 2.712.9 4 MDS 7.217.4 11136 9.819.9 5 MDS 8.117.1 60150 2.011.9 6 MDS 8.318.5 36132 4.214.4 7 MDS 6.0162 23140 3.513.2 Tx = blood transfusions in unitslmonth Ptsldisease

Hb g/l

~c~io9n

mi90 31125 93/90 3601370 80170 4391569 1201loo

Tx no 21110 414 414 212 no 413

We conclude, that Epo can be useful in the treatment of anemia in PNH pts and may permit regular phlebotomies to reduce the iron overload due to previous blood transfusions. The role in MDS pts needs yet to be. defined.

Molecular Biology of Hematopoiesis

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Role of Interleukin 6 in Plasma Cell Neoplasis Toshio Himno Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, 4-3-57,Nakanoshima, Kita-ku, Osaka 530,Japan Interleukin 6 (IL-6) is a multifunctional cytokine which regulates the immune responses, the acute phase reactions and hematopoiesis. The relationship between IL-6and polyclonal plasma cell abnormalitieshas been demonstrated, since the abnormal production of IL-6 was first suggested to be related to polyclonal B cell activation with autoantibody production in patientswith cardiac mymma. The role of IL-6 in the generation of plasmacytoma has also been suggested. In supportof these clinical and experimental observations, IL-6 transgenic C57BU6 mice showed a massive polyclonal plasmacytosis with production of autoantibodies. However, these plasma cells were not transplantable to syngeneic animals. It has been known that the susceptibility to pristane-induced plasmacytomagenesis is genetically determined; pristane can induce plasmacytomain BALB1c but not in C57BL16 mice. To elucidatethe genetic influence on plasmacytomagenesis, IL-6 hamgenic C57BL16mice were backcrossed to BALBlc mice. It was found that transplantable monoclonal plasmacytoma with t(I2;fi) chromosomal translocation was generated under the influence of BALBlc genetic background. The data indicated that IL-6 play a key role in the multistep oncogenesis of plasma cell neoplasia. The IL-6 receptor (ILdR) was molecularly cloned and found to consist of two domains, one is an immunoglobulin like domain and the other is a domain belonging to the cytokinereceptor family. The intracytoplasmicdomain of the ILdR is not necessary for the signal transduction of IL-6, suggesting the presence of a molecule involved in the signal transduction. Upon the binding of IL-6 to IL-6R. a second molecule, gpU0 associates with IL-6R. Molecular cloning of gpU0 indicated that gp130 molecule is involved in the formation of high affinity binding site and signal transduction. Furthermore it was demonstrated that IL-6activated tyrosine kinase activity andjrcnB expression.

Development and Function d T Cells In Mice Rendered Interleukin 2 M c i e n t by Gene lsrgeting lwn H o d , Hubert Schorle, l % o mHolischke, Tlromas Hiiig, Anncliese Schimpl Institute of Virology and Immunobiology, University of Wunburg, Versbacher Strasse 7, 8700 Wunburg. Federal Republic of Germany Interleukin 2 (IL-2) deficient mouse mutants were generated by mgetedrecombination in pluripe tent mouse embryonic stem cells. Mice homoeygous for the IL-2 gene mutation (IL-2-17 were normal with regard to thymocyte and peripheral T cell subset composition as defined by the cell surface markers CD4, CDB, and aIfl-WR, indicating that IL-2 is not essential for T cell development. The effectivenessof gene disruption was confirmed by analysis of IL-2 activity in supernatantsof C o d activated spleen and lymph node cells. A dysregulationof the immune system in IL-2-I- animals was manifested by reduced polyclonal in vitm T cell responses and by changes in isotype levels of serum immunoglobulins. In contrast to normal levels of IgM, drastic differenceswere observed in the serum concentration of IgGI, which was 20- to IMfold elevated in mutant animals as compared to control littermates. The IL-2'1- animals provide an excellent tool to evaluate various proposed pleiotropic effects of IL-2 and its role in innate and adaptive immunity to pathogens.

Regulation of Hematopoiesis: Growth FactordReceptors

360

TGF-j3l Inhibits the Proliferative Yet Not the IGdInducing Activity of Murine IL9 Exerted on Mouse Bone Marrow-Derived Mast Cells L. Hiiltner, J. Moeller GSF-Institut fir Experimentelle Himatologie. Munich, Germany Murine interleukin 9 (mIL-9) has been previously identified as a new T cell growth factor (WOIXGFIII) for certain long-term cultured mouse T cell clones and as a navel mast cell growthenhancing activity (MEA) for mouse bone marrow-derived mast cell (BMMC) lines. Recently we demonstrated that mIL-9 can induce IL-6production in a murine factordependent, IL-3IIL4IL-9responsive BMMC line (L138.U) and in a malignant subline(Ll38Cauto). Here we show that human transforminggrowth factor j31 (TGF-j31) which is known to inhibit the IL-3-induced growth of mouse BMMC in vitro, efficiently can block the proliferative actions of IL-3, IL-4, or IL-9 in cultures of L138.U mast cells without affecting the IL-6-inducing capacity of IL-9 at doses of 2.5 to 10.0 ng TGF-Pllml. 'IGF-Bl was also able to significantly inhibit the autonomous growth of Ll38Cauto cells cultured in the absence of exogenous mast cell growth factors. However. TGF-fl at 10 nglml did not significantlyinfluence the IL-g-dependent proliferation of mouse T cell line ISl.C3. From our results we conclude that TGF-j31 can provide anti-proliferative signals which are potentidy involved in the regulation of normal and malignant BMMC gmwth. Moreover, the action of TGF-fl on Ll38.U mast cells shows that the proliferative and the IL-6-inducing pathways of ILL-9in this cell l i e are differently regulated.

A Novel Derivative of Human Granulocyte Colony-Stlmulating Factor with N-Linked Sugar Chains S. Itoh, K. Sasaki, E Kbmatsu, M. Okabe Kyowa Hakko Kogyo Co., Ltd., Tokyo Research Laboratories, 3-6-6 Asahi-machi, Mahcidashi, Tokyo lpQ, Japan

Many potential functionshave been suggestedfor the carbohydrateside chains of glycoproteins. Therefore, we have investigated the effect of the addition of the new oligosaccharide to the protein. As a model protein, we have chosen human granulocyte colony-stimulatingfactor (hG-CSF) which is a glycoprotein with one 0-linked and no N-linked sugar chains. KW2228 is a hG-CSF derivative in which N-terminal amino acids were replaced at 5 positions (l,3.4,5 and 17) and its N-terminus were found to be susceptibleto pmteolysis. So, we introduced the new site for N-glycosylation(AsnX-ThrlSer)to KW2228 by replacing Ala-6 with Asn, to construct a new mutein, N6. cDNA encoding N6 WBS expressed in Chaninese hamster avary cells and it was revealed that the new site (Asn-6)was successfully glycosylated. Although specific activity of N6 was almost identical with KW2228, it showed the increased thermal stability and protease resistance, comparing with KW2228. These results suggest that the addition of the new oligosaccharide might be useful to improve the protein natures.

Molecular Biology of Hematopoiesis

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Studies on the Long Term Hematopoietic Repopulating Ability of Murine Bone Marrow Sca-l+,Lin-, Rh do Cells and Their Stimulation In Mtro By Hematopoietic Growth Factors G.R.Johnson, C.L. Li The Walter and E l k Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, 3050, Melbourne, Australia Although pluripotential hematopoietic stem cells are known to exist, the properties and controlling mechanisms for their proliferation and differentiation remain obscure. We have used fluorescence activated cell sorting to isolate murine bone Sca-1'. Lin-, Rh 123" cells and the Ly-5 marker to distinguish donor from host. In lethally irradiated transplanted hosts, the progeny of 100 or less sorted cells were able to reconstitutegreater than 95% of the cellularity of both myeloid and lymphoid tissues, for at least 52 weeks. Survival and reconstitution for this period of time using such low numbers of sorted bone marrow cells, without addition of filler cells, has not previously been reported. When cultured in vitro, colony formation by Sca-1', Lin-, Rh 123" cells was not simulated by G-, GM- and M-CSF, IL-1, IL-6 or Lif and less than 5%of the cells were stimulatedby IL. However, synergy for colony stimulation was observed between various combinationsof those factors although maximal numbers of colonies (20 - 30%cloning efficiency)were observed when IL-I, IL-3, G-, GMand M-CSF were added simultaneouslyto cultures. Although stimulatingproliferation and differentiation of the Sca-l', Lin-, Rh 123" cells, none of these growth factor combinationswas able to sup port the maintenance in vitro (7days) of the long term hematopoieticrepopulatingability of these cells. The implications of these findings on the so called "self-renewal" ability and differentiative capacity of pluripotential hematopoieticcells will be discussed as will recent observationsof the effects of the kit ligand (SI product) on the Sca-1', Lin-, Rh 123" cells.

Expression of the M-CSF Receptor in Hematopoietic Stem Cell Lines Is Upregulated After Induction of Macrophage-Granulocyte DifIerentiation by GM-CSF LI. Just! C.Heberlein! M. Baccarinif 1.Friell, W Ostemg' lHeinrich-Pette-Institut fiir Experimentelle Viiologie und Immunologie, Hamburg, Germany; 2Fraunhofer Institut, Hannover, Germany Multipotent murine stem cell lines (FDC-Pmix) dependent on IL-3 and horse serum for self renewal and proliferation can be induced to differentiate unidirectionally into granulocytes (G) and macrophages (M) after infection with retroviralvectors expressing GM-CSF (GMV). Mutants of GMVinfected FDC-Pmix cells have been selected that do not diffenxtiateterminally when kept in the presence of IL-3, but differentiate synchronously into G and M cells when IL-3 is removed. These mutants were used to analyze the expressionof the lineage-specificreceptor for M-CSF on differentiation induction by GM-CSF. When grown in IL-3 GMV-infected FDC-Pmix cells express low levels of both c-fmr mRNA and protein as determined by PCR analysis and imrnunoprecipitation.Differentiation induced by withdrawal of IL-3 caused a rapid increase of c-fms mRNA reaching levels up to 20-fold higher within 4 days. c-fms glycoprotein expression levels were elevated from day 2 until terminal differentiation. In contrast to previous reports by Gliniuk and Rohrschneider (Cell 63, 1990) who observed an almost complete suppression of c-fms expression after GM-CSF induction of differentiation of late macrophage precursor cells,the activated GM-CSF receptor functionally upregulates the M-CSFrecep tor in our system. These data are in accord with our proposed hybrid model of differentiation (Just et al.; Cell 64, 1991).

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Radioprotective Effect of Human Tumor Necrosis FactorlCachectin for Hemopoietic Pmgenitor Cells (HPC) From Human Long-Term Bone Marrow Cultures (LTBMCs) L.K Karkanitsa, S.I. Krivenko Institute of Hematology and Blood Transfusion, Minsk, Byelorussia, USSR A radioprotective effect of human recombinant TNF (bNF) for LTBMCs was studied. One part of LTBMCs on day 24 were treated with rTNF (50,200, and 400U/ml)and immediately X-ifiadiated (4590, and 900 cGy). The second part of LTBMCs were treated with ANF before irradiation. One hour exposure of LTBMCs to b N F was used. Number of survived colony-forming cells (CFC) was measured in semisolid agar cultures. Recombinant human IL-3 was used to stimulate the CFC proliferation. Our data suggest that b N F pretreatmentprovided suppressionof colony formationby CFC from unirradiated LTBMCs. X-irradiation provided dose-dependent suppression of colony formation. At 900 cGy there were no colonies in all experiments. We found significant radioprotective action of all doses of b N F for 45 and 90 cGy-irradiated LTBMCs. The effect was dose-dependent. Pretreatment of LTBMCs with b N F was better then analogical post-irradiationprocedure. though, the data did not quite differ. No radioprotectionof bNF was observed at 900 cGy. Our data suggest that ANF can protect human CFC from LTBMCs from ionizing radiation. Acknowledgment. rhIL-3 was kindly donated by Dr. G. Ufigemaker from TNO Radiobiological Institute (Rijswijk, the Netherlands).

Regulation of GCSF Expression in Human Endothelial Cells B. Kerner, M. Szamel, K. W l t e Medical School Hannover, Germany Granulocyte-colonystimulating factor (G-CSF) is one of the hematopietic growth tictors pivotal for proliferation and function of neutrophil cells. It is produced in endothelial cells aftex stimulation with various stimuli like IL-1, TNF, LPS, or TPA. Nevertheless, the regulationof expmsion is poorly understood. All different stimuli seem to act independent from each other. First, the kinetics of G-CSF expression varies slightly with each stimulus used. Second, when used together all stimuli were additive in their effect on G-CSF production. Third, endothelial cells can be made selectively refractory to IL-1, TNF, or TPA without cross-refractoriness of these stimuli. Independent ways of regulation could be demonstratedby different effectsof anti-inflammatorypharrnaconson the induction by various stimuli. Dexamethasone could inhibit G-CSF expression after TNF stimulation, but had no effect on IL-1 or LPS induced G-CSF. Indomethacin could markedly enhance GCSF release 24 hrs after IL-1 stimulation, an effect that could not be demonstrated after TNF Indomethacin.

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Receptor Mediated Uptake of Ferritin by Human Erythroid R e e u r s o ~in Culture A.M. hn$t", E.C. hkymn-Holrz",E. Fibachb

'Department of Nutrition, The Hebrew University-HadassahMedical School; bDepartmentof Hematology, Hadassah University Hospital, Jerusalem, Israel

In order to investigate into the possible role of ferritin and especially acidic isofemtin in erythroid development we studied the binding of lzsII-ferritinto normal, developing human erythroid cells in culture. Early erythroid progenitors, the burst-formingunits (BFU-e) were obtained from peripheral blood and cultured in liquid medium supplementedwith a combinationof htmopoietic growth hctors not including erythropoietin (Epo). This primary culture was followed by a secondary culture containing Epo. The cells were able to grow and to develop, in culture, to the stage of orthochromatic normoblasts. Incubation of the cells, after day eight following the addition of Epo, with **? acidic placental isoferritin (containing approximately47% H-subunits) showed considerable specific lZ'I-femtinbinding by the cells. Uptake of ferritin was substantiallyhigher at 3PC than at 4°C. All of the 12JIacidic ferritin specific bound at 4°C was dislodged by the addition of a 500% excess of non-labeled acidic placental isoferritin. Acidic isoferritin specific bound at 3PC could only be detached partly by the addition of a surplus of non-radiolabeled acidic isoferritin. The results point to specific binding, and subsequent internalization of ferritin by erythroid cells.

IblycythemiaWra Blood Burst-Forming Unitp-ErYthroidAre Hypersensitiveto Interleukin 3 S.B. Kmrrc. C.H. Dai. R.Z Means, Jr., S.Z Horn,H.S.Gilbert Hematology Division, Department of Veterans Affairs Medical Center and Vanderbilt University School of Medicine, Nashville, TN and Albert Einstein College of Medicine, Bronx, NY Pblycythemia Vera (PV) is a clonal hematopoietic stem cell disease with a trilineagehyperplasia, and interleukin 3 (IL-3) stimulates trilineage hematopoiesis. We have studied the response of highly purified PV blood burst-forming units-erythroid (BFU-e) to recombinant human IL-3 ( r - 3 ) and recombinant human Epo (rEpo). Dose-response experiments showed a 117-fold increase in PV BFU-e sensitivity to rIL-3, and a 6.5-fold increase in sensitivity to rEpo, compared to normal BFU-e. Blood BFU-e from patients with secondary polycythemia responded to rIL-3 and rEpo like normal BFU-e endogenous erythroid colony (EEC) formation, which is independent of the addition of rEpo, was reduced by 50% after erythroid colony-formingcells were generated from PV BFU-e in vitro without rIL-3 for 3 days, while rEpo-stimulated erythroid colonies were unaffected. Thus the period of increased sensitivity to rIL-3 appears to be largely responsible for EEC formation. Specific binding of IzJI-rIL-3 to PV BFU-e revealed no difference in the percentage of positive cells, or the amount of specific binding per cell. compared to normal BFU-e as measured by autoradiography. These studies demonstrate a striking hypersensitivityof PV blood BFU-c to rIL-3, which may be the major factor in the pathogenesisof increased erythropoiesiswithout increased Epo concentrations. However, increased binding of lZ9-rIL-3to PV BFU-e is not present and the mechanism for the increased hypersensitivity to rIL-3 still remains to be determined.

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Hemopoietic Inhibitors in Mouse Serum A.B. Kriegfer, LR. Bmdley, XM. Yerschoor, D. Bemardo Cell Biology Unit, Peter MacCallum Cancer Institute, Melbourne, Australia Serum is known to contain substancesthat inhibit hemopoieticcell growth in vitro. In vivo treatments that increase the proliferative activity of murine bone marrow cells, such as IL-1, G-CSF, lipopolysaccharideand 5-fluomuracil, have also been found to result in a concomitant reduction in serum levels. As also observed by other workers, ultracentrifugation of mouse serum indicates that the inhibitory activity is associatedwith the serum lipoproteins. Dextran sulphateor Heparin precipitations and DEAE-cellulose chromatographyof whole serum similarly indicate that the inhibitory activity is associated with the lipoproteins. The levels of serum inhibitory activity have been found to vary from batch to batch. These variations in inhibitor levels do not correlate with the gender of the mice, the time of serum collection or the lipoprotein content. A fractionation scheme involving chromatographythrough CM-cellulose followed by phenyl sepharose using a stepwise isopropanol gradient has recently resulted in the separation of the inhibitory activity from the lipoproteins. The fact that this inhibitorh can be isolated from lipoproteins and the observation that the levels in serum can be reduced by treatments, which have stirnulatoryeffects on BM cells, suggeststhat this inhibitorls is a distinct molecular type with a physiological role.

hssible Role of IGl and I M in Disorder of Hematopoiesis 0. Kupriyanowa, !l Mordvimv, I. Ivonova, 0.Krugleeva, Ci Gzlov Institute of Clinical Immunology, Novosibirsk, USSR Hematopoiesis is regulated by a network of cytokines with pleiotropic effects. We investigated expression of IL-1and IL-6 genes in allergy to pollen. The pollinosis is associated with such disorders as eosinophiiiaand lymphaytosis. We registered the increasedlevel of IL-lS mRNA hneutropMs from the peripheral blood of some patients suffering from the pollinosis for a long time. There was a decreased level of IL-1mRNA in neutrophils activated with LPS or with culturing on plastics of those. patients as well as in patients who had a normal level of IL-1 mRNA in freshly isolated neutrophils. IL-1 is known to be able to induce production of IL-6 in a range of cell types. Hybridization analysis showed that increased levels of IL-1 mRNA in freshly isolated neutrophils were associated with increased levels of IL-6 mRNA. The levels of IL-6 mRNA were decreased like those of IL-1 mRNA in activated neutrophils of pollinosis patients. We have supposed that the changed gene expression of IL-1 and IL-6 having the synergistic effect with IL-3 might contribute to hematopoiesis disorder in allergy to pollen.

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Interactions Between the Erythmpoletin Receptor and the Friend SFFVp Virus C. Lacornbe, S. Chretien, N . Casadevall, I. Dusanter. S. Gisselbrecht, I! Mayewr INSERM U 152 and U76, ICGM Hcpital Cochin, 75014 Paris, France The Friend SFFVp defective virus causes erythroleukemia by stimulating the late erythmid progenitor cells. We observed that two Friend erythroleukemia cell lines overexpressederythropoietin receptor (EpoR) mRNA. Abnormal transcription in these cell lines resulted from the rearrangement of one EpoR allele by insertion of a retroviral LTR within the first exon without altering the EpoR coding sequence. Such altered receptor gene activation may provide a positive pressure in the development of erythroleukemia. Furthermore, the SFFVp-induced erythroleukemia cells have been shown to be Epo independent due to the associationof the SFFVp envelope (Env) protein with the Epo-R. We have performed Epo cross-linkingin cells expressing the SFFVp Env and we showed that Epo was mainly cross-linked to a cell surface 63 kDa protein identified as the SFFVpEnv, associated with the cloned chain of the EpoR. Thus, the Friend SFFVp retrovirus could stimulate erythroid cells by combiningoverexpression of the Epo-R by viral insertion and stimulationof the Epo-R by association with SFFVp Env (either in the endoplasmic reticulum or in the plasma membrane or both).

Bone Marrow Failure Induced by the H2-Receptor Antagonist Famotidine I: Liersch, I.-H. Beyer, K. Vehmeyer Department of Internal Medicine, Division of HematologylOncology,D 3400 Gijttingen. FRG Severe agranulocytosisthat was observed in five cases clearly correlated with the application of famotidineas an ulcero-protectivemeasure. Under famotidine treatment a strongly inhibited progenitor cell growth (GM-CFU-c) was detected in the bone marrow and peripheral blood samples of the patients. One to five days after withdrawal of farnotidine, remarkable increases of GM progenitor cells in the bone marrow occurred in all cases, followed by the complete recovery of hematopoiesis after 7 to 17 days. Shortly after the removal of farnotidine accelerated colony growth was observed showing strong dependency on IL-3 until the full regeneration of hematopoiesis. In vitro studiesrevealed a dose-dependent suppression of CFU-mix. BFU-e and GM-CFU-c by famotidine. Removal of accessory cells did not affect these results. The data presented here show that although hematological side effects by famotidinehave not yet been reported, farnotidine is capable of participating in severe bone marrow alterations. The inhibition of the IL-3-induced histamine release or the blockade of the histamine-mediated(H1-recepton) proliferation signal on hematopoietic progenitor cells should be discussed as a possible mechanism of the famotidine-inducedagranulocytosis.

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Levels and Intranuclear Localization of myc,fos and jun Proteins During the Cell Cycle F? Loidl, G. Ldpa-Rodas, R. Greil, U! Mia University of Innsbruck-Medical School, 6020 Innsbruck, Austria Using polyclonal and monoclonal antibodies we demonstrate the presence of c-myc, c-fos and c-jun proteins in nuclei of Physantrn polycephalum, a muitinuclear organism with a naturally synchronous cell cycle. Whereas c-fos and c-jun protein levels fluctuate periodically during the cell cycle, c-myc protein levels remain invariant throughout interphase. c-fos protein is not associated with the nuclear matrix regardless of the cell cycle stage. In contrast, c-myc protein is firmly boundto the nuclear matrix in preparations obtained by a variety of matrix isolation procedures. This matrix-association is cell cycle dependent, since the total nuclear c-myc protein can be recovered from S-phase matrix preparations, but only 25% from G2-periodmatrices. Using immunoelectronrnicroscopy we demonstrate a significant variation of the intranuclear localization of the c-myc protein; in early S-phase it is exclusively located in the nuclear periphery (presumably nuclear lamina), during all other stages of the cell cycle it is uniformly distributed in the whole nuclear matrix. Among a variety of tested drugs only the histone deacetylase inhibitor butyrate was able to completely abolish the association of c-myc pmtein with the nuclear matrix.Togetherwith other data rhis indicates that the post-hanslational acetylation of core histones (in the nuclear matrix) may modulate the binding of the c-myc protein to DNA/chromatin components of the nuclear matrix.

Identificationof a New HematopoieticStem Cell, HighproliferstivePotential Megakmyocyte Mixed (HPP-Meg-Mix) Colony-Forming Cell P A . b w t y , H.E. McGmth, D.D.Deacon, undPJ. Quesenberry University of Virginia, Charlottemille, VA, USA We have previously reported synergisticinteractions of pairs of growth factors in the simulation of megakaryayte colonies from mixed mamw populations and observed augmentationof these colonies in populationspartially depleted of accessorycells. Using immuno-magentic and fluorescent activated cell sorting techniquesas originallydescribed by Spungnufeand Scollay (Exp Heme 18:920), we have isolated a highly purified murine stem cell progenitor pool identified by expression of an early stem cell antigen (SCA*)and lack of terminal differentiation markers (Lid). This population has shown 100-fold enrichment for High Proliferative Potential Colony Forming Cells (WP-CFC) over whole marrow when stimulated with three to five factor combinations of CSF-1, G-CSF, GM-CSF, IL-lor, and IL-3. Morphologicanalysis of the colonies produced shows that a sip5cant fractioncontain h t y l Cholinesterasepositive cells, a marker of murine megakaryocytes. Megakaryocytesare present in pure colony and admixed with, populationsof monocytesand granulocytes. A subpopuhon of highly pmlifkrative colonies meet traditional criteria for HPP-CFC but additionally contain over 100 megakaryocytes per colony. This population is tentatively designatedthe HPP-Meg-Mix colony-formingcell and is derived from highly purified (SCA'Lin') murine marrow populations under conditions of multiple factor stimulation.

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Long-Term Bone Marrow Hemstopietic Effectsof Benzene Exposure J.D. Lutton. N.G. Abmham New York Medical College, Valhalla, NY,USA The hematotoxic effects of benzene exposure in vivo (300ppm, 6 hr/day, 5 dayslweek for 2 weeks) was assessed with bone marrow cells from benzene-exposedmice grown in long-term bone marrow culture (LTBMC). LTBMC initiated one day after the last benzene exposure did not produce adequate numbers of hematopoieticcells during three weeks, and in most cases,no erythmid or myeloid clonogenic cells could be recovered. The adherent cell layer of these cultures had a lower capacity to support in vitro hematopoiesis after the second seeding with n o d cells as compared with controls. Two weeks after the last benzene exposure,weight of animals, hematocrit, hemoglobin, bone marrow ceUuIacity and cornmined hematopoieticprogenitor content (BFU-e and CFU-gm) wue regenerated to normal or subnormal values. However, hematopoiesisin LTBMC from exposed mice was very poor and during eight weeks, little or no progenitor production was observed. Treatmentof benzeneexposed mice with hemin (3 pg/g body wt, i.v.. 2 timedweek, for a total of three times), partially overcame the toxic effect as measured by LTBMC. Cultures from hemin-treatedanimals had a modest recovery of BFU-e and CFU-gm clonogenic potential after 5-6 weeb in LTBMC. In contrast, little or no recovery was obtained for the adherent cell layer clonogenic capacity, even after hemin treatment. These results clearly indicate a strong long-lasting toxic effect on the bone marrow stroma, and a limited recovery of hematopoietic potential by clonogenic cells of the nonadherent population after in vivo hemin treatment.

Quantitative Determination of the Hybrid b c d Z mRNA in Patients with Chronic Myelogenous Leukemia M.C.Malinge. EX. Mahon, M.H.Delfarc, E Ghuilbor, E Taner, B. Gmndchamp Facult6 X . Bichat. h i s and CHU de Poitiers, France

In vitro amplification of the bcr-abl cDNA has widely been used to assessthe presenceof minimal residual disease in CML patients achieving complete clinical and *genetic remission. However, the level of sensitivity achieved in diffennt laboratories remained largely unknown. Moreover, the results are usually expressed as positive or negative malung difficult a precise follow-up of the patients. In an attempt to overcome these limitations, we devised a quantitativemethod to measure the amount of bcr-abl mRNA in clinical samples. This methodologyinvolves a single reverse transcription step, followed by a separate amplification of bcr-abl and total abl -A. These two amplifications are performed in the presence of different dilutions of a same internal standard. This standard consists of bcr-abl sequences with a deletion of 8 bases in exon 2 of abl. One of the primers used in each separate reaction is labelled with fluomcein. Following amplification, PCR products derived from cellular RNA and those form the internal standard are separated and their relative fluorescence is determined using a laser fluorescent DNA sequencer (ALF, Phannacia). The number of bcr-ubf and total abf mRNA molecules initially present in each sample is than calculated. The accuracy and reproducibility of this method was assessed by studying serial dilutions of K562 mRNA in normal mRNA. Blood samples from 13 patients in CyGenetic remiasion under interkron therapy wcre studied. Only one sample was found negative while the others contained between 5 and Z103hybrid bcr-abl mRNA molecules per LO3molecules of abl mRNA. These results suggest that a Mliable number of malignant cells are present in the majority of CML patient in cytogenetic remission following inten%ron therapy.

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Genetic Transfer of the Human Interleukin 6 Receptor Gene (huIMR) in Leukemic Cells to Serve as 'hrget for the Fusion Protein IG6PseudomoMs Exotoxin (IL6PE40) for k t ment of Leukemia Anton C.M.Martens! hn-Fang Ren2, Kees J. de Grootl, Ivo I! Touw? Anton Hagenbeek1.3 lInstituteof Applied Radiobiology and Immunology/TNO, Rijswijk, The Netherlands; 2Institute of Basic Medical Sciences, Ekijing, People's Republic of China; 3TheDr. Daniel den Hoed Cancer Center, Rotterdam, The Netherlands

Human acute myeloCytic leukemias are found to express multiple hemopoietic gmwth factor (GF) receptors (R), e.g., the GM-CSF-R, IL-3-R, G-CSF-R and IL-6-R. The receptors have a high afinity for their ligands representingideal targets for the delivery of toxic molecules to cells. In addition, binding of GF-toxins to the receptors is followed by rapid internalizationof the complex allowing the toxin to start its cell-killing action. This offers a rationale for development of new leukemia treatment strategies. The human IL-6 receptor gene (huIL-6R)WBS tmsferred using the plasmid pXM6NmdL-6R and Ca-precipitationto LT12 cells (an in v i m growing sublineof the Brown Norway acute myelocytic leukemia model in the rat). Clones resistant to the neomycin analogueG418 were analyzed for expression of the IL-6R using the specific monoclonal antibody MP1 recognizing the huIL-6 receptor. Clones were found with stable expression of the IL-6R gene during long term in vitro culture as well as after in vivo transfer of the leukemic cells. Scatchard analysis with 12%diielabeled huIL-6 revealed the presence of 4OoO-So00 high affinity receptors per cell. The fusion protein between IL-6 and Pseudom o w exotoxin (IL-6PE40) WBS produced with molecular engineeringtechniques (Dr. D.FilrGemld, NIH,Bethesda, MD) and is now availablefor use in the LTl2IL-6R cell lines. The differential killing potential of leukemic cells and normal hemopoieticprogenitorcells with JL6PE40 can now be studied serving as a model for the general applicability of GF-toxin fusion pmteins for the treatment of leukemia.

Recombinant Human Leukemia Inhibitor (rhLIF) is an Inductor of Acute Phase Proteins and Raises the Blood Platelets in Nonhuman Primates I? Mayer", K. Geisslerb, P. %lentb, E! Bettelheimb,E. Liehl'. M. Mrd', D. Metcafl 'Sandoz Research Institute, Vienna; blst Medical Department, University of Vienna, Austria; 'AMRAD CorporationLtd., Australia; ? h e Walter and E l i Hall Institute, Victoria, Australii Human leukemia inhibitory factorproduced by molecular cloning WBS administered to nonhuman primates to assess its biological activity in vivo. Rhesus monkeys were treated S.C. with rhLIF at 2, 10 and 50 pglkg body weighthy for 14 days. Serum levels of positively regulated acute phase proteins (APP) (C-reactive protein, cr-antitrypsh,haptoglobin and ceruloplasmin) increased whereas negatively regulatedAPP @realbumin)d d in response to mLIp treatment in a dmdepedent mmner. After a latent period of 4 - 10 days, platelet counts rose following the onset of rhLIP treatment, resulting in a maximum two-fold increase above normal levels severaldays after the terminationof the rhLF treatment. No changes were seen in the progenitorcell levels in blood throughout the r h J S treatment and post-treatment observation period. In conclusion, rhLIF was shown to stimulate APP and was able to increase the number of platelets in nonhuman primates.

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Identification of Intracellular Receptom for Porphyrlns: The Pharmacology and Purification of the Mitochondria1 Eknzodiazepine Receptor M.W McEnery, A. &ma, S.H.Snjder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 The anti-anxiolytic benzodiazepine. [3H]-diazepam(Valium), has been shown to interact with two distinct classes of receptors which can be distinguished by pharmacology and localization. The therapeuticallyrelevant central benzodiazepine(Bz)receptor, is localized to the GABA-A type chloride channel of neurons. The second class of Bz receptors, enriched in adrenal, kidney, testis, and ovary, is termed the peripheral or mitochondrial Bz receptor. The affinity of [3H]-diazepam for the central and mitochondrial Bz receptors is identical, so at therapeutic doses of this dmg, both sites would be affected. An imporrant difference between Bz receptors classes is the effect of porphyrins on ligand binding (&m ef al. PNAS 84, 2256-2260, 1987). Porphyrins (protoporphyrin IX and mesoporphyrinIX)have been shown to inhibit benzodiazepinebinding at nanomolar concentrations to only the mitochondrialBz receptor. The mitochondrialBz receptor (mBzR) has been purified from rat kidney mitochondria, and has been shown to be comprised of three non-identical proteins which have been positively identified via specific antibodies. The 32 kDa subunit has been identified as the voltagedependent anion channel (VDAC). VDAC functionsas a pore in the mitochondrialouter membrane for the passage of solutes from the cytoplasm to the mitochondrial inner membrane and matrix. As the initial and f d steps in porphyrin biosynthesistake place within the mitochondria,the precursors and products must traverse the outer mitochondrial membrane. The effects of porphyrins on [3H]benzodiazepine binding to mBzR identifies porphyrins as endogenous ligands for the receptor.

Interactions Between Stem Cell Factor and ColonyStimulating Factors D. MetcaK N.A. Nicoia, K. neb0 The Walter and Eliza Hall Institute, Melbourne, Australia; Amgen, Thousand Oaks, CA Stem cell factor (SCF) is the ligand for the c-/&-encoded membrane receptor defective in W mutant mice. Purified recombinant SCF has been examined to determinewhether it can act as a directproliferative stimulus for normal murine marrow cells. Although thousand-fold higher concentrations of SCF arc required than for the CSFs, SCF stimulated the formationof blast cell coloniesand colonies composed of immature or mature granulocytes. This appeared to be a direct action because of the linearity of colony formation with cultured cell numbers and the proliferative action of SCF on transferred clones, resuspended colony cells and on FACS-purified stem cells. When mixed with purified colony stimulating factors, a 5-20-fold potentiation of colony size was observed with G-CSF, GM-CSF and Multi-CSF but not M-CSF. In addition, combination with CSFs induced a marked rise in the progenitor cell content of blast colonies but there was no evidence that SCF was capable of stimulating self-generation by blast colony-forming cells or their progeny. Unusually large numbers of high-affinity -tors for SCF arc present on blast cells in the marrow and these may be the primary targets of SCF. The amplification of granulocytecolony formation observed with combination of SCF and GCSF may explain the ability of injected G-CSF to induce high neutrophil levels in vivo.

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Molecular Mechanisms Underlying the Erythroid Specificity of the Cellular Response to Erythropoietin (Epo) A . R. Migliaccio, G. Migliaccio, S. Ottolenghi. J. W Adamson Laboratory of HematopoieticGrowth Factors, NY Blood Center, New York, NY 10021, USA; Dipartimento di Genetica e di Biologia dei Microrganismi, Universiti di Milano, Milano, Italy The cellular response to Epo is almost completely restricted in vivo and in vitro to cells of the erythroid lineage. Since Epo initiates its biological action by binding to a specific receptor (EpoR), the erythroid-restrictedaction of Epo could be achieved by specificregulation of the EpoR gene which results in a functional EpoR. It is unlikely that such a specificity is mediated by lineage-restricted gene activation sincethe expression of GATA-1, the major transcription factor involved in EpoR gene activation, is not restricted to erythroid cells. To understand how lineage-specific Epo response is achieved, we have analyzed the expression and function of EpoR in the E-3-dependent cell line 32D, which has the potential to respond to Epo, and one of its subclones, 32D Epo, which grows in Epo and differentiatesinto erythroidcells. For controls, we analyzed Friend-inducederythroleukemiacells (MEL) and GM-CSF (32D GM) or G-CSF-dependent subclones of 3 2 4 (32D GM and 32D G, respectively), which do not respond to Epo. EpoR mRNA (by RNAase protection analysis) and protein (by EpoR-specificantibody immunoprecipitationof metabolically-labelledproteins) were detectable not only in 32D Epo but also in the original 32D and in 32D GM. EpoR mRNA was also detectable by PCR in 32D G.In contrast, only the erythroid 32D Epo cells bound 125-I-Ep0(400 - 800 binding sites/cell; Kd 0.4 - 0.7 nM) and expressed EpoR protein on the surface (by immunoprecipitation of surface-labelledproteins). These resultsindicate that the major regUratory step determiningthe erythroidspecific response to Epo is the efficiency of EpoR protein translocation to the cell surface. Among the mechanisms which could regulate such lineage-specific translocationis the presence of an erythroidspecificchaperoneprotein and/or post-transcriptionalmodification of EpoR mRNA to produce a functional EpoR.

Human Stem Cell Factor (SCF): A Hematopoietic Growth Factor Targeting Human Multilineage Colony-Forming Cells (CFC) and Capable of Generating CFC from F’re-CFC G. Migliaccio, A.R. Migliaccio, J. Egrie, K. ZFebo, J.W -on New York Blood Center, New York, NY 10021; Amgen, Thousand Oaks, CA 91320, USA SCF is the growth factor encoded in the Steel locus of the Sl/Sldmouse. Affected animals have altered coat color, macrocytic anemia, and primordial germ cell defects. Thus, SCF is a growth factor potentially affecting the organization of a number of tissues including early hemopoietic progenitor cells. We have studied the effects of recombinanthuman SCF alone and in combination with interleukin 3 (IL-3). granulocyte/macrophagecolony-stimulating hctor (GM-CSF), G-CSF and erythropoietin (Epo) on progenitor cell growth under serum-deprived (FBS-) culture conditions. Target cells included non-adherent human marrow or CD34+, soybean agglutinin (SBA)- cord blood cells. Against these target cells, SCF had littJe colony-stimulating activity but it synergized with IL-3, GM-CSF, G-CSF and Epo to promote a variety of colony types including up to 50 large ( > lo4cells) mixed-cell colonies per los cells plated. Such colonies are observed infrequently (1 - 5/105cells) in FBS+ cultures and only rarely in FBS- cultures. IL-3, GM-CSF and Epo, alone or in combination, yielded virtually no mixed-cell colonies. Thus, human SCF represents a multilineagehematopoietic growth factor whose specificitydiffers from that of IL-3 and GM-CSF. In suspensionculture of 8000 CD34+, SBA- cord blood cells, SCF plus IL-3 induced (from

Second International Symposium on the Molecular Biology of Hematopoiesis. Innsbruck, Austria, July 14-18, 1991. Abstracts.

301 Second International Symposium on the Molecular Biology of Hematopoiesis University of InnsbrucWNew York Medical College Innsbruck, Austria, July...
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