Riitta Alitalo

Induced differentiation of K562 leukemia cells: A model for studies of gene expression in early megakaryoblasts

The K562 leukemia cell line has been extensively used in studies of erythroid differentiation but it has been less well appreciated that treatment of K562 cells with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) leads to loss of their erythroid properties and to acquisition of several megakaryoblastoid characteristics. These include synthesis and surface expression of glycoprotein IIIa, an increase in platelet peroxidase positivity, enhancement of thromboxane A2 receptors, and increased cell volume and DNA ploidy. TPA-treated K562 cells also synthesize and secrete platelet derived growth factor (PDGF), transforming growth factor beta 1 (TGF beta 1), urokinase-plasminogen activator (u-PA) and its specific inhibitor, type 1 plasminogen activator inhibitor (PAI-1). Induction of all these proteins, which have also been found in platelet granules (u-PA on platelet surface receptors) occurs at the level of mRNA accumulation. Therefore, in addition to facilitating studies and cloning of genes specific for erythroid differentiation, the K562 cells offer a tool to approach early steps of megakaryoblast commitment and differentiation. Observations made with the K562 cell line and several other leukemia cell lines co-expressing erythroid and megakaryocyte markers suggest that the erythroid and megakaryocyte lineages diverge from a common bipotent precursor cell.

ACUTE MYELOGENOUS LEUKAEMIA WITH C-MYC AMPLIFICATION AND DOUBLE MINUTE CHROMOSOMES

Cytogenetic analysis of bone-marrow cells from a woman with preleukaemia showed numerous mitoses with trisomy 4 and double minute chromosomes. These abnormalities were later seen in blood cells during subsequent acute myeloid leukaemia (AML). Complete remission was achieved with three courses of doxorubicin, cytosine arabinoside, and prednisone. A further clonal abnormality, trisomy 6, was seen in leukaemic cells after the first relapse. Analyses of total DNA from the peripheral-blood cells during relapse showed that the c-myc oncogene was amplified about 30-fold in the leukaemic cells. The N-myc, c-mos, and c-myb oncogenes showed only single-copy signals. On average about two copies of c-myc resided on each dmin chromosome. The finding of amplification of a cellular oncogene (c-myc) in fresh AML cells containing double minute chromosomes suggests that clonal evolution of some leukaemia cell populations may involve selection for increased dosage of oncogenes.

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.

Analysis of Tie receptor tyrosine kinase in haemopoietic progenitor and leukaemia cells

We generated a panel of monoclonal antibodies against the extracellular domain of the Tie receptor tyrosine kinase and studied its expression in human haemopoietic and tumour cell lines and in samples from leukaemia patients. Most of the erythroblastic/megakaryoblastic (6/8), 2/7 myeloid and 3/6 B‐lymphoblastic leukaemia cell lines were Tie‐positive. The erythroblastic/megakaryoblastic leukaemia cell lines also expressed the related Tie‐2/Tek gene and, surprisingly, its recently cloned ligand gene angiopoietin‐1, which was located in chromosome 8q23.1. In addition, 16% of freshly isolated leukaemia samples were Tie positive. Peripheral blood mononuclear cells were Tie negative, but a few Tie positive cells were found in immunoperoxidase staining of mobilized peripheral blood stem cells. Long‐term culture of isolated umbilical cord blood CD34+Tie+ and CD34+Tie−cells indicated that the Tie+ fraction contained a slightly higher frequency of cobblestone area forming cells (CAFC). Thus, Tie is expressed on haemopoietic progenitor cells and some leukaemic blasts. The coexpression of Tie‐2 and angiopoietin‐1 in megakaryoblastic leukaemia cell lines suggests the existence of an autocrine ligand/receptor signalling loop in these cells.

Induction of platelet-derived growth factor gene expression during megakaryoblastic and monocytic differentiation of human leukemia cell lines

Platelet‐derived growth factor (PDGF) is one of the most important polypeptide growth factors in human serum. It is composed of two polypeptide chains linked by disulfide bonds. The B‐chain is encoded by the c‐sis proto‐oncogene, which is expressed in several malignant and non‐malignant cells including K562 cells differentiating towards megakaryoblasts. Expression of the A‐chain has been reported to occur in human solid tumor cell lines independently of c‐sis expression. We report here the non‐coordinate expression of the A‐ and B‐chains in human leukemia cell lines. The PDGF‐A and B‐chain (c‐sis) RNA expression as well as secretion of PDGF polypeptides are induced in the K562 cell line upon induction of megakaryoblastic differentiation with 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) whereas erythroid differentiation induced with sodium butyrate is accompanied by c‐sis expression only. Simultaneously with megakaryoblastic differentiation the RNA level for another platelet protein, the transforming growth factor‐beta was also increased, but in a complex manner. The promyelocytic leukemia cell line HL‐60 does not express PDGF‐A RNA, whereas the promonocytic cell line U937 does. Preferential induction of the A‐chain RNA is obtained in both cell lines after treatment with TPA which causes monocytic differentiation. PDGF‐A expression in HL‐60 cells is also observed after treatment with the tumor necrosis factor‐alpha but granulocytic differentiation of HL‐60 cells induced with dimethyl sulfoxide or the granulocyte colony‐stimulating factor is not associated with PDGF gene expression.

Production of an active urokinase by leukemia cells: a novel distinction from cell lines of solid tumors

A new screening test is described which enabled rapid determination of the proportion of single-chain and two-chain urokinase produced in the culture supernatants of 18 human cell lines. A clear distinction was found between two groups of cell lines: cells derived from ten solid tumors produced almost exclusively single-chain proenzyme, while the majority of the enzyme found in cultures of eight leukemia cell lines was in the active, two-chain form.

Podocalyxin in human haematopoietic cells

Podocalyxin‐like protein (PCLP) is a sialomucin‐type membrane protein structurally related to CD34 and endoglycan. It was first described in glomerular podocytes and endothelial cells. In mice, PCLP is present in haemangioblasts, and in both chicken and mice it is a marker of early haematopoietic stem cells and lineage‐restricted haematopoietic progenitors. Its expression decreases during differentiation of haematopoietic cells. Of mature blood cells, only chicken and rat thrombocytes express PCLP protein. PCLP expression in human haematopoietic cells has not been studied. Here we demonstrate PCLP mRNA in human CD34+ cells, in lineage committed erythroid, megakaryocyte and myeloid progenitors, in K562 leukaemia cells, and in peripheral blood leucocytes. The mRNA expression level was higher in developing cells than in mature leucocytes. By Northern blotting and cDNA sequencing, the haematopoietic and renal PCLP mRNAs were identical. Of the mobilized CD34+ cells, 28% (mean; range 14–61%) expressed PCLP protein and the majority of PCLP+ cells were CD117+. Almost all of the K562 cells expressed PCLP protein. Surprisingly, PCLP protein was not detected in any mature blood cells. These results suggest that human PCLP may be a valuable marker for a subset of haematopoietic stem cells.

Bone marrow mesenchymal stem cells undergo nemosis and induce keratinocyte wound healing utilizing the HGF/c‐Met/PI3K pathway

We previously showed cell–cell contacts of human dermal fibroblasts to induce expression of the hepatocyte growth factor/scatter factor (HGF) in a process designated as nemosis. Now we report on nemosis initiation in bone marrow mesenchymal stem cells (BMSCs). Because BMSCs are being used increasingly in cell transplantation therapy we aimed to demonstrate a functional effect and benefit of BMSC nemosis for wound healing. Nemotic and monolayer cells were used to stimulate HaCaT keratinocyte migration in a scratch‐wound healing assay. Both indicators of nemosis, HGF production and cyclooxygenase‐2 expression, were induced in BMSC spheroids. When compared with a similar amount of cells as monolayer, nemotic cells induced keratinocyte in vitro scratch‐wound healing in a concentration‐dependent manner. The HGF receptor, c‐Met, was rapidly phosphorylated in the nemosis‐stimulated keratinocytes. Nemosis‐induced in vitro scratch‐wound healing was inhibited by an HGF‐neutralizing antibody as well as the small molecule c‐Met inhibitor, SU11274. HGF‐induced in vitro scratch‐wound healing was inhibited by PI3K inhibitors, wortmannin and LY294002, while LY303511, an inactive structural analogue of LY294002, had no effect. Inhibitors of the mitogen‐activated protein kinases MEK/ERK1/2 (PD98059 and U0126), and p38 (SB203580) attenuated HGF‐induced keratinocyte in vitro scratch‐wound healing. We conclude that nemosis of BMSCs can induce keratinocyte in vitro scratch‐wound healing, and that in this effect signaling via HGF/c‐Met is involved.

L-myc and N-myc in Hematopoietic Malignancies

The myc proto-oncogenes encode nuclear DNA-binding phosphoproteins which regulate cell proliferation and differentiation. The c-myc gene is implicated in hematopoietic malignancies on the basis of its frequent deregulation in naturally occurring leukemias and lymphomas. Recent evidence suggests that also the N-myc and L-myc genes may have a role in normal and malignant hematopoiesis. N-myc and to a certain degree L-myc can substitute for c-myc in transformation assays in vitro, and their overexpression can block the differentiation of leukemia cell lines. Immunoglobulin heavy chain enhancer (IgH) -driven overexpression of N-myc or L-myc genes cause lymphatic and myeloid tumors, respectively, in transgenic mice. Furthermore, the L-myc and N-myc genes are expressed in several human leukemias and leukemia cell lines, L-myc predominantly in myeloid and N-myc both in myeloid and in some lymphoid leukemias. All N/L-myc positive leukemias and leukemia cell lines coexpress the c-myc gene, thus exemplifying a lack of negative cross-regulation between the different myc genes in leukemia cells. Taken together, these data suggest that L-myc and N-myc may participate in the growth regulation of hematopoietic cells.

Plasminogen activation in human leukemia and in normal hematopoietic cells.

The active process of pericellular proteolysis is central in tumor invasion, and in particular the essential role of the urokinase‐type plasminogen activator (uPA) is well established. uPA‐mediated plasminogen activation facilitates cell migration and invasion through extracellular matrices by dissolving connective tissue components. uPA, its receptor (uPAR) and plasminogen activator inhibitor‐1 (PAI‐1) are enriched in several types of tumors. The importance of proteolysis and especially plasminogen activation is less clear in hematopoietic malignancies than in solid tumors. However, patients with leukemia have an increased tendency to bleeding, not always attributable to thrombocytopenia, and tissue infiltration by leukemic cells, processes in which plasminogen activation may be involved. Several studies have indicated that plasminogen activators (PAs) are highly expressed by cultured leukemia cells. Furthermore, differing from adherent tumor cells, leukemic cells have an enhanced capacity to activate pro‐uPA and mainly the active form of uPA is released to culture medium. Ex vivo studies have shown that uPAR, uPA and its inhibitors can be found on the surface of normal blood cells and on the blast cell surfaces from patients with acute leukemia as well as from plasma samples. Elevated levels of PAs and their inhibitors have been detected in leukemic cell lysates. Few studies have tried to demonstrate a correlation between prognosis of leukemia and levels of plasminogen activators. More in vivo studies are needed to show, if any of the factors of the plasminogen activation process can be used as tools in subclassification or as markers for prognosis in leukemia. This review article will focus on the in vivo studies of plasminogen activation in leukemia and will present several in vitro findings on PAs in normal leukocytes and leukemic cell lines.