Absence of p53 permits propagation of mutant cells following genotoxic damage.

Much evidence has been gathered in support of a critical role for p53 in the cellular response to DNA damage. p53 dysfunction is associated with progression and poor prognosis of many human cancers and with a high incidence of tumours in p53 knockout mice. The absence of a p53-dependent G1 arrest that facilitates DNA repair or apoptosis might impact critically on clinical cancer in two ways. First, by abrogating the impact on therapy that operates via genotoxic damage and apoptosis; and second, by encouraging progression either by inducing genomic instability and DNA mis-repair or by permitting survival of mutants. However, experiments examining the relationship between p53 deficiency and mutation frequency have so far failed to confirm these predictions. The precise role played by p53 is therefore unclear. We now report use of a short term in vitro approach to assess the influence of p53 on radiation-induced mutations at the hprt locus in murine B cell precursors that are normally radiation ultrasensitive. We find a high number of hprt mutants among X-irradiated p53 null cells, which results from preferential survival as clonogenic mutants rather than from a p53-dependent increase in mutation rate. This result has important implications for genotoxic cancer therapy.

Clonal characteristics of acute lymphoblastic cells derived from BCR/ABL p190 transgenic mice.

The clonal and immunophenotypic characteristics of blood leukemic cells from BCR/ABL p190 transgenic mice were investigated. All cell populations evaluated in vivo and in vitro had B-lymphocyte progenitor immunophenotypes. Immunoglobulin (JH) rearrangement patterns provided evidence for clonal diversification at different sites in vivo. Multiple clones were established in vitro from two of these mice (nos. 730 and 753). These cells expressed BCR/ABL p190 protein tyrosine kinase (PTK) and were highly malignant on transfer to secondary recipients. Cells independently cloned in vitro shared identical immunophenotypes and clonal IgH rearrangements, but these were distinct from those of the dominant clones in the mouse from which they were derived. Nevertheless, in vitro clones from mouse no. 753 had an abnormal karyotype (chromosome 14 trisomy) in common with the dominant clone in blood, providing evidence for a hierarchy or clonal selection in vivo and in vitro. Two sets of in vitro clones proliferated independently of exogenous growth factors and stroma and released autocrine interleukin 7 growth factor activity. These data provide evidence for rapid divergent clonal evolution and selection of B-cell progenitors initiated by BCR/ABL p190, followed by other, secondary genetic events mirroring similar changes in the equivalent, highly malignant human leukemia Philadelphia (Ph)-positive/B-precursor acute lymphoblastic leukemia (ALL).

Neonatal mixed lineage acute leukaemia.

We report here an uncommon case of neonatal acute leukaemia that presented concomitant with serological evidence of rubella infection. The clinical course was aggressive and the patient died 5 days after diagnosis from septicaemia. Leukaemic blasts had a mixed lineage immunophenotype co-expressing a constellation of B-lymphoid (CD19, cytCD22, TdT) and myeloid (CD13, CD33, CD14, anti-MPO) markers, as well as multiple adhesion molecules and markers associated with early lympho-myeloid progenitor cells (CD34, CD7, HLA-DR). A previously unrecorded discordant expression of different CD10 and CD34 epitopes was identified using different monoclonal antibodies. The karyotype was 46,XX t(4;11)(q21;q23) and molecular analysis confirmed rearrangement of the trithorax-related oncogene HRX at 11q23. There was a clonal biallelic rearrangement of the immunoglobulin heavy-chain gene. The features of this rare case have implications for possible aetiological events leading to leukaemia.

Distribution and epitope analysis of the cell membrane glycoprotein (HPCA-1) associated with human hemopoietic progenitor cells.

Monoclonal antibodies My10, BI.3C5, 12.8, and ICH3 identify a monomeric cell surface glycoprotein (HPCA-1) of 100-120 kD, which is selectively expressed on human hemopoietic progenitor cells. Other tissues are nonreactive with the exception of capillary endothelia and basement membrane in some sites. In addition, the antigen can be detected on cell lines that exhibit characteristics associated with early T cell precursors. HPCA-1 is therefore associated with myeloid, B, and T lineage precursors. Sequential immunoprecipitation and Western blotting studies demonstrate that BI.3C5, ICH3, My10, and an antibody directed against endothelial cells, 188.27, all react with the same glycoprotein species, although the epitopes involved may be distinct. The epitope recognized by BI.3C5 is sialic acid dependent, whereas that recognized by ICH3 is not. The My10 epitope has partial sensitivity to neuraminidase. Competitive/additive binding experiments suggest that these epitopes, although probably distinct, may be closely associated.

In vitro proliferation by cells mobilized into the peripheral blood for collection and autologous transplantation.

We have investigated the properties of mobilized, cryopreserved, peripheral blood stem cells (PBSC), collected by leukapheresis over a period of 5 days, from eight myeloma patients in clinical remission. Cells were mobilized by treatment with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF), and each day's collection was evaluated for its content of CD34+ cells, colony-forming units granulocyte/macrophage (CFU-GM), and plastic-adherent pre-CFU-GM. Peak values for these three parameters were observed at different times in different patients. There was no correlation between CD34+ content and CFU-GM, but there was some (r = 0.65) between CD34 numbers and colonies generated from a delta assay initiated using plastic-adherent pre-CFU-GM. In suspension cultures, the cells grew exponentially for 50 days. Thereafter, they did not divide, although they remained viable in culture for up to 1 month longer. Suspension cultures of PBSC grown with interleukin-3 (IL-3) displayed a predominantly myelomonocytic phenotype, but some megakaryocytes and erythroid cells were observed consistently. These results indicate that pre-CFU-GM in PBSC collections are capable of generating large numbers of clonogenic progeny in liquid culture and are capable of producing multiple lineages of differentiation.

An in vitro model for the production of committed haemopoietic progenitor cells stimulated by exposure to single and combined recombinant growth factors.

A plastic-adherent mononuclear cell population in human bone marrow produces non-plastic-adherent nucleated cells in liquid cultures. These cells can be harvested from the culture medium and a proportion of them can be identified as granulocyte-macrophage colony-forming cells (GM-CFC) by plating them in semi-solid cultures with granulocyte-macrophage colony-stimulating factor (GM-CSF). The generation of GM-CFC from their plastic-adherent precursors can be amplified considerably by adding 5637 conditioned medium (CM) to the liquid phase of the adherent cell cultures. This effect of 5637 CM cannot be reproduced by recombinant (r) GM-CSF or interleukins (ILs) 1, 3 or 6 if they are added singly to the culture medium. In contrast, the combination of GM-CSF + IL-1 equalled or surpassed the activity of 5637 CM. The combinations of rGM-CSF + rIL-3 and rGM-CSF + IL-6 also mimicked the activity of 5637 CM but less effectively than GM-CSF + IL-1.

Extent of variability inherent in measurements of CD34-positive cells in different human haemopoietic tissues.

Estimation of CD34 expression is widely used to detect and quantify progenitor cells in haemopoietic tissues used as stem cell sources for transplantation. Mouse monoclonal antibodies to CD34 recognise different epitopes of the mucin-like sialoglycoprotein. These epitopes can be grouped into three classes by their differing sensitivities to the enzymes: neuraminidase, chymopapain and glycoprotease. We have compared the expression, by flow cytometry, of the three CD34 epitopes on normal adult and fetal haemopoietic tissue and in chronic myeloid leukaemia, and have used four antibodies from each class to assess variability of staining within and between epitope classes. The results reveal variable expression of CD34 both within and between tissue types and antibody classes. As a result of the different levels of detection by different antibodies, the apparent number of CD34-positive cells vary by approximately 6-fold. Enrichment for CD34 cells using magnetic bead technology shows a significant difference in the percentage of CD34 cells detected for two of the epitope types.

The second ETV6 allele is not necessarily deleted in acute leukemias with a ETV6/ABL fusion.

The ETV6 (TEL) locus at chromosome band 12p 13 is a major site of translocations in acute leukemia, particularly in childhood acute lymphoblastic leukemia (ALL). In cases with translocations involving ETV6, the normal ETV6 allele is often deleted. In addition, loss of heterozygosity of ETV6 is frequently observed in childhood'ALL. Thus, it has been suggested that ETV6 may have an anti-oncogenic role to play, in addition to its oncogenic role. We have described an unusual case of ALL in which ETV6 is found fused to the ABL gene; ABL is normally activated by fusion to the BCR gene in the 9:22 translocation. We expanded the primary cells from this ETV6/ABL rearranged case of ALL in SCID animals and analyzed them for expression of both ETV6/ABL and the normal ETV6 mRNA. We found that both the rearranged and normal ETV6 mRNAs are expressed in the expanded cell population. Furthermore, sequence analysis of the ETV6 PCR product revealed no point mutations which would influence the amino acid sequence. Thus, deletion of the second ETV6 allele is not necessary for the transformation to leukemia by ETV6/ABL.

Immunoglobulin heavy-chain and CD3 delta-chain gene enhancers are DNase I-hypersensitive in hemopoietic progenitor cells.

Multipotential interleukin 3-dependent non-immortalized murine hemopoietic progenitor cells have DNase I-hypersensitive sites in the immunoglobulin heavy-chain and CD3 delta enhancers and transcribe germ-line T-cell antigen receptor gamma-chain (TCR gamma), but not IgM or TCR beta, genes. Induction of myeloid differentiation in these cells clones down expression and/or transcriptional accessibility of the immunoglobulin heavy-chain and TCR gamma genes. The CD3 delta enhancer region remains DNase I-hypersensitive but closes down in B cells. In embryonic stem cells and pan-mesodermal cells, these genes or enhancer regions are neither expressed nor DNase I-hypersensitive. These data suggest that lineage potential may be programmed, at least in part, by alterations in the accessibility or conformation of regulatory regions of genes and that some promiscuity of gene expression and/or accessibility can precede lineage commitment and maturation in progenitor cells induced to self-renew by interleukin 3.

Cloning of human thymic subcapsular cortex epithelial cells with T-lymphocyte binding sites and hemopoietic growth factor activity.

The thymic microenvironment involves complex cell interactions among different types of epithelial cells, macrophages, tissue histiocytes, and immature and maturing T cells. We describe the isolation of a subset of thymic epithelial cells by selective primary culture followed by cotransfection with a simian virus 40 replication-origin-defective mutant and pSV2neo plasmid. The cloned cells have the composite immunophenotype that is unique to thymic subcapsular epithelial cells, suggesting that they may provide a model system in vitro for analyzing the earliest steps in T-cell differentiation. This possibility is supported by the finding that these epithelial cells express LFA-3-associated binding sites for T cells, secrete a macrophage hemopoietic growth factor, and synergize with macrophages in the production of interleukin 1.

Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.

Multilineage phenotypes of interleukin-3-dependent progenitor cells.

Interleukin-3 (IL-3)-dependent murine FDCP-mix cells have multilineage differentiation capacity; they are nonleukemic, have a normal karyotype, and are nonimmortalized. These cells coexpress on their cell surface the "early" B-lineage marker B220/CD45R and the myeloid marker Mac-1/iC3b receptor (CR3), transcribe germline T-cell receptor gamma genes, and express the macrophage lineage growth factor receptor gene c-fms as a predominant 8.4-kb transcript. They do not detectably express at the stable mRNA or protein level other lymphoid precursor cell genes including CD2, TdT, lambda 5, and BP1. Induction of granulocyte/macrophage differentiation in these cells closes down expression of the lymphoid genes and activates stable expression of genes specific to the myeloid lineage, including myeloperoxidase. Expression of the c-fms gene at the mRNA level is upregulated and the dominant stable transcript is now in the 4.1-kb form typical of the macrophage lineage. These data provide a plausible explanation for the coexpression of lymphoid and myeloid lineage markers on human leukemic cells of stem cell or progenitor cell origin and have implications for the programming of lineage potential in normal multipotential hematopoiteic progenitor cells.

Circulating stem cells in mice treated with cyclophosphamide.

Chemotherapy has been used clinically to mobilize hematopoietic progenitor cells into the peripheral blood so that they can be harvested for autologous transplantation. In humans, this is demonstrated by the presence of circulating granulocyte-macrophage colony-forming cells (CFU-GM) and CD34-positive cells, but it has not been possible to confirm the presence of marrow-repopulating stem cells. In this study, we treated mice with 200 mg/kg cyclophosphamide (CY) and measured the numbers of white blood cells, day 12 CFU-S (CFU-S12), and CFU-GM in the peripheral blood. There was a peak in the numbers of CFU-S12 and CFU-GM 8 days after treatment with cyclophosphamide. Peripheral blood cells taken at this time rescued lethally irradiated mice and engraftment of donor cells was confirmed after 140 days in sex mismatched recipients using a Y chromosome-specific probe. In vitro culture of the blood cells harvested after cyclophosphamide showed that they proliferated in suspension cultures for at least a year in the presence of interleukin-3. The cultured cells rapidly lost their abilities to rescue irradiated mice and to form colonies in vitro, but they did not become leukemic. Also, CY-treated mice were irradiated with a leukemogenic dose of x-rays to coincide with peak circulating cell numbers but these animals did not develop an excess of leukemias over mice given irradiation alone.

Cloning, chromosomal localization and expression pattern of the POU domain gene Oct-11.

POU domain genes encode a family of highly conserved transacting factors that influence the transcriptional activity of several cell type-specific and ubiquitous genes. We have cloned and sequenced cDNAs encoding a novel mouse POU domain protein, Oct-11, that is closely related within the POU domain to the POU class II proteins, Oct-1 and Oct-2. Recombinant Oct-11 protein binds specifically to an octamer sequence in vitro. The Oct-11 gene is expressed during mouse embryogenesis and in the adult thymus and testis. In addition, it is abundant in the myeloma cell line P3/NS-1/1-Ag4.1. We describe the structure of Oct-11 and its chromosomal localization, and discuss the evidence that the POU class II gene family has evolved by duplication and divergence of a common ancestral gene.

Establishing a KSHV+ cell line (BCP-1) from peripheral blood and characterizing its growth in Nod/SCID mice.

Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) sequences are present in primary effusion lymphomas (PEL). KSHV+ cell lines have been established from such lymphomas. Here we report the first description of the establishment of a KSHV+, EBV- cell line (BCP-1) from the peripheral blood of a patient with PEL. Using this cell line and a KSHV+, EBV+ PEL cell line (HBL-6) previously established from ascitic fluid, we investigated whether in nonobese diabetic/severe combined immunodeficiency disease (Nod/SCID) mice tumors representing PEL can be established. When injected intravenously (IV) into Nod/SCID mice, BCP-1 and HBL-6 infiltrated organs, with only occasional macroscopic tumor formation. Intraperitoneal injections (ip) led to the development of ascites and diffuse infiltration of organs, without obviously solid lymphoma formation, resembling the diffuse nature of human PEL. To investigate a possible mechanism for the peculiar phenotype of PEL, we examine the presence of adhesion molecules and homing markers on PEL cells before and after growing in mice. Both BCP-1 and HBL-6 cells lack expression of important cytoadhesion molecules including CD11a and CD18 (LFA1 alpha and beta chains), CD29, CD31, CD44, CD54 (ICAM-1), and CD62L and E (L and E selectins).