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R K Humphries

Publications and source records attributed to R K Humphries.

At least 91 records · Page 5Linked to original sources
PubMed1992

The human hematopoietic stem cell in vitro and in vivo.

A quantitative assay for a primitive human hematopoietic cell has been developed. The cell identified has been assigned the operational designation of long-term culture (LTC)-initiating cell based on its ability when cultured on supportive fibroblast monolayers to give rise to daughter cell(s) detectable by standard in vitro colony assays. Three lines of evidence support the view that the LTC-initiating cell assay may allow the relatively specific enumeration of totipotent cells with in vivo reconstituting potential. These involve the demonstration: (1) that conditions in analogous murine long-term cultures stimulate the extensive amplification (self-renewal) of some totipotent long-term repopulating cells, (2) that most of the LTC-initiating cells in normal human bone marrow are phenotypically different from most of the colony-forming cells present in the same cell suspensions in their possession of a number of characteristics specifically associated with transplantable stem cells; and (3) that cultured marrow cells from patients with chronic myeloid leukemia which, after maintenance under LTC conditions for 10 days contain some normal LTC-initiating cells but no detectable leukemic LTC-initiating cells, can after autografting reconstitute the hematopoietic system with normal cells.

Animals↗
PubMed1991

CD24, a signal transducer modulating B cell activation responses, is a very short peptide with a glycosyl phosphatidylinositol membrane anchor.

CD24 is a signal-transducing molecule on the surfaces of most human B cells that can modulate their response to activation signals by antagonizing IL-induced differentiation into antibody-forming cells and inducing proliferation in combination with signals generated by Ag receptors. A cDNA that directs the expression of CD24 on the surfaces of transfected COS cells was cloned by its homology to a cDNA encoding the murine M1/69-J11d heat stable Ag. The CD24 cDNA encodes a mature peptide of only 31 to 35 amino acids that is extensively glycosylated and is attached to the outer surface of the plasma membrane by a glycosyl phosphatidylinositol lipid anchor. Although CD24 is structurally similar to M1/69-J11d, and the two Ag appear to have a common genetic ancestry, the homology of CD24 to the M1/69-J11d Ag is confined to a small cluster of amino acids comprising potential N-linked glycosylation sites. Combined with the differences in expression patterns of the human and murine Ag, this indicates that CD24 and M1/69-J11d may not have equivalent functional roles in lymphoid development. The novel structure of CD24 suggests that signaling could be triggered by the binding of a lectin-like ligand to the carbohydrates projecting from the CD24 peptide, and transduced through the release of second messengers derived from the glycosyl phosphatidylinositol membrane anchor of CD24.

Antigens, CD↗
PubMed1991

Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte "homing" receptor expressed by hemopoietic cells.

In addition to the 85-95 kD CD44 species found on most hemopoietic cell types, the human myelomonocytic cell line KG1a expresses proteins of approximately 115 kD and 130 kD that react with monoclonal antibodies belonging to CD44. The possibility that these higher molecular weight species may represent novel CD44 isoforms containing additional protein sequence was investigated. CD44 cDNA clones were isolated from a plasmid-based expression library prepared from KG1a mRNA. One of the three clones obtained (clone 2.3) was found to encode a CD44 molecule of approximately 130 kD in transfected COS cells. Sequences analysis indicated that the molecule encoded by this cDNA clone, designated CD44R1, was essentially identical to CD44 except for the presence of an additional 132 amino acids inserted into the extracellular domain. This inserted region is rich in serine and threonine residues that may serve as sites of O-linked glycosylation, and contains a potential site of N-linked glycosylation and a potential site of chondroitin sulphate attachment. PCR analysis using primers that flank the inserted region present within CD44R1 identified an additional CD44 isoform, designated CD44R2, that contains only the last 69 amino acids present within the unique region of CD44R1. Peripheral blood mononuclear cells and granulocytes from normal individuals and patients with chronic myelogenous leukemia, polycythemia vera, or acute myelomonocytic leukemia, express both CD44R1 and CD44R2. In contrast, CD44R1 and CD44R2 appear to be differentially expressed in various CD44-positive cell lines. Thus KG1a, and the Epstein-Barr Virus-transformed B cell lines WalkDR4 and Way-1 express both CD44 and the CD44 isoforms CD44R1 and CD44R2, while the myeloid cell lines HL60 and U937 express high levels of CD44, but only very low levels of CD44R1 and CD44R2. The CD44-negative cell lines DHL-4, DHL-10, Jurkat, and K562 are also negative for CD44R1 and CD44R2.

Amino Acid Sequence↗
PubMed1991

Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer.

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified, but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved, we performed Northern analyses on RNA extracted from human marrow LTC adherent layers, or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta), IL-6, and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF, GM-CSF, and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast, transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC, and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures, respectively. Together, these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC, with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF, GM-CSF, and IL-6. Given the similarities between the LTC system and the marrow microenvironment, it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells.

Blotting, Northern↗
PubMed1991

Differential and synergistic effects of human granulocyte-macrophage colony-stimulating factor and human granulocyte colony-stimulating factor on hematopoiesis in human long-term marrow cultures.

The ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF to influence hematopoiesis in long-term cultures (LTC) of human marrow was studied by cocultivating light density normal human marrow cells with human marrow fibroblast feeders engineered by retroviral infection to constitutively produce one of these growth factors. Feeders producing stable levels of 4 ng/mL GM-CSF or 20 ng/mL G-CSF doubled the output of mature nonadherent cells. The numbers of both colony forming unit-GM (CFU-GM) and erythroid burst forming unit (BFU-E) in the G-CSF LTC were also increased (twofold and fourfold, respectively, after 5 weeks in culture), but this effect was not seen with the GM-CSF feeders. At the time of the weekly half medium change 3H-thymidine suicide assays showed primitive adherent layer progenitors in LTC to be quiescent in both the control and GM-CSF cultures. In contrast, in the G-CSF cultures, a high proportion of primitive progenitors were in S-phase. A single addition of either recombinant GM-CSF or G-CSF to LTC in doses as high as 80 ng/mL and 150 ng/mL, respectively, failed to induce primitive progenitor cycling. However, three sequential daily additions of 150 ng/mL G-CSF did stimulate primitive progenitors to enter S-phase and a single addition of 5 or 12.5 ng/mL of G-CSF together with 10 ng/mL GM-CSF was able to elicit the same effect. Thus, selective elevation of G-CSF in human LTC stimulates proliferation of primitive clonogenic progenitors, which may then proceed through to the terminal stages of granulopoiesis. In contrast, the effects of GM-CSF in this system appear limited to terminally differentiating granulopoietic cells. However, when both GM-CSF and G-CSF are provided together, otherwise biologically inactive doses show strong stimulatory activity. These findings suggest that the production of both of these growth factors by normal stromal cells may contribute to the support and proliferation of hematopoietic cells, not only in LTC, but also in the microenvironment of the marrow in vivo.

Bone Marrow↗
PubMed1991

Continuous activation of primitive hematopoietic cells in long-term human marrow cultures containing irradiated tumor cells.

Human hematopoietic cells can be maintained in vitro for many weeks in the absence of exogenously provided hematopoietic growth factors if an adequate stromal cell containing adherent layer is present. We have now extended the use of this type of long-term culture (LTC) system to create a model of perturbed hematopoiesis in which human tumor cells that constitutively produce a variety of factors are co-cultured together with normal human marrow cells. In the present study, we used the human bladder carcinoma cell line (5637) because these cells were known to produce not only a variety of factors active directly on hematopoietic cells but also factors that can stimulate hematopoietic growth factor production by human marrow stromal cells. Analysis of mRNA extracted from the adherent layer and measurement of growth factor bioactivity in the medium of established LTC of human marrow containing irradiated 5637 cells, showed increased levels of interleukin-1 and -6, as well as granulocyte and granulocyte-macrophage colony-stimulating factor production by comparison to control cultures. As in normal cultures, high proliferative potential clonogenic hematopoietic cells were found almost exclusively in the adherent layer of these co-cultures, but these primitive cells were maintained in a state of continuous turnover, in contrast to control cultures where the same cell types showed the expected oscillation between a quiescent and a proliferating state following each weekly change of the medium. A similar perturbation of primitive progenitor cycling was achieved by adding medium conditioned by 5637 cells twice a week to otherwise normal LTC. The presence of irradiated 5637 cells in the LTC or the addition of 5637 conditioned medium also resulted in modest (2- to 3-fold) but sustained increases in the total hematopoietic progenitor population, as well as in the final output of terminally differentiated granulocytes and macrophages. These findings indicate that primitive hematopoietic cells in LTC can be kept in a state of continuous activation for many weeks by appropriate endogenous or exogenous hematopoietic growth factor provision and that this does not necessarily lead either to their rapid exhaustion or to a large amplification in output of mature progeny.

Blotting, Northern↗
PubMed1991

Molecular analysis of primitive hematopoietic cell proliferation control mechanisms.

Cells at two distinct early stages in the development of mature human blood cells from primitive totipotent hematopoietic stem cells can now be defined and quantitated by separate in vitro assays. Current evidence suggests that most, if not all, colony-forming cells--that is, cells that give rise to colonies of mature progeny within one to three weeks in semisolid culture systems, represent an intermediate stage of hematopoietic progenitor. These cells are not self-sustaining; if they are used to initiate hematopoiesis on competent marrow stromal layers, they rapidly disappear as they differentiate or die. However, clonogenic cells can be generated in such cultures from another cell type over a period of four to eight weeks. We have, therefore, assigned the term long-term culture initiating cell (LTC-IC) to this latter type of clonogenic precursor cell. The production and differentiation of cells in both of these compartments in LTC are dependent on, and regulated by, nonhematopoietic "stromal" cells that form a heterogeneous adherent layer in which close-range interactions with hematopoietic cells take place. The use of separate endpoints to monitor the maintenance, differentiation, and reversible activation or arrest of cycling of these cells has recently revealed different molecular mechanisms regulating their respective functions. However, an important common feature appears to be the relative local concentration of positive and negative regulators to which the target hematopoietic cell is exposed. Both gene expression and growth factor release measurements as well as results obtained using genetically engineered stroma and repeated soluble growth factor addition implicate G-CSF as an endogenous positive regulator of primitive hematopoietic cells. Similarly, gene expression, factor production, factor addition, and neutralizing antibody experiments implicate TGF-beta as an endogenous inhibitor of primitive hematopoietic cells.

Bone Marrow Cells↗
PubMed1991

Lack of evidence for abnormal autocrine or paracrine mechanisms underlying the uncontrolled proliferation of primitive chronic myeloid leukemia progenitor cells.

Previous studies have revealed a consistent defect in the cycling behavior of primitive neoplastic progenitor cells in patients with Philadelphia chromosome (Ph1)-positive chronic myeloid leukemia (CML). This is manifested both in vivo and in long-term cultures of CML cells as an increased rate of turnover amongst Ph1-positive progenitor cell types whose counterparts in normal individuals are mainly quiescent. To determine whether this deregulated proliferative activity of primitive Ph1-positive cells might be explained by a perturbation in the production of growth factors that regulate the turnover of primitive normal cells, the possibility of either autocrine or paracrine mechanisms of Ph1-positive cell stimulation was investigated. Northern blot analysis of total cellular RNA extracted from various CML blood cell populations showed no evidence of increased expression of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-3, IL-6, or tumor necrosis factor-alpha (TNF-alpha) compared with analogous normal peripheral blood cell populations in which transcripts for most of these growth factors are not detectable. A similar analysis of RNA extracted from the adherent layer of 4-week-old long-term cultures established from CML marrow (in which the Ph1-positive cells typically disappear) or from CML blood seeded onto normal marrow adherent layers (in which Ph1-positive cells typically persist) also revealed no difference in growth factor production compared with analogous cultures established with exclusively normal cells. For some of the growth factors studied, the assessment of bioactivity detectable in the medium confirmed the RNA data. There was also no evidence of a decreased production of putative inhibitors of primitive hematopoietic cells, i.e. transforming growth factor-beta and macrophage inflammatory protein-1 alpha by CML versus normal cells or cultures. These results do not support the existence of BCR-ABL induced autocrine or paracrine mechanisms in CML and suggest that constitutive activation of events normally dependent on growth factor receptor stimulation is more likely to underlie the lack of proliferation control exhibited by primitive Ph1-positive cells.

Bone Marrow↗
PubMed1991

Human hematopoietic stem cells in long-term culture: quantitation and manipulation.

These results suggest a potentially important role for the LTC system, not only in the development of a quantitative assay for hematopoietic cells, but also clinically as a procedure for manipulating autologous marrow in culture in a variety of ways for therapeutic advantage. These include strategies for immune modulation, gene marking and gene therapy, and perhaps eventually for stem cell amplification.

Bone Marrow Transplantation↗
PubMed1990

Detection of breakpoint cluster region- negative and nonclonal hematopoiesis in vitro and in vivo after transplantation of cells selected in cultures of chronic myeloid leukemia marrow.

Philadelphia (Ph1) chromosome-positive clonogenic progenitors usually disappear within 4 to 6 weeks in long-term cultures established from the marrow of patients with chronic myeloid leukemia (CML). In contrast, coexisting chromosomally normal hematopoietic cells are relatively well maintained. Thus, even though normal cells are initially undetectable, they may become the predominant population. Recently, we have begun to explore the potential of such cultures as a strategy for preparing CML marrow for autografting, and based on cytogenetic studies of the differential kinetics of Ph1-positive and Ph1-negative clonogenic cells, have chosen a 10-day period in culture to obtain maximal numbers of selectively enriched normal stem cells. Here we present the results of molecular analyses of the cells regenerated in vivo for the initial three CML patients to be treated using this approach by comparison with the differentiated cells generated by continued maintenance of an aliquot of the autograft in vitro (using a slightly modified culture feeding procedure to enhance the production and release of cells into the nonadherent fraction after 4 weeks) for the one patient whose genotype made molecular analysis of clonality status also possible. These analyses showed that cells with a rearranged breakpoint cluster region (BCR) gene were not detectable by Southern blotting in either in vitro or in vivo populations of mature cells that might be assumed to represent the progeny of primitive cells present at the end of the initial 10 days in culture. Production of BCR-negative cells was also shown to be temporally correlated with the appearance of nonclonal hematopoietic cells both in culture and in vivo. These findings provide support for the view that prolonged maintenance of CML marrow cells in long-term culture may allow molecular characterization of both the BCR-genotype and clonality status of cells with in vivo regenerative potential.

Bone Marrow↗
PubMed1990

Expression cloning of a cDNA encoding M1/69-J11d heat-stable antigens.

The differentiation Ag identified by the mAb M1/69 and J11d (commonly referred to as heat-stable Ag) are found in structurally heterogeneous forms on the surfaces of many types of murine hemopoietic cells. The extinction of expression of these antigens is associated with thymocyte maturation and Ig class switching in B cells, as well as terminal differentiation of macrophages. A cDNA encoding the M1/69-J11d peptide was cloned from a hemopoietic progenitor cell line by immunoselection of COS cells transfected with expression libraries. The cloned cDNA is a copy of a gene that is transcribed in M1/69-J11d+ lymphoid, myeloid, and erythroid cells. This gene could be responsible for the expression of all forms of the M1/69-J11d Ag, although there are homologous genes that may encode some forms of the Ag that are specifically expressed in bone marrow. The cloned cDNA encodes a surprisingly small peptide, predicted to contain only 30 amino acids after removal of a signal sequence and displacement of the C-terminal region by the glycosyl-phosphatidylinositol group that anchors the peptide to the cell surface. Almost all of the mass of the M1/69-J11d Ag accumulates through extensive N- and O-linked glycosylation at multiple sites in the short peptide. These carbohydrates are likely to execute the functions of M1/69-J11d Ag, which could be specialized to each cell type as a consequence of differential glycosylation.

Amino Acid Sequence↗
PubMed1990

Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells.

A large number of biologic, technological, and clinical studies await the development of procedures that will allow totipotent hematopoietic stem cells to be expanded in vitro. Previous work has suggested that hematopoiesis can be reconstituted using transplants of cells from long-term marrow cultures. We have used retrovirus mediated gene transfer to demonstrate that marked totipotent hematopoietic stem cells are both maintained and can be amplified in such cultures, and then subsequently regenerate and sustain lympho-myeloid hematopoiesis in irradiated recipients. Marrow cells from 5-fluorouracil-treated male mice were infected with a recombinant virus carrying the neomycin resistence gene and seeded onto irradiated adherent layers of pre-established, long-term marrow cultures of female origin. At 4 weeks, cells from individual cultures were transplanted into single or multiple female recipients. Southern blot analysis of hematopoietic tissue 45 days posttransplantation showed retrovirally marked clones common to lymphoid and myeloid tissues in 14 of 23 mice examined. Strikingly, for 3 of 4 long-term cultures, multiple recipients of cells from a single flask showed marrow and thymus repopulation with the same unique retrovirally marked clone. These results establish the feasibility of retroviral-marking techniques to demonstrate the maintenance of totipotent lympho-myeloid stem cells for at least 4 weeks in the long-term marrow culture system and provide the first evidence of their proliferation in vitro. Therefore, such cultures may serve as a starting point for identifying factors that stimulate totipotent hematopoietic stem cell expansion.

Animals↗
PubMed1990

Detection and isolation of the erythropoietin receptor using biotinylated erythropoietin.

Procedures have been developed to label human erythropoietin (Ep) with biotin to detect and isolate the Ep-receptor. The labeling method used the abundant carbohydrate groups on Ep and resulted in biologically active biotin-Ep (b-Ep) containing 8 to 10 biotins per Ep molecule. Specific binding of b-Ep to cells from spleens of mice made anemic by phenylhydrazine injections was demonstrated using 125I-labeled streptavidin. B-Ep, together with fluorescently tagged streptavidin, was found to specifically detect Ep-receptor-bearing cells by flow cytometry. This was demonstrated in several ways. First, approximately 90% of nucleated spleen cells from phenylhydrazine-treated mice were clearly fluorescent after staining with b-Ep and streptavidin-phycoerythrin, whereas only background fluorescence was detected using spleen cells from untreated mice. In addition, Ep-receptors were detected on 5% to 10% of normal mouse bone marrow cells, and these cells could be identified as erythroid in nature by separating the cells into subpopulations based on light-scatter properties. Third, Ep-receptor expression was found to correlate positively with expression of transferrin receptors, confirming the erythroid nature of these cells. B-Ep was also used to isolate Ep-receptors from monkey COS cells transfected with the murine Ep-receptor cDNA. In these experiments a cell-surface-bound protein of approximately 65 Kd and an intracellular protein of approximately 60 Kd were isolated from these cells. The procedures described in this report for detecting Ep-receptor expressing cells and for isolating the Ep-receptor should be valuable for purifying erythroid cells from heterogeneous cell populations, for elucidating the structure of the Ep-receptor, and for studying the biological activities of Ep at the cellular and molecular level.

Animals↗
PubMed1990

Heterogeneous modifications of the 114/A10 protein of interleukin-3-dependent cells are concentrated in a highly repetitive amino-terminal domain.

The protein 114/A10 is expressed in structurally heterogeneous forms on the surfaces of all murine hemopoietic cells that are responsive to the growth factor interleukin-3, including multipotent progenitors. Despite their structural diversity, all forms of 114/A10 appear to be expressed from a single gene that encodes a peptide with a potential transmembrane segment, three sequences with homology to epidermal growth factor, and an N-terminal domain consisting of eight perfect or near perfect tandem repeats of a 27-amino acid peptide that has a very high content of serine and threonine. Constructed cDNAs that encode deleted or hybrid forms of the 114/A10 protein have been expressed on COS cells in order to localize sites of post-translational modification. The results demonstrate that the structural diversity of 114/A10 is confined to its N-terminal repeat domain and probably arises through extensive O-linked glycosylation within each of the repeats in that domain. The highly modified N terminus of 114/A10 projects from the outer surface of the cell and could serve as a ligand for lectin-like proteins or could modulate the activities of the adjacent epidermal growth factor-like domains of 114/A10.

Amino Acid Sequence↗
PubMed1990

Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy.

Although hematopoiesis is known to originate in a population of very primitive cells with both lymphopoietic and myelopoietic potential, a procedure for enumerating such cells has to date not been available. We now describe a quantitative assay for long-term repopulating stem cells with the potential for reconstituting all hematopoietic lineages. This assay has two key features. The first is the use of competitive repopulation conditions that ensure not only the detection of a very primitive class of hematopoietic stem cells but also the survival of lethally irradiated mice transplanted with very low numbers of such cells. The second is the use of a limiting-dilution experimental design to allow stem cell quantitation. The assay involves transplanting limiting numbers of male "test" cells into lethally irradiated syngeneic female recipients together with 1-2 x 10(5) syngeneic female marrow cells whose long-term repopulating ability has been compromised by two previous cycles of marrow transplantation. The proportion of assay recipients whose regenerated hematopoietic tissues are determined to contain greater than or equal to 5% cells of test cell origin (male) greater than or equal to 5 weeks later is then used to calculate the frequency of competitive repopulating units (CRU) in the original male test cell suspension (based on Poisson statistics). Investigation of this assay system has shown that all three potential sources of stem cells (test cells, compromised cells, and the host) can under appropriate circumstances contribute to long-term hematopoietic regeneration, thus establishing both the competitive pressure of hematopoietic stem cells in the cotransplanted compromised population and in the host, and the need to use genetic markers to track the specific contribution of the injected test cells. Analysis of the frequency of CRU in test marrow suspensions that varied widely in their CRU content gave similar values when endpoints of either 5 or 10 weeks posttransplantation were used and when either recipient marrow or thymus was used to identify progeny populations. In addition, repopulation of marrow and thymus was found to be associated in most mice injected with limiting numbers of test cells. These findings are consistent with the conclusion that the assay is highly selective for a very primitive, totipotent, reconstituting hematopoietic stem cell and should therefore be particularly useful in future gene therapy-oriented research as well as for more basic studies of hematopoietic stem cell regulation and differentiation.

Animals↗
PubMed1990

An 80,000-kd glycoprotein cell surface antigen found only on nonhematopoietic cells in human bone marrow.

The murine IgG2a monoclonal antibody (MoAb) 6-19 binds to the surface of human nonhematopoietic cells but not to hematopoietic cells. As previously described, it can be used with complement to selectively kill nonhematopoietic cells prior to culture of human bone marrow. It is now shown that the 6-19 MoAb recognizes a specific antigenic determinant of apparent molecular mass 80 kd, detected by western blotting on nonhematopoietic tumor cell lines, endothelial cells, and bone marrow stromal cells. Mouse L cells were transfected with human DNA, and the 6-19 monoclonal antibody selected cells that expressed an antigen with similar characteristics. The antigenic determinant is absent in hematopoietic tumor cell lines, peripheral blood cells, and bone marrow hematopoietic cells. Investigation of the biochemical nature of the antigen, using various enzymes, lectin-binding studies, and western blotting suggests that the 6-19 epitope is at least partially carbohydrate and that the protein has N-linked sugars but not O-linked. The affinity (Ka approximately 10(9) M-1) of MoAb 6-19 binding is similar in tumor cells and normal fibroblasts and endothelial cells. The identification of a specific antigenic determinant of 80 kd may help to discriminate between hematopoietic and nonhematopoietic cells in human bone marrow.

Animals↗