Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia.

Familial platelet disorder with predisposition to acute myelogenous leukaemia (FPD/AML, MIM 601399) is an autosomal dominant disorder characterized by qualitative and quantitative platelet defects, and propensity to develop acute myelogenous leukaemia (AML). Informative recombination events in 6 FPD/AML pedigrees with evidence of linkage to markers on chromosome 21q identified an 880-kb interval containing the disease gene. Mutational analysis of regional candidate genes showed nonsense mutations or intragenic deletion of one allele of the haematopoietic transcription factor CBFA2 (formerly AML1) that co-segregated with the disease in four FPD/AML pedigrees. We identified heterozygous CBFA2 missense mutations that co-segregated with the disease in the remaining two FPD/AML pedigrees at phylogenetically conserved amino acids R166 and R201, respectively. Analysis of bone marrow or peripheral blood cells from affected FPD/AML individuals showed a decrement in megakaryocyte colony formation, demonstrating that CBFA2 dosage affects megakaryopoiesis. Our findings support a model for FPD/AML in which haploinsufficiency of CBFA2 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia.

Growth factor-dependent inhibition of normal hematopoiesis by N-ras antisense oligodeoxynucleotides.

To determine whether N-ras expression is required at specific stages of the process of in vitro normal human hematopoiesis, adherent- and T lymphocyte-depleted mononuclear marrow cells (A-T-MNC) or highly purified progenitors (CD34+ cells) were cultured in semisolid medium, under conditions that favor the growth of specific progenitor cell types, after exposure to N-ras sense and antisense oligodeoxynucleotides. N-ras antisense, but not sense, oligodeoxynucleotide treatment of A-T-MNC and CD34+ cells resulted in a significantly decreased number of granulocyte/macrophage colony-forming units (CFU-GM) induced by interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and of macrophage colonies (CFU-M) induced by M-CSF, but not of granulocytic colonies induced with G-CSF or IL-5. However, the same treatment significantly inhibited colony formation induced by each of the above factors in combination with IL-3. Megakaryocytic colony (CFU-Meg) formation from A-T-MNC or CD34+ cells in the presence of IL-6 + IL-3 + erythropoietin (Epo) was also markedly decreased after antisense oligodeoxynucleotide treatment. Erythroid colonies derived from A-T-MNC in the presence of Epo (CFU-E) were not inhibited upon antisense treatment, whereas those arising from A-T-MNC or CD34+ cells in the presence of IL-3 + Epo (BFU-E) were markedly affected. These results are consistent with the hypothesis that distinct signal transduction pathways, involving N-ras or not, are activated by different growth factors in different hematopoietic progenitor cells.

A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis in vitro.

The nuclear protein encoded by the proto-oncogene c-myb has been hypothesized to play an important role in the process of hematopoiesis, but direct proof of this function has been lacking. To address this issue, normal human bone marrow mononuclear cells were exposed to c-myb sense and antisense synthetic oligodeoxynucleotides, and the effects on hematopoietic colony formation and maturation were examined. Exposure of these cells to c-myb antisense, oligodeoxynucleotides resulted in a decrease in both colony size and number, without apparent effect on the maturation of residual colony cells. Exposure to c-myb sense, or irrelevant antisense, oligonucleotides had no such effect. These results show that (i) c-myb plays a critical role in regulating normal human hematopoiesis and (ii) the combined use of antisense oligodeoxynucleotides and hematopoietic cell culture techniques will provide a powerful tool for studying the role of proteins encoded by proto-oncogenes, or other specific genes, in normal human hematopoiesis.

G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb.

Exposure of peripheral blood mononuclear cells (PBMC) to an 18-base c-myb antisense oligomer before mitogen or antigen stimulation resulted in almost complete inhibition of c-myb messenger RNA and protein synthesis and blockade of T lymphocyte proliferation. Expression of early and late activation markers, interleukin-2 receptor and transferrin receptor, respectively, by PBMC was unaffected by antisense oligomer exposure as was the expression of c-myc messenger RNA. In contrast, histone H3 messenger RNA levels and DNA content were selectively decreased. These results suggest that c-myb protein deprivation does not perturb T lymphocyte activation or early molecular events that may prepare the cell for subsequent proliferation. Rather, it appears to specifically block cells in late G1 or early S phase of the cell cycle.

Lineage-specific requirement of c-abl function in normal hematopoiesis.

Structural abnormalities of the c-abl proto-oncogene are found in hematopoietic cells of more than 90 percent of individuals with chronic myelogenous leukemia. Therefore c-abl may be important in normal as well as malignant hematopoiesis. Normal human hematopoietic progenitor cells were exposed to three different c-abl sense or antisense oligodeoxynucleotides, and the effects on myeloid and erythroid colony formation were examined. The c-abl antisense oligodeoxynucleotides inhibited myeloid, but not erythroid, colony formation. The c-abl sense oligodeoxynucleotides and bcr sense and antisense oligodeoxynucleotides were not inhibitory in this assay. These data show that c-abl is critical in normal myelopoiesis and may explain the relatively selective expansion of leukocytes in patients with chronic myelogenous leukemia.

Selective inhibition of leukemia cell proliferation by BCR-ABL antisense oligodeoxynucleotides.

To determine the role of the BCR-ABL gene in the proliferation of blast cells of patients with chronic myelogenous leukemia, leukemia blast cells were exposed to synthetic 18-mer oligodeoxynucleotides complementary to two identified BCR-ABL junctions. Leukemia colony formation was suppressed, whereas granulocyte-macrophage colony formation from normal marrow progenitors was unaffected. When equal proportions of normal marrow progenitors and blast cells were mixed, exposed to the oligodeoxynucleotides, and assayed for residual colony formation, the majority of residual cells were normal. These findings demonstrate the requirement for a functional BCR-ABL gene in maintaining the leukemic phenotype and the feasibility of gene-targeted selective killing of neoplastic cells.

2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) modified oligonucleotides (ON) effect highly efficient, and persistent, gene silencing.

To be effective in vivo, antisense oligonucleotides (AS ON) should be nuclease resistant, form stable ON/RNA duplexes and support ribonuclease H mediated heteroduplex cleavage, all with negligible non-specific effects on cell function. We report herein that AS ONs containing a 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) sugar modification not only meet these criteria, but have the added advantage of maintaining high intracellular concentrations for prolonged periods of time which appears to promote longer term gene silencing. To demonstrate this, we targeted the c-MYB protooncogene's mRNA in human leukemia cells with fully phosphorothioated 2'F-ANA-DNA chimeras (PS-2'FANA-DNA) and compared their gene silencing efficiency with AS ON containing unmodified nucleosides (PS-DNA). When delivered by nucleofection, chemically modified ON of both types effected a >90% knockdown of c-MYB mRNA and protein expression, but the PS-2'F-ANA-DNA were able to accomplish this at 20% of the dose of the PS-DNA, and in contrast to the PS-AS DNA, their silencing effect was still present after 4 days after a single administration. Therefore, our data demonstrate that PS-2'F-ANA-DNA chimeras are efficient gene silencing molecules, and suggest that they could have significant therapeutic potential.

Stage-related proliferative activity determines c-myb functional requirements during normal human hematopoiesis.

To determine if MYB protein is preferentially required during specific stages of normal human hematopoiesis we incubated normal marrow mononuclear cells (MNC) with c-myb antisense oligodeoxynucleotides. Treated cells were cultured in semisolid medium under conditions designed to favor the growth of specific progenitor cell types. Compared with untreated controls, granulocyte-macrophage (GM) CFU-derived colonies decreased 77% when driven by recombinant human (rH) IL-3, and 85% when stimulated by rH GM colony-stimulating factor (CSF); erythroid burst-forming unit (BFU-E)- and CFU-E-derived colonies decreased 48 and 78%, respectively. In contrast, numbers of G-CSF-stimulated granulocyte colonies derived from antisense treated MNC were unchanged from controls, though the numbers of cells composing these colonies decreased approximately 90%. Similar results were obtained when MY10+ cells were exposed to c-myb antisense oligomers. When compared with untreated controls, numbers of CFU-GM and BFU-E colonies derived from MY10+ cells were unchanged, but the numbers of cells composing these colonies were reduced approximately 75 and greater than 90%, respectively, in comparison with controls. c-myc sense and antisense oligomers were without significant effect in these assays. Using the reverse transcription-polymerase chain reaction, c-myb mRNA was detected in developing hematopoietic cells on days 0-8. At day 14 c-myb expression was no longer detectable using this technique. These results suggest that c-myb is required for proliferation of intermediate-late myeloid and erythroid progenitors, but is less important for lineage commitment and early progenitor cell amplification.

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis.

Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.

An enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques. Chemical cross-linking studies with a biotin-labeled P-ODN (bP-ODN), revealed the existence of five major cell surface P-ODN binding protein groups ranging in size from approximately 20-143 kD. Binding to these proteins was competitively inhibited with unlabeled P-ODN, but not free biotin, suggesting specificity of the interactions. Additional experiments suggested that binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding. Uptake studies with 35S-labeled P-ODN revealed that endocytosis, mediated by a receptor-like mechanism, predominated at P-ODN concentrations < 1 microM, whereas fluid-phase endocytosis prevailed at higher concentrations. Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes. Labeled ODN were also found in significant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER). Since nuclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import process is suggested. These data imply that antisense DNA may exert their effect in the nucleus. They also suggest rational ways to design ODN which might increase their efficiency.

A reappraisal of the role of insulin-like growth factor I in the regulation of human hematopoiesis.

IGF-I has been reported to increase hematopoietic progenitor cell cloning efficiency. To investigate this phenomenon, we studied the IGF-I responsiveness of human marrow cells expressing IGF-I receptor (IGF-IR), a direct strategy not used previously. IGF-IR+ and control CD34+ marrow cells were isolated using immunoaffinity methods. Then, the cells were cloned in methylcellulose containing variable amounts of serum- and lineage-appropriate growth factors supplemented with recombinant human IGF-I. In contrast to CD34+ cells, IGF-IR+ cells never gave rise to CFU-Blast, CFU-Mix, CFU-GM, BFU-E, or CFU-E. To substantiate the suggestion that CD34+ and IGF-IR+ cells were distinct populations, we used reverse transcription PCR to detect IGF-I, EpO, and KIT receptor mRNAs in these cells. The mRNA phenotype of CD34+ cells was EpO (+), KIT (+), and IGF-IR (-), while IGF-IR+ cells were IGF-IR (+), EpO (-), and KIT (-). These results suggested that IGF-IR is either not expressed or expressed at low levels on normal hematopoietic progenitor cells. Functional significance of the latter possibility was tested by exposing CD34+ cells to IGF-IR antisense oligodeoxynucleotides. Colony formation was unaffected by oligodeoxynucleotide disruption of IGF-IR, suggesting that, even if expressed at low level, the receptor's functional significance was doubtful. Nevertheless, when cultured in the presence of IGF-I, IGF-IR+ cells elaborated an activity with mild BFU-E stimulatory effects. Accordingly, if IGF-I plays a role in hematopoietic colony formation, it is probably and results from stimulation of IGF-IR-positive ancillary cells to secrete growth factors. Studies carried out with human leukemia cells yielded similar results.

Progress in the development of nucleic acid therapeutics.

Abnormal gene expression is a hallmark of many diseases. Gene-specific downregulation of aberrant genes could be useful therapeutically and potentially less toxic than conventional therapies due its specificity. Over the years, many strategies have been proposed for silencing gene expression in a gene-specific manner. Three major approaches are antisense oligonucleotides (AS-ONs), ribozymes/DNAzymes, and RNA interference (RNAi). In this brief review, we will discuss the successes and shortcomings of these three gene-silencing methods, and the approaches being taken to improve the effectiveness of antisense molecules. We will also provide an overview of some of the clinical applications of antisense therapy.

Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules.

Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression. The ODNs were targeted to two different sites within the c-myb mRNA. One site was chosen arbitrarily. The other was a 'rational' choice based on predicted hybridization accessibility after physical mapping with self-quenching reporter molecules (SQRM). The Myb mRNA and protein levels were equally diminished by OXE and PS ODNs, but the latter were delivered to cells with approximately six times greater efficiency, suggesting that OXE modified ODNs were more potent on a molar basis. The rationally targeted molecules demonstrated greater silencing efficiency than those directed to an arbitrarily chosen mRNA sequence. We conclude that rationally targeted, OXE modified ODNs, can function efficiently as gene silencing agents, and hypothesize that they will prove useful for therapeutic purposes.

Myb targeted therapeutics for the treatment of human malignancies.

For the past several years, we have been engaged in developing a therapeutically effective strategy for disrupting gene function with reverse complementary, or so called 'antisense', oligodeoxynucleotides (ODN). This pursuit has focused on finding appropriate diseases in which to apply this approach, and suitable gene targets. Of the genes that we have targeted for disruption using the antisense ODN strategy (Clevenger et al., 1995; Gewirtz and Calabretta, 1988; Ratajczak et al., 1992c; Small et al., 1994) one that has been of particular interest, and one where therapeutically motivated disruptions are now in clinical trial, is the myb gene (reviewed in Lyon et al., 1994). These trials involve treatment of human leukemias. These diseases are a logical choice for developing oncogene targeted therapies because of easy access to tissues, and the abundance of knowledge about the cell and molecular biology of these diseases. Nevertheless, as will be touched on below, other malignancies have also been examined as models for Myb targeted therapy with some surprisingly encouraging results. Finally, while we have focused our efforts on the ODN strategy, I will allude briefly to other strategies for disrupting Myb function with therapeutic intent.

Oligonucleotide therapeutics for hematologic disorders.

During the last decade, the catalogue of known genes responsible for cell growth, development, and neoplastic transformation has expanded dramatically. Attempts to translate this information into new therapeutic strategies for both hematologic and non-hematologic diseases have accelerated at a rapid pace as well. Inserting genes into cells which either replace, or counter the effects of disease causing genes has been one of the primary ways in which scientists have tried to exploit this new knowledge. Strategies to directly downregulate gene expression have developed in parallel with this approach. The latter include triple helix forming oligonucleotides (ODN) and 'antisense' ODN. The latter have already entered clinical trials for a variety of disorders. In this monograph, we review the use of these materials in the treatment of hematologic diseases, particularly myelogenous leukemias. Problems and possible solutions associated with the use of ODN will be discussed as well.

Rationally targeted, conformationally constrained, oxetane-modified oligonucleotides demonstrate efficient gene-silencing activity in a cellular system.

Antisense oligodeoxynucleotides (AS ODN) have been employed as gene-silencing agents in the laboratory and, in the clinic. The in vivo use of these molecules has been facilitated by chemical modifications to the DNA backbone which augment their nuclease stability. Attempts to further improve the efficacy of AS ODN have largely focused on 2' alterations of the ribose sugar that make the molecules more RNA like in structure. This increases the T(m) of formed DNA/RNA hybrids but simultaneously prevents binding of RNaseH which is important for ODN effectiveness. Herein, we demonstrate the use of AS ODN containing nucleosides with a novel oxetane (OXE) modification [oxetane, 1-(1', 3'-O-anhydro-beta-D-psicofuranosyl nucleosides)] which augments Tm, enhances nuclease stability, and is permissive of RNaseH activation. We also illustrate herein the value of rational targeting of OXE modified, and by analogy, AS ODN of any chemical modification.

CD34+, kit+, rhodamine123(low) phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells.

We hypothesized that human hematopoietic cells displaying a CD34+, kit-, rhodamine123(low) phenotype would be highly enriched for cells with stem-like properties. To test this hypothesis, we employed fluorescence activated cell sorting (FACS) to isolate cells with this phenotype from normal light density marrow mononuclear cells (MNC). CD34+, kit+, rhodamine123(low) cells comprised from 0.05-0.01% of the total MNC population. They were small, had scant cytoplasm, and contained nuclei with dense, hyperchromatic chromatin and inconspicuous nucleoli. Additional immunophenotyping revealed that these cells were CD33-, CD38-, CD20-, and glycophorin A-. When plated in semisolid cultures containing optimal concentrations of IL-3, GM-CSF, KL, EPO, IL-6, and IL-1 these cells did not form colonies. However, when cultured over irradiated stromal cells, cobblestone areas were observed to form after 3 weeks, and harvested cells were able to initiate long-term cultures. To further demonstrate that these cells were indeed stem like, we also tested their ability to engraft and mature in immunocompromised (SCID) mice. Irradiated (400 cGy) SCID mice were transplanted with 2 x 10(3) candidate stem cells which were then injected with recombinant human growth factors every other day. Two months post-transplant the animals were sacrificed. PCR and FACS analysis of marrow and spleen cell samples revealed the presence of cells expressing human CD45 consistent with engraftment of human stem cells and the establishment of murine-human chimerism. Moreover, MNC isolated from transplanted mice formed unambiguously human BFU-E, CFU-GM and B cell colonies when stimulated with the appropriate growth factors. Accordingly, we have identified a relatively rapid and simple mechanism for isolating primitive human hematopoietic cells with stem cell-like properties. We anticipate that this strategy will be useful for experimental and therapeutic applications that require human stem cells in quantity.

Effect of phorbol myristate acetate on c-myc, beta-actin, and FV gene expression in morphologically recognizable human megakaryocytes: a kinetic analysis employing in situ hybridization.

Phorbol esters are reported to modulate numerous megakaryocyte maturation maturation parameters, but the effects of these compounds on normal human megakaryocyte gene expression are unknown. We therefore determined if phorbol myristate acetate (PMA)-induced changes in megakaryocyte gene expression could be detected and semi-quantified in single cells using the techniques of in situ hybridization and photodensitometry. Megakaryocytes were isolated from normal human bone marrow by counterflow centrifugal elutriation. After 2, 15, and 24 h of culture in 8 nM PMA, megakaryocytes were probed by in situ hybridization with biotin-11-dUTP-labeled cDNA probes for human c-myc, beta-actin, and coagulation cofactor V (FV) mRNA. Hybridization was detected by an enzyme-catalyzed colorimetric reaction that was photometrically quantified as an inverse function of light transmission through labeled cells. In control megakaryocytes, steady state levels of c-myc and FV mRNA did not change over the 24-h observation period, whereas that of beta-actin appeared to increase slightly. In contrast, after 2 h of exposure to 8 nM PMA, a statistically significant increase (p less than 0.001) in c-myc and beta-actin mRNA expression was observed, whereas FV mRNA expression appeared to be unchanged (p = 0.207). These inductions were transient, however, because by 15 or 24 h, beta-actin and c-myc expression levels, respectively, no longer exceeded those measured in untreated controls. However, in the presence of higher PMA doses (160 nM), beta-actin mRNA levels remained elevated at 24 h. The relationship between megakaryocyte maturation and apparent level of beta-actin mRNA expression was also examined. Although cell size and stage, and size and beta-actin mRNA levels showed a modest relationship, mRNA levels and morphologic maturation stage were poorly correlated. These results demonstrate that PMA has complex effects on gene expression in morphologically recognizable human megakaryocytes. Of equal importance, they also demonstrate that in situ hybridization can be employed as a useful tool for studying human megakaryocyte cell biology at the molecular level.

Occurrence of platelet basic protein, a precursor of low affinity platelet factor 4 and beta-thromboglobulin, in human platelets and megakaryocytes.

beta-thromboglobulin antigen, a platelet-specific secreted protein, occurs in three forms: platelet basic protein, low affinity platelet factor 4, and beta-thromboglobulin. The combined level of beta-thromboglobulin antigen in megakaryocytes, measured by radioimmunoassay, was 13 +/- 7 micrograms/10(6) cells (SD, n = 6). The relative proportions of the three forms of beta-thromboglobulin antigen present within platelets and megakaryocytes were determined in cells lysed with trichloroacetic acid to minimize artifactual proteolysis. Samples were analyzed by isoelectric focusing in polyacrylamide gel with quantitative immunological detection on a nitrocellulose transfer of the gel. In platelets, the major species found was low affinity platelet factor 4 with precursor platelet basic protein as only 25% +/- 11% (SD, n = 16) of total beta-thromboglobulin antigen. In megakaryocytes partially purified both from normal bone marrow aspirates and from whole marrow specimens obtained after surgery, platelet basic protein was a higher proportion of beta-thromboglobulin antigen (49% +/- 13% SD, n = 11) than was the case in platelets. beta-thromboglobulin itself was never detected under the conditions of cell lysis used. Our results suggest that platelet basic protein is synthesized in megakaryocytes and that its cleavage is associated with an earlier stage of cell development than simply maturation to platelets. Further support for the precursor status of platelet basic protein was found in the expression of predominantly this antigenic form in a human erythroleukemia cell line.

In vitro sensitivity of human hematopoietic progenitor cells to 4-hydroperoxycyclophosphamide.

Protracted engraftment and prolonged thrombocytopenia remain problems when marrow purged with 4-hydroperoxy-cyclophosphamide (4-HC) is employed for autotransplants. Toxic effects of 4-HC on early progenitor and stem cells might play an important causative role. Surprisingly, few investigations have examined the effects of 4-HC on progenitor cells other than colony-forming units-granulocyte/macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E), and only one study could be found where 4-HC exposure was carried out on cells purified beyond the buffy coat stage. Since the cellular milieu, and in particular the red blood cell count, can ameliorate 4-HC toxicity, the suppressive effect of this agent on marrow stem cells might be underestimated. We therefore investigated the relative 4-HC sensitivity of different human bone marrow progenitor cells in vitro using partially purified adherent cell- and T lymphocyte-depleted bone marrow mononuclear cells (A-T-MNC). Cells were exposed to increasing doses (10 to 200 micrograms/mL) of 4-HC using a standard purging protocol established for bone marrow buffy coat cells. Effects on mixed (CFU-Mix), erythroid (CFU-E and BFU-E), granulocyte-macrophage (CFU-GM), and stromal cell (CFU-F) progenitors were determined. In addition, we examined the 4-HC sensitivity of megakaryocyte progenitors (CFU-Meg) since, to our knowledge, this has not been reported before. As expected, increasing doses of 4-HC led to progressive inhibition of hematopoietic colony formation. CFU-F, CFU-Mix, and CFU-Meg appeared most resistant to 4-HC exposure, while CFU-E, BFU-E, and CFU-GM appeared most sensitive. At doses over 100 micrograms/mL, the usual concentration recommended for purging of buffy coat cells, hematopoietic colony formation was completely inhibited. These data suggest that if more highly purified marrow cells are employed for purging, lower 4-HC doses may need to be used. They also suggest that the thrombocytopenia that frequently accompanies 4-HC purging is not likely due to loss of CFU-Meg.