Isolation and purification of a substance immunologically cross-reactive with insulin (SICRI) from tumor tissue

1. A substance immunologically cross-reactive with insulin (SICRI) was isolated and purified from murine melanoma B16 and myeloid leukemia. 2. Monospecific anti-insulin immunoglobulin G was coupled with CNBr-activated Sepharose 4B to yield an adsorbent. The immunoaffinity column was used to isolate SICRI from tumor tissue. 3. Purified SICRI yielded a single band with mol. wt 158,000 on SDS-PAGE. After non-denaturing conditions SICRI appeared again in one single peak. 4. Affinity purified SICRI was shown to be a potent growth factor for different human and murine transformed and normal cells. 5. Biochemical and biological data provide evidence that SICRI and insulin are two distinct biologically active agents.     

Growth factors and proto-oncogenes in early mouse embryogenesis and tumorigenesis.

Growth factors and proto-oncogenes play an important role in the regulation of embryonic growth and differentiation as well as in tumorigenesis. Insulin and insulin-like growth factor I (IGF I) are secreted by embryonic tissues during the prepancreatic stage of mouse development. Measureable amounts of these factors were found in 8- to 12-day-old embryos. Embryonic cells derived from 8- to 10-day-old embryos secrete insulin and IGF I in serum-free medium. Relatively high levels of c-myc, c-fos and c-H-ras oncoproteins were also detected in 8- to 12-day-old embryos. Insulin and IGF I, when added to the culture of embryonic cells, stimulate their proliferation. Similar results were obtained in some animal or human tumors. Murine myeloid leukemias and melanoma B 16 secrete a substance immunologically cross reactive with insulin (SICRI) both in vivo and in serum-free media. In culture, the DNA synthesis rate per leukemic or melanoma cell is proportional to cell density and is reduced by antiinsulin serum in case of leukemic cells. Human hemangiosarcoma secrete IGF I, which also plays a role as an autocrine factor. Purified IGF I efficiently induce c-myc and c-fos mRNA, which is among the earliest events following growth factor stimulation, leading to mitosis. These results lead us to the conclusion that IGF I and insulin together with oncoproteins stimulate the growth of embryonic and tumor cells, which is indirect evidence for a paracrine (or autocrine) type of action.     

Purification of a murine leukemia inhibitory factor from Krebs ascites cells

A factor capable of inducing terminal differentiation in the murine myeloid leukemia cell line M1 has been purified to apparent homogeneity from the medium conditioned by Krebs II ascites tumor cells. The factor, termed leukemia inhibitory factor (LIF) is a single chain glycoprotein of apparent Mr 58,000 which induces differentiation and inhibits proliferation of the M1 cell line but not the WEHI-3B D+ murine myeloid leukemic cell line and has no detectable proliferative activity on normal myeloid progenitor cells. It was purified using four successive high-efficiency purification steps--anion-exchange chromatography on DEAE-Sepharose; cation-exchange chromatography on CM-Sepharose; affinity chromatography on lentil lectin-Sepharose; and reverse-phase high-performance liquid chromatography on a phenyl-silica matrix--to a specific biological activity of approximately 1.25 X 10(8) units/mg with an overall purification of 12,000-fold and a yield of 73% for the activity failing to bind to DEAE-Sepharose. Sufficient quantities of the factor (12 micrograms, 200 pmol) have been purified to allow structural and functional analysis of the molecule and comparison with other know differentiation inducers.     

Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.     

Role of pescadillo and upstream binding factor in the proliferation and differentiation of murine myeloid cells

Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.     

DMSO-induced terminal differentiation and trisomy 15 in myeloid cell line transformed by the Rauscher murine leukemia virus

A new myeloid cell line was isolated from a myeloid leukemia obtained after infection of BALB/c mice with Rauscher murine leukemia virus (R-MuLV). After syngeneic transplantation of leukemic cells tumor formation was induced. Of one of these tumors a permanent cell line could be established. The cells grow in suspension culture with a doubling time of 18 h and morphologically and cytochemically show all the characteristics of myelocytes. The cells carry trisomy of chromosome 15. These cells prove to be completely independent of colony stimulating activity (CSA) regarding both their growth and their differentiation capacity.One of the main characteristics of this cell line is its inducibility for terminal differentiation after treatment with dimethylsulfoxide varying in concentrations from 0.5% to 1.5%. After two days metamyelocytes and after three to four days granulocytes and macrophages formed. The differentiation of these cells goes together with an increase of lysosomal enzyme activities like β-N-acetylglucosaminidase and lysozyme.     

鼠粒细胞集落刺激因子受体的纯化及鉴定

粒细胞集落刺激因子受体(G-CSFR)在鼠NFS-60细胞中有较高的含量,通过对NFS-60细胞的大规模培养,用CHAPS及超速离心抽提G-CSFR, 经G-CSF亲和层析纯化获得G-CSFR, 采用ABC-ELISA进行鉴定.     

Protein kinase C-mediated phosphorylation of the leukemia-associated HOXA9 protein impairs its DNA binding ability and induces myeloid differentiation

HOXA9 expression is a common feature of acute myeloid leukemia, and high-level expression is correlated with poor prognosis. Moreover, HOXA9 overexpression immortalizes murine marrow progenitors that are arrested at a promyelocytic stage of differentiation when cultured and causes leukemia in recipient mice following transplantation of HOXA9 expressing bone marrow. The molecular mechanisms underlying the physiologic functions and transforming properties of HOXA9 are poorly understood. This study demonstrates that HOXA9 is phosphorylated by protein kinase C (PKC) and casein kinase II and that PKC mediates phosphorylation of purified HOXA9 on S204 as well as on T205, within a highly conserved consensus sequence, in the N-terminal region of the homeodomain. S204 in the endogenous HOXA9 protein was phosphorylated in PLB985 myeloid cells, as well as in HOXA9-immortalized murine marrow cells. This phosphorylation was enhanced by phorbol ester, a known inducer of PKC, and was inhibited by a specific PKC inhibitor. PKC-mediated phosphorylation of S204 decreased HOXA9 DNA binding affinity in vitro and the ability of the endogenous HOXA9 to form cooperative DNA binding complexes with PBX. PKC inhibition significantly reduced the phorbol-ester induced differentiation of the PLB985 hematopoietic cell line as well as HOXA9-immortalized murine bone marrow cells. These data suggest that phorbol ester-induced myeloid differentiation is in part due to PKC-mediated phosphorylation of HOXA9, which decreases the DNA binding of the homeoprotein.     

Intracellular Restriction of Ecotropic Murine Leukemia Virus in Rat NRK Cells and Its Abolishment by Adaptation

Ecotropic murine leukemia viruses, both N-tropic FN-2 (purified helper component of Friend leukemia virus) and B-tropic WNB-2 (purified WN1802B BALB/c-derived endogenous virus), were partially restricted in rat NRK cells. In NRK cells, they produced obscure small plaques at reduced efficiencies relative to their plaque-producing efficiencies in mouse SC-1 cells (10-fold for FN-2 and 100-fold for WNB-2). After three or four passages in NRK cells, the plaquing efficiencies of the viruses in NRK cells increased to levels close to their efficiencies in mouse cells, and the plaques in NRK cells became larger and clearer. The adaptation was more complete with FN-2 than with WNB-2. The adaptation was not due to simple selection of a virus in the FN-2 stock, but was host induced, as the viruses had been submitted to successive limiting dilutions in SC-1 cells before propagation in NRK cells. Possible commitment of xenotropic virus in the adaptation was excluded. The change was stable, even if the adapted viruses were propagated back into SC-1 cells. The NRK-adapted viruses were restricted in other rat cell lines of different origins, and the virus adapted in another rat cell line, RFL, was still restricted in NRK cells. The adaptation was mainly brought about by increased viral growth within the rat cells and not by an increased efficiency of viral penetration into the rat cells. This inversely suggests that the restriction of the ecotropic murine leukemia viruses in NRK cells was a mainly intracellular event. The mobilities of gp69/71 and p30 in sodium dodecyl sulfatepolyacrylamide gel electrophoresis remained unchanged after adaptation of FN-2 in NRK cells.     

Purification of a novel growth inhibitory factor for partially differentiated myeloid leukemic cells

A novel factor termed growth inhibitory (GI) factor, which specifically inhibits the growth of mouse monocytic leukemia cells including monocytic cell lines (Mm-A and J774.1) and other partially differentiated myeloid leukemic cells, has been purified from conditioned medium of some clones of mouse myeloblastic leukemia M1 cells. The procedure for purification of the GI factor included ammonium sulfate precipitation, CM-Sepharose CL-6B and Sephadex G-200 chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The purified factor gave a single band of protein with a molecular weight of 25,000 on sodium dodecyl sulfate-polyacrylamide gel. A concentration of 8 X 10(-10) M GI factor was required for 50% inhibition of growth of Mm-A cells. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 8.2-8.4. The purified GI factor markedly inhibited growth of mouse bone marrow cells stimulated by macrophage colony-stimulating factor. The GI factor appeared to be a unique cytokine unrelated to known cytokines such as the tumor necrosis factor, interferons, and oncostatin M.     

The suppressive effects of recombinant human tumor necrosis factor-alpha on normal and malignant myelopoiesis: synergism with interferon-gamma

The modulation of growth of normal and leukemic myeloid progenitor cells in soft agar cultures by recombinant human tumor necrosis factor-alpha (TNF alpha) and recombinant human interferon-gamma (IFN gamma) was investigated. TNF alpha inhibited colony formation of all colony types representing different maturational stages of normal progenitor cells committed to the myeloid lineage with different orders of sensitivity. Blast-type colonies derived from patients with acute myelogenous leukemia were more sensitive to TNF alpha inhibition than progenitor cells purified from normal bone marrow or bone marrow from patients with stable-phase chronic myelogenous leukemia. The response of most colony types to IFN gamma was poor. However, when IFN gamma was administered together with TNF alpha, synergistically enhanced antiproliferative effects were detected in all colony types tested. The antiproliferative action of IFN gamma on myelopoiesis was enhanced in culture by the presence of autologous monocytes, presumedly by inducing endogenous production of TNF alpha. However, TNF alpha seemed to act directly on the progenitor cells themselves to suppress their clonal growth, rather than involving accessory marrow elements such as monocytes and/or T lymphocytes.     

Hematopoietic stem cell expansion and distinct myeloid developmental abnormalities in a murine model of the AML1-ETO translocation

The t(8;21)(q22;q22) translocation, which fuses the ETO gene on human chromosome 8 with the AML1 gene on chromosome 21 (AML1-ETO), is one of the most frequent cytogenetic abnormalities associated with acute myelogenous leukemia (AML). It is seen in approximately 12 to 15% of AML cases and is present in about 40% of AML cases with a French-American-British classified M2 phenotype. We have generated a murine model of the t(8;21) translocation by retroviral expression of AML1-ETO in purified hematopoietic stem cells (HSC). Animals reconstituted with AML1-ETO-expressing cells recapitulate the hematopoietic developmental abnormalities seen in the bone marrow of human patients with the t(8;21) translocation. Primitive myeloblasts were increased to approximately 10% of bone marrow by 10 months posttransplant. Consistent with this observation was a 50-fold increase in myeloid colony-forming cells in vitro. Accumulation of late-stage metamyelocytes was also observed in bone marrow along with an increase in immature eosinophilic myelocytes that showed abnormal basophilic granulation. HSC numbers in the bone marrow of 10-month-posttransplant animals were 29-fold greater than in transplant-matched control mice, suggesting that AML1-ETO expression overrides the normal genetic control of HSC pool size. In summary, AMLI-ETO-expressing animals recapitulate many (and perhaps all) of the developmental abnormalities seen in human patients with the t(8;21) translocation, although the animals do not develop leukemia or disseminated disease in peripheral tissues like the liver or spleen. This suggests that the principal contribution of AML1-ETO to acute myeloid leukemia is the inhibition of multiple developmental pathways.     

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.     

Maturation inducer activity and the process of human myeloid leukemia cell differentiation

The process and mechanism of human myeloid leukemia cell differentiation induced by T-cell lymphokine maturation inducer activity was investigated. The maturation inducer activity was purified from conditioned medium of normal peripheral blood lymphocytes and shown to be a 50,000 M.W. protein. The degree of maturation of myeloid cell cultures was directly related to the dosage of the inducer. The interaction of the leukemia cells with the inducer led to initiation of terminal differentiation to monocytic cells. Proliferation cessation of the leukemia cells and the expressions of mature monocytic cells indicated a continuous and multistaged process.     

Antibody-producing human-human hybridomas. III. Derivation and characterization of two antibodies with specificity for human myeloid cells

Peripheral blood mononuclear cells from a patient with acute myeloid leukemia (AML) and spleen cells from a patient with chronic myeloid leukemia (CML) were fused with HAT-sensitive human B lymphoma cells (RH-L4) in attempts to generate human monoclonal antibodies (Mab) against antigens with high specificity for myeloid leukemia cells. Forty-seven of 246 hybridomas secreted Ig that bound to AML cell surface constituents, as determined by FACS analysis of viable cells that were FITC-stained with the human Mab as the first-step reagent and FITC-conjugated rabbit anti-human Ig as second-step. Two of the 47 human Mab (one from each patient and designated AML-19 and CML-20, respectively) bound to both autologous and allogeneic myeloid leukemia cells. No significant binding was observed to cell surface constituents on human bone marrow cells, granulocytes, lymphocytes, erythrocytes, thymocytes, monocytes, lymphoblastic leukemia cells, fibroblasts, malignant B and T lymphocytic cell lines, and murine bone marrow cells. Both human Mab were IgG and were cytotoxic to myeloid leukemia cells in the presence of complement. About 70% of peripheral blood cell samples from 46 AML patients contained AML-19- and CML-20-positive cells, but the reactivity pattern had no correlation to the morphologic FAB classification of the samples. The promyelocytic HL60 cell line and the K562 cell line reacted with the two antibodies. Dot blot analysis of binding of AML-19 and CML-20 to cellular extracts immobilized on nitrocellulose paper showed that both human Mab in this assay also reacted with normal bone marrow cells. This was supported by microscopic immunofluorescence because both human Mab stained intracytoplasmatic structures in normal bone marrow cells, but both intracytoplasmatic and cell surface components stained in myeloid leukemia cells. Moreover, immunoblotting demonstrated that both human Mab in leukemia cells reacted with two cellular proteins with Mr approximately 14,500 and 18,000, and in normal bone marrow cells with a molecule with Mr approximately 20,000. Immunoprecipitation of cell membrane molecules with both the AML-19 and CML-20 antibody precipitated from leukemic cells only the molecule with Mr approximately 18,000 and no components from normal bone marrow cells. It is concluded that myeloid leukemogenesis may result in generation of cell surface expression of either new or abnormally processed molecules that are immunogenic in the autochthonous host. These molecules may also be useful as markers in diagnosis of myeloid leukemia.     

Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: role in leukemogenesis.

The c-myb proto-oncogene is abundantly expressed in tissues of hematopoietic origin, and changes in endogenous c-myb genes have been implicated in both human and murine hematopoietic tumors. c-myb encodes a DNA-binding protein capable of trans-activating the c-myc promoter. Suppression of both of these proto-oncogenes was shown to occur upon induction of terminal differentiation but not upon induction of growth inhibition in myeloid leukemia cells. Myeloblastic leukemia M1 cells that can be induced for terminal differentiation with the physiological hematopoietic inducers interleukin-6 and leukemia inhibitory factor were genetically manipulated to constitutively express a c-myb transgene. By using immediate-early to late genetic and morphological markers, it was shown that continuous expression of c-myb disrupts the genetic program of myeloid differentiation at a very early stage, which precedes the block previously shown to be exerted by deregulated c-myc, thereby indicating that the c-myb block is not mediated via deregulation of c-myc. Enforced c-myb expression also prevents the loss in leukemogenicity of M1 cells normally induced by interleukin-6 or leukemia inhibitory factor. Any changes which have taken place, including induction of myeloid differentiation primary response genes, eventually are reversed. Also, it was shown that suppression of c-myb, essential for terminal differentiation, is not intrinsic to growth inhibition. Taken together, these findings show that c-myb plays a key regulatory role in myeloid differentiation and substantiate the notion that deregulated expression of c-myb can play an important role in leukemogenicity.     

Cell cycle dependence of calmodulin levels during HL-60 proliferation and myeloid differentiation: No changes during pre-commitment

The putative role of Ca²⁺ and calmodulin in regulating cell proliferation and differentiation was tested in HL-60 human promyelocytic leukemia cells. The dependence of retinoic acid (RA)-induced terminal myeloid differentiation of HL-60 promyelocytic leukemia cells on calmodulin levels and calcium ion flux was ascertained. RA-treated and untreated control cells were stained for cellular DNA with a Hoechst dye. Populations of G1/0, S and G2+M phase cells were isolated by fluorescence activated cell sorting (FACS). Cytosolic calmodulin levels were then measured as a function of cell cycle phase for RA-treated and untreated cells using a radioimmunoassay. RA-treated cells were measured at early times, corresponding to the precommitment state, and late times, when significant cell differentiation had occurred. Cellular calmodulin levels increased with progression through the cell cycle. In contrast, no difference in calmodulin levels was observed between RA-untreated or -treated cells in the same cell cycle phases at early or late times. RA-induced HL-60 terminal myeloid differentiation was thus apparently not regulated by cellular cytosolic calmodulin levels. These conclusions were supported by the effects of calmodulin antagonists and calcium flux inhibitors. The calmodulin antagonists trifluoperazine and compound 48/80 both retarded cell growth in a concentration-dependent manner. But at concentrations where cellular effect was evidenced by slight growth inhibition, neither antagonist inhibited RA-induced cell differentiation or G1/0 growth arrest. The same was true of the gated calcium channel inhibitors, verapamil and nitrendipene, and the passive calcium flux inhibitor, CoC1₂. RA-induced HL-60 cell differentiation and arrest in G0 was thus apparently not strongly dependent on cellular calmodulin levels or calcium flux. This is in strong contrast to murine erythroleukemia cells. The results argue against a central regulatory role for calmodulin or calcium flux in control of HL-60 growth arrest or differentiation.