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M Valtieri

Publications and source records attributed to M Valtieri.

At least 55 records · Page 3Linked to original sources
PubMed1991

Two-step differentiation of AML-193 leukemic line: terminal maturation is induced by positive interaction of retinoic acid with granulocyte colony-stimulating factor (CSF) and vitamin D3 with monocyte CSF.

The human AML-193 cell line requires exogenous granulocyte-monocyte colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) for growth in liquid or semisolid medium. However, these CSFs do not stimulate the differentiation of the cell line. We show that addition of all-trans retinoic acid (RA) or 1,25 dihydroxyvitamin D3 (D3) induces AML-193 cells to differentiate into the granulocytic or monocytic lineage, respectively. On the other hand, addition of either G- or M-CSF alone exerts virtually no differentiative effect. Terminal granulocytic or monocytic differentiation was observed when AML-193 cells were treated with RA and G-CSF, or D3 and M-CSF, respectively, as evaluated by cell morphology, analysis of surface antigens, and phagocytic functions. These positive interactions indicate that the differentiating activity of G- and M-CSF on leukemic cells may be unmasked by preliminary treatment with RA and D3, respectively, ie, the physiologic inducers override the leukemic differentiation blockade and CFSs exert their differentiative activity on the unblocked leukemic cells. These preliminary observations on a single cell line may pave the way for the designing of clinical protocols combining physiologic inducer(s) and hematopoietic growth factor(s) in the treatment of acute leukemia.

Antibodies, Monoclonal↗
PubMed1991

Normal and leukemic hematopoietic cells manifest differential sensitivity to inhibitory effects of c-myb antisense oligodeoxynucleotides: an in vitro study relevant to bone marrow purging.

The c-myb protooncogene is preferentially expressed in hematopoietic cells, and its encoded protein, Myb, is required for hematopoietic cell proliferation. To analyze the relative Myb dependence of normal and leukemic human hematopoietic progenitor cells, normal bone marrow cells, several types of leukemic blast cells, and 1:1 mixtures of normal and leukemic cells were cultured in the presence of c-myb sense or antisense oligodeoxynucleotides; cell viability and cloning efficiency were then assessed. c-myb sense oligomers had negligible effects on normal and leukemic cells. In contrast, c-myb antisense oligomers strongly inhibited or completely abolished clonogenic growth of a T-cell leukemia line, 78% (18 of 23) of primary acute myelogenous leukemia cases examined, and 4 of 5 primary chronic myelogenous leukemia (CML) cases in blast crisis. In three of the latter patients, polymerase chain reaction analysis of a 1:1 mixture of c-myb antisense-treated normal and CML cells revealed a complete absence of bcr-abl expression, suggesting that the CML clonogenic units had been completely eliminated from the cultures. At antisense doses that inhibited leukemic cell growth, normal hematopoietic progenitor cells survived. Thus, normal and leukemic hematopoietic cells show differential sensitivity to the toxic effects of c-myb antisense DNA. Perturbation of c-myb function with antisense oligodeoxynucleotides might eventually form the basis for a molecular approach to leukemia therapy, perhaps most immediately as ex vivo bone marrow purging agents.

Base Sequence↗
PubMed1991

Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha.

These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited (73.2% +/- 10.4% and 74.2% +/- 12.7%). Using highly purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of highly purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase. Under the same experimental conditions a progressive increase of the mRNA level of DNA polymerase alpha was observed. These observations suggest that in early erythroid differentiation c-myb activation is associated with the progression of progenitors into the S phase of the cell cycle, as well as to the synthesis of DNA polymerase alpha.

Antisense Elements (Genetics)↗
PubMed1991

Pure human hematopoietic progenitors: direct inhibitory effect of transforming growth factors-beta 1 and -beta 2.

TGF-beta 1 and TGF-beta 2 are effective inhibitors of hematopoiesis. We report that colony formation by pure peripheral blood CD34+CD33- BFU-E and CFU-GM (100 cells/dish) is effectively inhibited by both molecules, although TGF-beta 1 is up to 10-fold more potent than TGF-beta 2. Therefore, the effect of these molecules is apparently direct, rather than mediated by accessory cells. The maximal inhibitory activity of TGF-beta is exerted essentially at the early progenitor level, whereas BFU-E/CFU-GM primed for 48 h and IL-3, GM-CSF, and erythropoietin become insensitive to its action. In addition, [3H]TdR suicide experiments indicate that TGF-beta 2 blocks the IL-3-induced progression of early progenitors into the S phase of the cell cycle, whereas IL-6 and bFGF potentiate their entry into the mitotic process. Altogether, these results are compatible with the hypothesis that TGF-beta plays a relevant regulatory role in the homeostasis of early hematopoietic proliferation/differentiation.

Cell Cycle↗
PubMed1990

Down-regulated c-myb expression inhibits DNA synthesis of T-leukemia cells in most patients.

We have investigated the functional relevance of c-myb expression for DNA synthesis in patients' T-leukemia cells. [3H]Thymidine incorporation assays of 32 patients' leukemia cells exposed in vitro to c-myb sense or antisense oligodeoxynucleotides served to define two groups of patients: a responder group whose leukemia cells showed 2- to 16-fold lower levels of [3H]thymidine incorporation in c-myb antisense-treated cultures than in c-myb sense-treated cultures (20 patients) and a nonresponder group whose cells showed comparable [3H]thymidine incorporation levels in either c-myb sense- or antisense-treated cultures (12 patients). Down-regulation of c-myb mRNA levels in cells exposed to c-myb antisense oligodeoxynucleotides was comparable in both groups of patients, indicating that differential sensitivity to c-myb antisense oligodeoxynucleotides was not due to differential uptake of these oligodeoxynucleotides. DNA polymerase alpha mRNA levels were down-regulated in cells from the responders but were unaffected in the nonresponder group. These results suggest that c-myb is required for DNA synthesis in cells of many but not all T-leukemia patients and that leukemia cells in which DNA synthesis is not inhibited despite down-regulation of c-myb expression may have undergone some genetic change(s) that obviate(s) the requirement for myb protein.

Base Sequence↗
PubMed1990

Molecular mechanisms underlying erythropoiesis: cycling activity of adult BFU-e relates to their requirement for c-myb function and potential for HbF synthesis.

Highly purified erythroid burst-forming units (BFU-e) from human embryonic liver, adult marrow and blood were manipulated in vitro by cytokine addition in order to explore their requirements for c-myb function and potential for fetal hemoglobin (HbF) synthesis, particularly as related to their cycling activity. c-myb is expressed at a minimal level and functionally required to a limited extent in quiescent adult BFU-e. However, c-myb is actively transcribed and stringently required for differentiation of actively cycling progenitors (embryonic BFU-e, embryonic and adult erythroid colony-forming units). The cycling activity of highly purified adult BFU-e, gradually enhanced by interleukin 3 (IL-3) addition, is strictly and directly related to both their functional requirements for c-myb and the level of myb mRNA expression in the progenitor population. It may be concluded that the transcriptional activity and the functional role of c-myb in early erythropoiesis are dependent upon the cycling activity of the erythroid progenitors. The reactivation of HbF synthesis in normal adult bursts, observed in the standard fetal calf serum-rich (FCS+) clonogenic system, is suppressed in cultures with a drastically limited growth of accessory cells (i.e., in FCS- or FCS+ Mo- conditions). In these cultures, addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) or IL-3 induces a dose-related rise of gamma-chain synthesis, at least in part via a direct action at the BFU-e level. Preliminary studies involving priming of adult BFU-e with IL-3 in liquid phase suggest that the HbF potential is relatively low in quiescent BFU-e, but distinctly higher in actively cycling ones. It is postulated that the in vivo reactivation of HbF synthesis in bone marrow regeneration may be mediated via increased IL-3 and GM-CSF activity, leading to enhanced cycling and differentiation of BFU-e.

Adult↗
PubMed1990

Reactivation of HbF synthesis in normal adult erythroid bursts by IL-3.

Reactivation of HbF synthesis has been reported in normal adult erythroblast colonies ('burst') generated by erythroid progenitors (BFU-E) after seeding peripheral blood mononuclear cells (PBMC) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated by erythropoietin (Ep). Reactivation is almost totally suppressed when: (i) PMBC are grown in optimized FCS- culture or (ii) PBMC are first stringently depleted of monocytes and then plated in FCS+ medium (i.e. BFU-E growth in FCS+Mo- culture). In either case, addition of biosynthetic granulocyte-macrophage colony stimulating factor (GM-CSF) induces a dose-related increase of relative HbF synthesis up to the level in FCS+ culture. We report that, in FCS- culture of partially purified adult blood BFU-E, treatment with biosynthetic interleukin 3 (IL-3) causes a dose-related rise of relative HbF production in the bursts. A similar phenomenon is observed in FCS+ culture of highly purified BFU-E. The rise of HbF synthesis is seemingly mediated, at least in part, by a direct effect of IL-3 at BFU-E level. It is tentatively concluded that reactivation of HbF in vitro, as well as in a variety of in vivo conditions (i.e. stress erythropoiesis, marrow regeneration), may be at least in part mediated by IL-3 and GM-CSF.

Adult↗
PubMed1990

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.

Base Sequence↗
PubMed1989

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.

Blood Cells↗
PubMed1989

Alteration of growth and differentiation factors response by Kirsten and Harvey sarcoma viruses in the IL-3-dependent murine hematopoietic cell line 32D C13(G).

32D C13(G) is an interleukin 3(IL3)-dependent non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocytic colony stimulating factor (G-CSF). Infections of 32D C13(G) cells with either Kirsten rat sarcoma virus or Balb murine sarcoma virus, both containing a v-ras oncogene, generates clones that can permanently grow in G-CSF without differentiation. 32D-Ki-ras cells show a heterogeneous morphology ranging from the promyelocytic to the myelocytic stage of differentiation, and express high levels of both myeloperoxidase (MPO) and lactoferrin (LF) mRNA. 32D-Ha-ras cells show a more immature phenotype and express MPO but no LF mRNA. The apparent differentiation block of both 32D Ki-ras and 32D Ha ras can be reversed by treatment with the chemical inducers retinoic acid, sodium butyrate or dimethylsulphoxide, which leads to terminal differentiation into granulocytes. When 32D-Ki-ras and 32D-Ha-ras cells are cultured in medium containing IL-3 they become adherent and express some monocyte-macrophage markers. Upon prolonged exposure to IL3, 32D-Ki-ras, but not 32D-Ha-ras, resume suspension growth. Both 32D-Ki-ras and 32D-Ha-ras rapidly die if grown in chemically defined medium in the absence of any growth factor and are non-tumorigenic in immunosuppressed mice. These findings indicate that ras activation may interfere with the normal response to growth and differentiation factors in cells of the granulocytic lineage. These alterations may represent a critical, although non-sufficient, step in leukemogenesis.

Butyrates↗
PubMed1989

Granulocyte-macrophage colony-stimulating factor reactivates fetal hemoglobin synthesis in erythroblast clones from normal adults.

Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in normal human adult erythroblast colonies ("bursts") generated by erythroid progenitors (BFU-E) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated with erythropoietin (Ep). Our studies focused on the reactivation of HbF synthesis in normal adult erythroid bursts generated by peripheral blood mononuclear cells (PBMCs) seeded in FCS+ methylcellulose culture. Reactivation is almost totally suppressed when (a) PBMCs are grown in optimized FCS- culture, or (b) PBMCs are first stringently depleted of monocytes and then plated in FCS+ medium (ie, BFU-E growth in FCS+ Mo- culture). In both experimental conditions, the proliferation of lymphocytes and macrophages interspersed among colonies is drastically reduced, and the cloning efficiency of granulocyte-macrophage (GM) progenitors is sharply diminished. In either case, addition of biosynthetic GM colony-stimulating factor (GM-CSF) induces a dose-related increase of HbF synthesis up to the level in FCS+ culture, with even more elevated values on delayed addition of Ep. A dose-related increase was also observed in erythroblast clones generated by highly purified BFU-E. These results suggest that reactivation of HbF synthesis in normal adults is at least in part mediated by GM-CSF. Furthermore, they imply intriguing hypotheses on the mechanism(s) of perinatal Hb switching. Finally, they raise the possibility of reactivation of HbF synthesis in beta-thalassemia and sickle cell anemia by GM-CSF therapy.

Biological Factors↗
PubMed1989

Erythropoietin alone induces erythroid burst formation by human embryonic but not adult BFU-E in unicellular serum-free culture.

Erythroid progenitors (BFU-E) from adult human peripheral blood generate erythroid bursts in semisolid culture supplemented with at least two growth factors, ie, erythropoietin (Ep) and interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). We have analyzed the hematopoietin(s) requirement of human embryonic BFU-E, as compared to that of adult peripheral blood progenitors: This was basically evaluated in fetal calf serum-free (FCS-) methylcellulose culture of partially or highly purified progenitors treated with human recombinant hemopoietins. At a low seeding concentration (2 x 10(3) cells/dish) purified embryonic BFU-E generated erythroid bursts when treated only with Ep: Further addition of IL-3 or GM-CSF had no effect on BFU-E cloning efficiency, although the size of bursts was increased in a dose-dependent manner, particularly with IL-3. At a similar seeding concentration (ie, 10(3) cells/dish), purified adult BFU-E efficiently generated erythroid bursts in the presence of Ep and GM-CSF or IL-3, while only few small erythroid colonies were observed in the presence of Ep alone. In a final series of experiments, unicellular FCS-cultures of purified embryonic BFU-E gave rise to erythroid bursts in the presence of Ep alone. Furthermore, the cloning efficiency induced by Ep was unmodified by further addition of GM-CSF or IL-3. Unicellular FCS- cultures of highly purified adult peripheral blood progenitors generated no erythroid bursts in the presence of Ep alone. The addition of GM-CSF or IL-3 was required to generate BFU-E colonies. These studies indicate that in human embryonic life, BFU-E require only Ep for efficient erythroid burst formation, while IL-3 and GM-CSF essentially enhance the proliferation of early erythropoietic precursors.

Cells, Cultured↗
PubMed1987

Cytokine-dependent granulocytic differentiation. Regulation of proliferative and differentiative responses in a murine progenitor cell line.

Human granulocyte colony stimulating factor (G-CSF) can support the survival and short term proliferation of the interleukin 3 (IL 3)-dependent diploid murine hemopoietic progenitor cell line 32D C13. After 8 days in the presence of 30 U/ml of G-CSF and in the absence of IL 3, the great majority of 32D C13 cells becomes positive for myeloperoxidase (a marker that appears at the promyelocytic stage of the granulocytic lineage) and progressively differentiates into lactoferrin-containing neutrophilic granulocytes. Myeloperoxidase mRNA rapidly increases after 24 to 48 hr of treatment with G-CSF, peaks at day 6 and is no longer detectable at day 9 and 12, paralleling the appearance of myeloperoxidase-positive promyelocytes and myelocytes in the culture. After 12 days, 100% of the cells terminally differentiate, and clonogenic assays in IL 3-containing semisolid media indicate that the whole population has irreversibly lost proliferative capability. By using varying concentrations of both murine IL 3 and recombinant human G-CSF, the cultures develop an heterogeneous population of cells representing all the differentiation stages of the myeloid lineage, and the relative ratios of immature proliferating precursors and terminally differentiated cells present in the cultures can be modulated by modifying the concentrations of IL 3 or recombinant human G-CSF. Isobolic curves indicate that IL 3 and G-CSF have an antagonistic effect on the proliferation of 32D C13 cells. Thus, these cells represent a simplified in vitro model of normal granulocytic differentiation whose extent may be modulated completely in the presence of serum by two well-defined growth and differentiation factors: IL 3 and G-CSF.

Animals↗
PubMed1987

Establishment and characterization of an undifferentiated human T leukemia cell line which requires granulocyte-macrophage colony stimulatory factor for growth.

A human leukemia cell line (TALL-101) was established from the bone marrow of a patient with an undifferentiated acute T cell leukemia using the conditioned medium (CM) of the human T cell leukemia virus (HTLV) II-transformed human cell line J-LB1. Immunofluorescence analysis on the original leukemic cells indicated the presence of T cell markers (Leu-1, Tdt, and T11); however, the established TALL-101 cell line expressed only antigens commonly present on progenitor cells, thymocytes, and myelomonocytic cells, but not on mature T cells. A high percentage of TALL-101 cells displayed the Tac antigen which was down-regulated upon incubation in the presence of recombinant human (rH) interleukin 2 (IL 2). Interferon (IFN)-gamma induced the appearance of class II histocompatibility leukocyte antigens (HLA) and of a T cell marker (3A1), and enhanced the expression of transferrin receptors on these cells. Further evidence for a T cell lineage of the TALL-101 cell line was provided by both chromosomic and genotypic analysis showing a translocation in chromosome 14 typical of T cell leukemias, and a rearrangement of the T-beta receptor locus. The growth-promoting activity in the J-LB1-CM was identified as granulocyte-macrophage colony stimulatory factor (GM-CSF), a growth factor which stimulates proliferation of normal myelomonocytic cells and other progenitor cells, but not known to have an effect on T cells. Dose response curves of [3H]thymidine incorporation and growth indicated that TALL-101 cells were sensitive to very low concentrations of rHGM-CSF, 5 ng/ml inducing maximal proliferation in chemically defined medium. The TALL-101 cell line is strictly GM-CSF-dependent for growth: upon depletion of GM-CSF from the culture medium, the cells stop proliferating immediately and die within 1 to 2 wk. The overall data, showing that GM-CSF is able to support the growth of a highly undifferentiated T cell leukemia, strongly suggests that this factor might have similar growth promoting effects on other immature T cell leukemias, and possibly, on normal T cell progenitors.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Tumor-derived growth factors that support proliferation and differentiation of normal and leukemic hemopoietic cells.

The conditioned media of 34 human tumor cell lines were screened for the ability to induce granulocyte-macrophage colonies in vitro in bone marrow cultures, to stimulate proliferation of a murine IL-3 dependent hemopoietic cell line (32D clone 3) and to stimulate thymidine incorporation in suspension cultures of acute myelogenous leukemia cells. Twelve tumor cell lines produced factors that were active in these assays. The conditioned medium of the glioblastoma cell line U87 MG was characterized in detail and found to contain G-CSF and GM-CSF. Cloning and sequencing of the U87 MG G-CSF indicated that it was derived from G-CSF b mRNA, which encodes a protein with a deletion of 3 amino acids at residues 36-38. The gene for G-CSF was mapped to human chromosome 17 band q21, a region involved in translocations frequently found in acute promyelocytic leukemia. G-CSF (U87MG) was able to induce granulocytic differentiation of the total population of a murine IL-3 dependent cell line, 32D clone 3; this effect was antagonized by IL-3. GM-CSF (U87-MG) supported the proliferation without inducing differentiation of two growth factor-dependent leukemic cell lines, TALL 101 and AML-193.

Animals↗
PubMed1987

Effect of Abelson murine leukemia virus on granulocytic differentiation and interleukin-3 dependence of a murine progenitor cell line.

The murine diploid hematopoietic cell line 32D Cl3 strictly requires interleukin-3 (IL-3) for proliferation. When 32D Cl3 cells are transferred to IL-3-free medium which contains recombinant human granulocyte colony stimulating factor (rhG-CSF), the cell number increases four- to five-fold, and after 14 days the whole cell population is differentiated into morphologically normal and myeloperoxidase- and lactoferrin-positive metamyelocytes and granulocytes. Infection with Abelson murine leukemia virus (A-MuLV) of 32D Cl3 cells growing in the presence of IL-3 induces, within 2 weeks, the appearance of cells that are IL-3-independent for growth. The latter cells lack myeloid, T and B cell markers, and are unable to differentiate, even in the presence of very high doses of rhG-CSF. However, once the 32D Cl3 cells have been exposed to G-CSF, they become resistant to the transforming effects of A-MuLV as judged by the appearance of the IL-3-independent clones. These findings suggest that the ability of Abelson virus to transform immature progenitor cells is due to interference of the v-abl gene product with the mechanisms that control the commitment of the cells to differentiate.

Abelson murine leukemia virus↗