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Biomedical subjects

G Rovera

Publications and source records attributed to G Rovera.

At least 109 records · Page 6Linked to original sources
PubMed1983

Differential expression of the normal and of the translocated human c-myc oncogenes in B cells.

We have investigated whether the translocated and the untranslocated human c-myc oncogenes of Burkitt lymphoma cells are equally or differentially expressed in host mouse B cells. The human c-myc mRNA levels in somatic cell hybrids between mouse plasmacytoma cells and Burkitt lymphoma cells with either the t(8;14) or the t(2;8) chromosome translocation were determined by using the nuclease S1 protection procedure. Although both the human parental lines and the hybrid cells carrying the translocated c-muc oncogene expressed high levels of human specific c-myc transcripts, the hybrid cells carrying the untranslocated c-myc gene on normal chromosome 8 did not contain human specific c-myc mRNA. These results suggest that the translocated human c-myc oncogene has escaped the normal transcriptional control to which the untranslocated c-myc gene remains subjected. This interpretation is also supported by the finding that the expression of the c-myc genes of lymphoblastoid cells and of HL-60 promyelocytic leukemia cells are repressed when they are transferred into a mouse plasmacytoma background. The ability of the translocated c-myc oncogene to escape the normal transcriptional control occurring in B cells may be important for the expression of B cell neoplasia in mouse and man. We have also transferred the Burkitt 14q+ chromosome carrying a translocated c-myc oncogene into mouse LM-TK- fibroblasts and studied the levels of human c-myc transcripts in the hybrids. Because the levels of human c-myc transcripts in the fibroblast hybrids are dramatically decreased in comparison to the plasmacytoma hybrids, we conclude that the levels of transcripts of the translocated c-myc oncogene depend on the differentiated state of the cells harboring the translocated chromosome.

Animals↗
PubMed1983

The structure and nucleotide sequence of the 5' end of the human c-myc oncogene.

We have established the structure and nucleotide sequence of the 5' end of the human c-myc oncogene, using a cloned genomic fragment isolated from a fetal liver library (clone lambda MC41) and cloned cDNA from the human leukemic cell line K562. The human c-myc oncogene consists of three exons and two introns. Primer extension of the human c-myc mRNA of three different cell lines and S1 nuclease protection experiments served to establish the position of two transcription initiation sites. The splicing site of the first exon-intron boundary was determined by comparative analysis of the sequences of the genomic and cDNA clones. The first exon contains termination codons in all three reading frames and no translation initiation signals, confirming our previous observation that the c-myc mRNA has a long 5' noncoding sequence. This first exon also was found to be utilized in the formation of c-myc mRNAs in a variety of human cell lines.

Base Sequence↗
PubMed1983

Amplified C lambda and c-abl genes are on the same marker chromosome in K562 leukemia cells.

The human leukemia cell line K562, derived from a patient with Philadelphia chromosome-positive chronic myelogenous leukemia, contains amplified c-abl oncogenes and unrearranged C lambda genes. Using in situ hybridization techniques, we have determined that the amplified c-abl and C lambda DNA sequences of K562 cells are both located on the same abnormal acrocentric marker chromosome, which may represent an altered Philadelphia chromosome.

Cell Line↗
PubMed1983

Target antigen of monoclonal reagent S5.7: comparison with T3 antigen.

S5.7 recognizes a 20 kD cell surface protein which is present on T lymphocytes. S5.7 binds to a nonglycosylated protein, which can be labeled by cell-surface radioiodination and by a hydrophobic reagent [125I]-iodo-5-naphthyl-1-azide (INA). As the T-lymphocyte-specific T3 complex was found to contain a nonglycosylated 20 kD species, and since this 20 kD T3 form can be labeled preferentially by INA, a comparison between T3 and S5.7 was made. Isoelectric focusing experiments showed, however, that the two proteins are different. Moreover, the S5.7 monoclonal antibody does not block CML, is not mitogenic, reacts with immature cells of several hemopoietic lineages, and differs in that respect from anti-T3 monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1983

Induction of differentiation of human myeloid leukemias: surface changes probed with monoclonal antibodies.

The surface changes occurring in three acute myeloid leukemia cell lines (HL60, ML3, and KG1) induced to differentiate by a variety of agents (dimethylsulfoxide, retinoic acid, 12-O-tetradecanoylphorbol-13-acetate, and factors present in lymphocyte conditioned medium) were probed using monoclonal antibodies that are differentiation stage- and lineage-specific. In all cases, the differentiated phenotype was defective and varied with the inducing agent and the cell line used. HL60 proved to be the most sensitive to the effect of the inducers. Retinoic acid was better than DMSO, and TPA was better than the medium factors in the ability to induce granulocytic and monocytic differentiation, respectively, in HL60 cells. These findings indicate that the differentiation block in acute myeloid leukemias is heterogeneous and that each cell line has different phenotypic characteristics that are responsible for the extent of differentiation obtained with a given inducer. These results also suggest that the extent of the differentiation response in vitro may be improved by the use of more suitable inducers for each specific leukemic line.

Antibodies, Monoclonal↗
PubMed1983

Differentiation antigens of human hemopoietic cells: patterns of reactivity of two monoclonal antibodies.

Two mouse anti-human monoclonal antibodies (S3.13 and S5.7) raised against cells of acute myelogenous leukemia were found to react with antigens expressed on the surface of subsets of monocytes and lymphocytes. S3.13 precipitates a peptide of Mr 29,000, and S5.7 precipitates a peptide of Mr 20,000 present on the surface of all the cell types tested. These two surface antigens were distributed on discrete subpopulations of normal hemopoietic cells. The antibodies reacted with all (S5.7) or a subpopulation (S3.13) of peripheral blood T-lymphocytes, and with a subset of monocytes. Both antibodies reacted with bone marrow blast cell progenitors of the myelomonocytes and erythroid lineage. S5.7 also reacted with non-T-lymphocytes and with cells of the eosinophilic lineage. Both antigens disappeared from the cell surface during normal myeloid and erythroid differentiation. Thus, these monoclonal antibodies define the molecular characteristics and the cellular distribution of two differentiation antigens present on cells of the hemopoietic lineage.

Animals↗
PubMed1983

Human granulocyte surface molecules identified by murine monoclonal antibodies.

We have investigated the nature of the antigens recognized by four classes of mouse anti-human monoclonal antibodies that characteristically reacted with neutrophilic granulocytes and their precursor cells, but not with monocytes or other normal hemopoietic cells. The antigenic targets of the majority (9/12) of the independently isolated monoclonal antibodies were present on two surface glycoproteins (Mr 145,000 and 105,000) and glycolipids. This antigen(s) was also detected on granulocyte precursor cells, including the bone marrow granulocyte/monocyte progenitor cells (CFU-GM). The same antigen(s) detected by these monoclonal antibodies was also present in non-hemopoietic cell lines (colon carcinoma and neuroblastoma). Three other antigens, defined by monoclonal antibodies AHN-8, L12.2, and L13.1 and present on granulocytes and their mid-late precursor cells, could not be identified as proteins but were detected in a protein-free glycolipid extract of these cells. The diversity of the antigens was confirmed by cross-competition experiments and by the identification of their different patterns of reactivity with cell lines and bone marrow cells.

Animals↗
PubMed1982

Induction of differentiation of human myeloid leukemias by phorbol diesters: phenotypic changes and mode of action.

Treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) of acute myeloblastic leukemia cells halts proliferation and induces expression of monocyte/macrophage markers. Surface characteristics of leukemic HL60 cells, as defined using a panel of monoclonal antibodies, were found to be similar to those of normal human promyelocytes. TPA treatment, however, induced a phenotype that, unlike normal monocytes, contained several myeloid-specific markers and lacked several monocyte-specific markers. TPA treatment of HL60 cells causes the rapid disappearance of the transferrin receptor from the cell surface. Because transferrin is essential for HL60 cell proliferation in culture, the disappearance of this receptor is followed by an irreversible accumulation of the cells in the G1 phase of the cell cycle. The TPA-induced arrest of cell proliferation suggests the potential of this agent in experimentally treating myeloblastic leukemias.

Animals↗
PubMed1982

Nuclear precursor molecules of the two beta-globin mRNAs in Friend erythroleukemia cells.

Processing of the beta major and beta minor globin pre-mRNAs has been compared in murine erythroleukemia cells induced to synthesize hemoglobin by dimethyl sulfoxide or hemin treatment, using both the Northern blot technique and S1 nuclease mapping with 3' and 5' end-labeled probes. The small intervening sequence of both beta-globin pre-mRNAs was removed in one step, although minor amounts of incompletely spliced RNA were detected. During the processing of the large intervening sequence of beta major globin pre-mRNA two internal splice sites were clearly detected. On the contrary, the beta minor globin pre-mRNA did not show any internal splice sites. A model of processing of the mouse adult beta major globin pre-mRNA is proposed.

Animals↗
PubMed1982

A monoclonal antibody with specificity for leukemic cells transformed by defective avian leukemia viruses.

Mouse anti-chicken monoclonal antibodies were raised against an avian myeloblastosis virus (AMV)-transformed myeloblastic leukemic cell line. One monoclonal antibody, S1-37 (IgG2a), reacted with producer and nonproducer myeloblastic leukemia cell lines transformed by AMV and by E-26 virus, but it did not react with chicken fibroblasts infected with RAV-2, MAV-2, MAV-1, or RAV-7. S1-37 also did not react with normal chicken hemopoietic cells, except for yolk sack macrophages and a small population of embryonal and adult bone marrow cells that morphologically resembled macrophages. Cytotoxicity studies of GM-CFU, the normal stem cell population of the granulocytic macrophage lineage, indicated that these cells lack the surface antigen recognized by S1-37. Immunoprecipitation studies of 125I surface-labeled myeloblastic leukemic cells indicated that S1-37 binds a 42,000 Mr polypeptide. The possible role of this polypeptide in the process of transformation and differentiation of chicken myeloid cells is discussed.

Animals↗
PubMed1982

Monoclonal antibodies that detect differentiation surface antigens on human myelomonocytic cells.

We describe here the production and characterization of several new monoclonal antibodies that recognize differentiation antigens present on human cells of the myelomonocytic lineage. The lineage and the stage specificities of our reagents (myeloid-, monocytic-, and myelomonocytic-specific) were determined on the basis of their reactivity with human cell lines and with human peripheral blood and bone marrow cells. Cross-competition experiments demonstrated that some of the antibodies react with the same or closely associated antigenic determinants. Five antigens have been identified in this way: one present on myeloid, one on monocytic, and three on both myeloid and monocytic cells. The possible relationship of our antibodies with other established monoclonal antibodies is discussed, in addition to their use in the in vitro study of the differentiation pathways of human hemopoietic cells and in the characterization of leukemias.

Animals↗
PubMed1982

A monoclonal antibody that detects expression of transferrin receptor in human erythroid precursor cells.

A monoclonal antibody, L5.1, obtained by immunizing a Balb/c mouse with HL60 human promyelocytic leukemia cells, was found to react with both HL60 cells and with the K562(S) cell line. This monoclonal antibody binds and immunoprecipitates a glycoprotein (Mr 87,000) present on the cell surface membrane of K562(S) as a disulfide bonded dimer. In competition experiments L5.1 competes with both transferrin and OKT9 (a known antitransferrin receptor antibody) for binding to target K562(S) erythroleukemia cells. Binding of both L5.1 and transferrin to the surface of K562(S) cells is inhibited by treatment with 12--O-tetradecanoyl-phorbol-13-acetate, and the extent and time course of inhibition is similar in both cases. Cell sorting analysis of normal human marrow cells incubated with L5.1 indicates that L5.1 reacts strongly with all the morphologically recognizable erythroid lineage precursors, from the pronormoblast to the orthochromatic normoblast, and with reticulocytes. Erythrocytes, myeloid elements, monocytes, megakaryocytes and platelets, peripheral blood B and T lymphocytes do not bind significantly with this antibody and only a small fraction of promyelocytes was reactive. Antibody L5.1 did not react with leukemic cells of patients with acute lymphoblastic, myeloblastic and promyelocytic leukemias, but it did react with some established B (1 of 5) and T (2 of 3) cell lines, and a myeloid (1 of 3) cell line, and with PHA-stimulated peripheral blood lymphocytes. The nonhemopoietic cell lines tested did not bind with L5.1 with the exception of a colorectal adenocarcinoma and a melanoma cell line, which were both strongly positive. The relationship of antibody L5.1 to other monoclonal antibodies that bind the transferrin receptor is discussed.

Animals↗
PubMed1981

Differential expression of the globin genes in human leukemia K562(S) cells induced to differentiate by hemin or butyric acid.

Human leukemia K562(S) cells were induced to differentiate by 50 microM hemin or 1.4 mM butyric acid, and the types of hemoglobins synthesized were compared. In both cases, embryonal hemoglobins [Portland, Gower 1, Hb X, and fetal hemoglobin (Hb-F)] were detected. Butyric acid-treated K562(S) cells contained mostly Hb Gower 1 (zeta 2 epsilon 2) and a hemoglobin with the electrophoretic characteristics of Portland (gamma 2 zeta 2). For hemin-treated K562(S), the most abundant hemoglobin synthesized by Hb X (epsilon 2 gamma 2), and the second most abundant was Bart's (gamma 4). Traces of Gower 1 were observed in nontreated K562(S) cells. The kinetics of hemoglobin induction as a result of the two treatments differed; increased hemoglobin synthesis was detected after only 24 hr of hemin treatment, whereas 4 days were required in butyric acid-treated cells. Both hemin and butyric acid were able to induce their respective patterns of hemoglobin synthesis independent of the presence of serum in the K562(S) growth medium. Analysis of the globin chains in induced K562(S) cells induced to differentiate indicated that, with both inducers, adult alpha- but not beta-globin chains were present. Karyotype analysis of K562(S) cells revealed a nearly triploid chromosome complement with a modal number of 68 chromosomes. Three copies of chromosome 11 and four copies of chromosome 16 (coding for the beta-like and alpha-like globin genes, respectively) were present. A large marked chromosome, involving chromosome 7, and a Philadelphia chromosome were also seen. These data characterize the K562(S) subline and also indicate that hemin and butyric acid differ in their effects on the expression of embryonal globin genes.

Butyrates↗
PubMed1981

Terminal differentiation surface antigens of myelomonocytic cells are expressed in human promyelocytic leukemia cells (HL60) treated with chemical inducers.

The expression of two surface antigens present on the cell membrane of both human granulocytes and monocytes was studied during the process of myelomonocytic differentiation using two monoclonal antibodies (B9.8.1 and B13.4.1). These surface antigens are not present on immature myeloid cells nor on nonmyeloid hematopoietic cells, but can be detected when the cells are terminally differentiated. Among the bone marrow cells, B13.4.1 binds to metamyelocytes and B9.8.1 to metamyelocytes and a fraction (30%) of myelocytes. HL60 human promyelocytic leukemia cells did not react with such monoclonal antibodies. However, when such cells were induced to differentiate in vitro into mature myeloid elements by treatment with retinoic acid or dimethyl sulfoxide, 70%--90% of the differentiated cells expressed both surface antigens. Cell sorting studies on these treated HL60 cells indicated that myelocytes and metamyelocytes were the most immature cells expressing such markers. Expression of the two surface antigens was also observed when HL60 cells were induced to differentiate into monocyte/macrophage cells by treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Thus, human promyelocytic leukemia cells induced to differentiate in vitro by treatment with specific chemical agents express membrane antigens in the same pattern as normal bone marrow myeloid cells at the corresponding stage of differentiation.

Animals↗
PubMed1981

Growth and differentiation of human and murine erythroleukemia cell lines in serum-free synthetic medium.

Only two chemicals (transferrin and selenium dioxide) are required to supplement serum-free Roswell Park Memorial Institute Medium 1640 for long-term growth and for spontaneous and induced differentiation of established lines of human and mouse erythroleukemia cells. We describe here two serum-free media (a minimal synthetic medium and a high-density synthetic medium) that support the growth and differentiation of human K562(S) and mouse clones 745, 707, and 3TCl 12 erythroleukemia cell lines in long-term culture. The doubling times of the erythroleukemic cell populations are longer in minimal synthetic medium that in serum-containing medium. Cell saturation density in minimal growth medium is one-half that obtained in serum-containing medium for clone 745, whereas for K562(S) it is approximately the same. Cell saturation density in high-density medium (containing albumin) is greater than that achieved in serum-containing medium for K562(S), whereas for clone 745 cell saturation density increases for cells in midlogarithmic growth, although not to the density of cells grown in serum-containing medium. The differences in saturation density are due to a decreased doubling time as well as to better survival of the cells 3 or 4 days after plating. The cells can grow in the synthetic media and be passaged for as many generations as desired without impairment of growth capabilities. In the minimal synthetic medium, spontaneous differentiation of erythroleukemia cells continues to occur, indicating that spontaneously differentiating cells are the result of intracellular mechanisms controlling the expression of a genetic program of some of the cells at any given time. Hemoglobin synthesis can be induced in cells growing in synthetic medium by using lower concentrations of the same inducers that are effective in serum-containing medium, indicating that these chemicals do not depend on serum factors to initiate the process of differentiation. The percentage of benzidine-positive cells and the concentration of hemoglobin per cell, however, are less in the synthetic medium than in serum-containing medium, suggesting that serum factors do play a role in modulating the extent of hemoglobin synthesis. The types of hemoglobins synthesized by cells in synthetic medium are identical to those reported in serum-containing medium.

Animals↗
PubMed1980

The beta major and beta minor globin nuclear transcripts of Friend erythroleukemia cells induced to differentiate in culture.

Friend erythroleukemia clone 745 cells were induced to differentiate by dimethyl sulfoxide (Me2SO) or by hemin treatment, and nuclear transcripts of the beta major and beta minor globin genes examined. The beta major and beta minor genomic sequences selectively labaeled in the nonhomologous part of the large intervening sequence 2 were hybridized to the nuclear RNA of Friend cells. The nuclear RNA of Me2SO-treated cells contained both beta major and beta minor nuclear transcripts, whereas hemin-treated cells contained only the beta minor transcripts. The beta minor nuclear transcripts were more abundant in the Me2SO-treated cells than in hemin-treated cells. Nuclear transcripts with the size of partially processed beta major globin mRNA precursors were also detected in the Me2SO-treated cells.

Animals↗
PubMed1980

The effect of tumor-promoting phorbol diesters on terminal differentiation of cells in culture.

Phorbol diesters with tumor-promoting activity, in particular, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), can induce or inhibit terminal differentiation in a variety of cell systems, with specificity for particular cell lineages. The phorbols are excellent tools to investigate the expression and control of differentiation in some cells and the mechanism by which oncogenic agents interfere with the process of terminal differentiation. The mechanism of action of the phorbols on different target cells is not understood at the present time. It is felt that the status of the cell is of major importance as, in some cases, opposite effects can be achieved by the same concentration of the phorbol diester used. Changes in membranes, receptors, in secretion of prostaglandins and in the level of cyclic AMP have all been reported. However, the relationship of these changes with the alterations in the genetic program involved in the differentiation process is not clear, and the recent report of a possible cell receptor for phorbol diesters should elucidate their mechanism of action. The findings on the effect of phorbol diesters on differentiation have suggested the testable hypothesis that promotion could be mediated through inhibition of cellular differentiation. It has also been suggested that changes in differentiating systems could be of future use in screening for unknown tumor promoters, however, this possibility seems quite remote. Finally, phorbol diesters with tumor-promoting activity appear to exert a specific effect on differentiation of leukemic cells of both mouse and human origin, and therefore, the application of this particular phenomenon in experimental therapy should be the subject of future investigations.

Adipose Tissue↗