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Biomedical subjects

B Calabretta

Publications and source records attributed to B Calabretta.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of BALB/c 3T3 fibroblast proliferation by B-myb is accompanied by selective activation of cdc2 and cyclin D1 expression.

The B-myb gene is expressed in many cell types at the G1/S transition of the cell cycle. Inhibition of B-myb expression in BALB/c 3T3 fibroblasts by introduction of a B-myb antisense construct greatly diminished cell proliferation, whereas constitutive expression of a human B-myb cDNA in these cells reduced their growth factor requirements and induced a transformed phenotype. Constitutive expression of B-myb cDNA was accompanied by activation of cyclin D1 and cdc2 expression but not of cyclin A and cyclin B. Transfection of BALB/B-myb cells (a cell line expressing high levels of exogenous human B-myb) with a cyclin D1 antisense construct drastically reduced cloning efficiency of these cells. These results suggest that the B-myb-encoded product regulates fibroblast proliferation by activating cdc2 and cyclin D1 gene expression and that abnormal expression of cyclin D1 might be a step in the process of transformation.

3T3 Cells↗
PubMed1992

The Jun family members, c-Jun and JunD, transactivate the human c-myb promoter via an Ap1-like element.

The c-myb protooncogene, which is preferentially expressed in hematopoietic cells at the G1/S boundary of the cell cycle, encodes a transcriptional activator that functions via DNA binding. The regulatory mechanisms governing this specific pattern of expression are not fully understood, although human c-myb expression appears to be positively autoregulated via myb-binding sites in the 5'-flanking region of the c-myb gene (Nicolaides, N. C., Gualdi, R., Casadevall, C., Manzella, L., and Calabretta, B. (1991) Mol. Cell. Biol. 11, 6166-6176). To determine the contribution of other transcription regulators such as JUN family members in the control of c-myb expression, transient expression assays were carried out which revealed a 6- to a 15-fold enhancement by c-Jun and JunD, but not JunB, in chloramphenicol acetyltransferase reporter gene expression driven by different segments of the human c-myb 5'-flanking region. An Ap1-like element located at nucleotide -149 from the c-myb initiation site appears to be required for this transactivation upon binding to a nuclear protein complex containing c-Jun and JunD, since site-directed mutations of this Ap1-like element abolished c-Jun and JunD binding and transactivation. Exposure of phytohemagglutinin-stimulated peripheral blood mononuclear cells to c-jun and junD antisense oligodeoxynucleotides resulted in a 46 and 43% inhibition of T-lymphocyte proliferation that was accompanied by a decrease in c-myb mRNA levels as compared with sense-treated cultures. Because T-lymphocytes induced to proliferate express c-jun and junD before c-myb, these data suggest a mechanism whereby c-Jun and JunD contribute to the transcriptional activation of c-myb that, in turn, is maintained at the G1/S transition and during S phase by positive autoregulation.

Base Sequence↗
PubMed1992

Inhibition of leukemia cell proliferation by receptor-mediated uptake of c-myb antisense oligodeoxynucleotides.

Exposure of human leukemia HL-60 cells to an oligodeoxynucleotide complementary to an 18-base sequence (codons 2-7) of c-myb-encoded mRNA has previously been shown to result in inhibition of cell proliferation. Because HL-60 cells express high levels of transferrin receptor we adapted a DNA delivery system based on receptor-mediated endocytosis to introduce myb oligomers complexed with a transferrin-polylysine conjugate into those cells. A DNA.RNA duplex resistant to S1 nuclease digestion was detected as early as 12 hr after culture of HL-60 cells in the presence of the myb antisense/transferrin-polylysine complex. Exposure of HL-60 cells to the myb antisense/transferrin-polylysine complex resulted in rapid and profound inhibition of proliferation and loss of cell viability much more pronounced than that occurring in cells exposed to free myb antisense oligodeoxynucleotides. The transferrin-polylysine/myb sense complex or the transferrin-polylysine conjugate alone had no effect on HL-60 cell proliferation and viability. These findings indicate that myb synthetic oligodeoxynucleotides enter efficiently into HL-60 by transferrin receptor-mediated endocytosis and exert a profound biological effect. Such a delivery system could exploit other ligand-receptor interactions for the selective delivery of oncogene-targeted antisense oligodeoxynucleotides.

Antineoplastic Agents↗
PubMed1992

Inhibition of proliferation by c-myb antisense RNA and oligodeoxynucleotides in transformed neuroectodermal cell lines.

Transfection of a neuroblastoma cell line with expression vectors containing two different segments of human c-myb complementary DNA in antisense orientation yielded far fewer transfectant clones than did the transfection with the identical segments in sense orientation. In cell clones expressing c-myb antisense RNA, levels of the c-myb protein were down-regulated and the proliferation rate was slower than that of cells transfected with sense constructs or the untransfected parental cell line. Treatment of neuroblastoma and neuroepithelioma cell lines with a c-myb antisense oligodeoxynucleotide strongly inhibited cell growth. These data indicate a definite involvement of c-myb in the proliferation of neuroectodermal tumor cells extending the role of this protooncogene beyond the hematopoietic system. The availability of cell clones that transcribe c-myb antisense RNA provides a useful tool to study the involvement of other genes in the proliferation and differentiation of neuroblastoma cells.

Animals↗
PubMed1992

Role of the KIT protooncogene in normal and malignant human hematopoiesis.

The role of the KIT protooncogene in human hematopoiesis is uncertain. Therefore, we examined KIT mRNA expression in normal human bone marrow mononuclear cells (MNC) and used antisense oligodeoxynucleotides (oligomers) to disrupt KIT function. KIT mRNA was detected with certainty only in growth factor-stimulated MNC. Expression was essentially abrogated by making MNC quiescent or by inhibiting myb gene function. Oligomers blocked KIT mRNA expression in a dose-response and sequence-specific manner, thereby allowing functional examination of the KIT receptor. In experiments with either partially purified or CD34(+)-enriched MNC, neither granulocyte nor megakaryocyte colony formation was inhibited by oligomer exposure. In contrast, KIT antisense oligomers inhibited interleukin 3/erythropoietin-driven erythroid colony formation approximately 70% and "stem cell factor"/erythropoietin-driven colony formation 100%. The presence of erythroid progenitor cell subsets with differential requirements for KIT function is therefore suggested. Growth of hematopoietic colonies from chronic myeloid leukemia and polycythemia vera patients was also inhibited, while acute leukemia colony growth appeared less sensitive to KIT deprivation. These results suggest that KIT plays a predominant role in normal erythropoiesis but may be important in regulating some types of malignant hematopoietic cell growth as well. They also suggest that KIT expression is linked to cell metabolic activity and that its expression may be regulated by or coregulated with MYB.

Antigens, CD↗
PubMed1992

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.

Antigens, CD↗
PubMed1992

Antisense oligonucleotides.

Growing evidence indicates that antisense oligodeoxynucleotides can specifically inhibit gene expression thereby providing an essential tool for understanding gene function and the potential to affect abnormal cell proliferation. Because oncogene activation is intimately involved in tumour initiation and progression, down-regulation of oncogene expression is associated with a selective or a preferential inhibition of tumour as compared to normal cell proliferation. Even though numerous studies attest the short-term in vitro efficacy of antisense oligodeoxynucleotides as inhibitors of tumour growth, the use of these compounds as therapeutic agents awaits a more rigorous demonstration of their long term effects and favourable pharmacological properties.

Antineoplastic Agents↗
PubMed1992

A model of Ph' positive chronic myeloid leukemia-blast crisis cell line growth in immunodeficient SCID mice.

A human Philadelphia-chromosome positive chronic myeloid leukemia-blast crisis (CML-BC) cell line BV173 proliferated in the hematopoietic tissues, infiltrated various organs and caused the death of immunodeficient SCID mice. Leukemia spreading was assessed with diminished number of bone marrow cells and caused splenomegaly. The leukemic colonies grew from single cell suspension of bone marrow, spleen and peripheral blood. Bcr-abl m-RNA was detectable in bone marrow, spleen, peripheral blood, liver, lungs and brain. Dying mice demonstrated severely hypoplastic bone marrow, splenomegaly and massive metastases in the liver and kidneys. The survival time of animals was dependent on the number of inoculated leukemia cells.

Animals↗
PubMed1992

c-myb and growth control.

The available evidence indicates that c-myb plays an important role in the proliferation of hematopoietic cells and in those nonhematopoietic cell types in which c-myb is expressed. A critical aspect in the regulation of c-myb expression rests in the positive autoregulatory mechanism, which is dependent on the interaction of myb protein with the 5' flanking region of the human c-myb gene. The positive autoregulation of c-myb, in conjunction with tissue-specific mechanisms that most likely involve efficient transcription beyond the site of "transcriptional pause" in the c-myb first intron, might allow the generation of c-myb transcripts at levels sufficiently high for optimal biological activity (e.g., at the G1/S transition of the cell cycle). Other transactivating factors, such as the Jun family members, also appear to be involved in regulating c-myb expression. Such factors might act to increase basal levels of c-myb expression to allow activation of the autoregulatory mechanism, or might cooperate with myb in transcriptional regulation of c-myb expression. The function of c-myb is ultimately dependent on the genes that are regulated by the myb product. Preliminary evidence suggests that DNA polymerase-alpha and cdc2, two genes that are critical for DNA synthesis, contain myb binding sites in their promoter region that appear to be required for myb transactivation of their expression. The paradox of the generality of the mechanisms by which c-myb affects cell proliferation and the apparent tissue-specific expression of this gene might be resolved by the growing evidence that the tissue distribution of c-myb is more general than previously appreciated, and that many cell types with no detectable c-myb expression contain a functional equivalent of this gene. For example, B-myb a gene that is homologous to c-myb in the DNA binding and transactivating domains and appears to be ubiquitously expressed, is also required for cell proliferation and, like c-myb, appears to regulate the expression of cdc2, a gene required for cell cycle progression. Together, these findings indicate a general role of members of the myb family in regulation of cell proliferation.

Cell Division↗
PubMed1991

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.

Base Sequence↗
PubMed1991

Chromosome locations of the MYB related genes, AMYB and BMYB.

The MYB related loci, AMYB and BMYB, were localized to specific human chromosome regions by Southern blot analysis of their segregation patterns in a panel of rodent-human hybrid DNAs using radiolabeled AMYB and BMYB probes. The AMYB locus was present in hybrids retaining the chromosome region 8cen----8q22 and was absent in hybrids which had lost this chromosome region. The presence of the BMYB locus in rodent-human hybrids correlated with, and only with, chromosome region Xq13. Chromosomal in situ hybridization refined the localization of AMYB to region 8q22-23 and confirmed the localization of BMYB to region Xq13. Chromosome region 8q22 is involved in recurrent translocations in malignant lymphoma and in acute myeloid leukemia (AML-M2); therefore AMYB is a candidate for involvement in such translocations. A region on Xq13 is also involved in chromosomal abnormalities in acute myeloid leukemia and myelodysplasias.

Chromosome Aberrations↗
PubMed1991

Inhibition of proliferation by c-myb antisense oligodeoxynucleotides in colon adenocarcinoma cell lines that express c-myb.

Steady-state mRNA levels of the protooncogene c-myb were measured by Northern blot analysis in the human colon carcinoma cell lines LoVo, the doxorubicin-resistant derivative LoVo/Dx, Colo 205, and HT 29. Overexpression of c-myb mRNA was detected in the Colo 205 cell line, probably because of gene amplification, while in human HT 29 cells c-myb was not expressed at a detectable level. Comparison between LoVo and LoVo/Dx cell lines showed that c-myb mRNA levels were much higher in the doxorubicin-resistant derivative than in the parental line. c-myb antisense oligodeoxynucleotides inhibited cell proliferation only in the cell lines with detectable mRNA c-myb (LoVo, LoVo/DX, and Colo 205). The dose of antisense exerting inhibitory effect was related to the levels of c-myb mRNA expression. Inhibition of c-myb expression in antisense-treated LoVo/DX cells was demonstrated by the reverse transcriptase polymerase chain reaction technique. LoVo/Dx cells were induced to differentiate by treatment with dimethylformamide to determine whether down-regulation of c-myb expression would accompany the process of differentiation. During the treatment with dimethylformamide the expression of c-myb decreased in parallel with the reduction of cell growth, while terminal differentiation of these cells was associated with changes in the expression of carcinoembryonic antigen and laminin receptor genes. Our findings demonstrate that the expression of c-myb is important for the proliferation of colon carcinoma cell lines and suggest that the role of this protooncogene is not restricted to cells of hematopoietic origin but is more general than previously thought.

Adenocarcinoma↗
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

Growth regulated expression of B-myb in fibroblasts and hematopoietic cells.

The B-myb cDNA has extensive sequence similarities to the c-myb proto-oncogene, but, at variance with c-myb, it is expressed in cells other than hematopoietic cells. In this paper, we show that (1) B-myb is expressed in mouse, human, and hamster fibroblasts; (2) B-myb mRNA levels are growth-regulated in both fibroblasts and peripheral blood mononuclear cells; (3) by its mode of growth regulation (peak of expression, behavior in G1-specific temperature sensitive (ts) mutants and in the presence of cycloheximide), B-myb can be classified, like c-myb, thymidine kinase, PCNA, and others, as a late growth-regulated gene; (4) B-myb mRNA levels decrease when HL-60 cells are induced to differentiate; and (5) the increase in mRNA levels in serum-stimulated cells is only partially explained by an increase in rate of transcription. The possibility that the B-myb gene may be the equivalent in fibroblasts and epithelial cells of the c-myb proto-oncogene of hematopoietic cells is discussed.

Animals↗