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B Calabretta

Publications and source records attributed to B Calabretta.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1991

Positive autoregulation of c-myb expression via Myb binding sites in the 5' flanking region of the human c-myb gene.

The nuclear proto-oncogene c-myb is preferentially expressed in lymphohematopoietic cells, in which it plays an important role in the processes of differentiation and proliferation. The mechanism(s) that regulates c-myb expression is not fully understood, although in mouse cells a regulatory mechanism involves a transcriptional block in the first intron. To analyze the contribution of the 5' flanking sequences in regulating the expression of the human c-myb gene, we isolated a genomic clone containing extensive 5' flanking sequences, the first exon, and a large portion of the first intron. Sequence analysis of a subcloned 1.3-kb BamHI insert corresponding to 687 nucleotides of the 5' flanking sequence, the entire first exon, and 300 nucleotides of the first intron revealed the presence of closely spaced putative Myb binding sites within a segment extending from nucleotides -616 to -575 upstream from the cap site. A 165-bp segment containing these putative Myb binding sites was linked to a human thymidine kinase (TK) cDNA driven by a low-activity proliferating cell nuclear antigen promoter and cotransfected into TK- ts13 cells with a plasmid in which a full-length human c-myb cDNA is driven by the early simian virus 40 promoter; Myb inducibility of TK mRNA expression was observed both in transient expression assays and in stable transformants. The highest level of inducibility was detected when the 165-bp fragment was placed 138 bp upstream of the proliferating cell nuclear antigen promoter-TK cDNA reporter unit or 3' of the TK cDNA. Mutation of the putative Myb binding sites greatly reduced c-myb transactivation of TK mRNA expression and specifically reduced the binding of in vitro-translated Myb protein at those sites. Finally, c-myb transactivated TK mRNA expression driven by a segment of the authentic c-myb 5' flanking region containing the Myb binding sites. These data suggest that human c-myb maintains high levels of Myb protein in cells that require this gene product for proliferation and/or differentiation by an autoregulatory mechanism involving Myb binding sites in the 5' flanking region.

Base Sequence↗
PubMed1991

Constitutively expressed c-myb abrogates the requirement for insulinlike growth factor 1 in 3T3 fibroblasts.

The proto-oncogene c-myb, whose expression is usually limited to cells of the hematopoietic lineages, can be expressed in fibroblasts if placed under the control of a constitutive promoter, such as the simian virus SV40 early promoter. 3T3 cells carrying a constitutively expressed human c-myb were found to grow in 1% serum or in a serum-free, platelet-derived growth factor-supplemented medium, whereas the parent cell line, BALB/c 3T3, needed insulinlike growth factor 1 (IGF-1) in addition to platelet-derived growth factor for growth. myb-carrying cells, however, could not grow in platelet-poor plasma. In fibroblasts, therefore, a constitutively expressed c-myb can abrogate the requirement for platelet-poor plasma or IGF-1. When 3T3 cells constitutively expressed both c-myc and c-myb, they could grow in serum-free medium without added growth factors. The ability of c-myb to abrogate in fibroblasts the IGF-1 requirement seems to be due to its ability to induce overexpression of IGF-1, as indicated by an increase in steady-state levels of IGF-1 mRNA. These results have some important implications; for instance, they suggest a commonality of pathways for entry into S phase in different cell types and the possibility of a myb-like or myb-equivalent gene product of critical importance for entry of fibroblasts into S phase.

Animals↗
PubMed1991

Effect of the myb gene product on expression of the PCNA gene in fibroblasts.

Tk-ts13 cells are BHK-derived fibroblasts that have a G1-specific temperature sensitive (ts) mutation so that the cells arrest in the G1 phase of the cell cycle at the restrictive temperature of 39.6 degrees. We have introduced into these cells a plasmid carrying the human c-myb cDNA under the control of the early SV40 promoter. The resulting cell line, ts13 myb cells, express the c-myb RNA and protein and, when serum-stimulated, they undergo one round of DNA replication even at the restrictive temperature. Under the same conditions, the parent cell line (tk-ts13 cells) are blocked in G1 and fail to replicate DNA. We have investigated the expression of two late growth-regulated genes, PCNA and histone H3, in tk-ts13 and in ts 13 myb cells, both at permissive and restrictive temperature. At the permissive temperature of 34 degrees, the mRNA levels for PCNA and histone H3 increase markedly after serum stimulation in both cell lines, reaching a peak at the time of DNA synthesis. At the restrictive temperature of 39.6 degrees, the mRNA's for PCNA, and histone H3 are detectable in serum-stimulated ts13 myb cells while they are not detectable in the parent tk-ts13 cell line. Run-on transcription assays indicate that these 2 genes are transcribed in tk-ts13 cells equally well at permissive and nonpermissive temperatures, and that the presence of the myb product does not significantly increase their rates of transcription. The stability of the respective mRNA's is roughly the same at either temperature and in both types of cells. These results indicate: (1) a constitutively expressed c-myb gene product confers to fibroblasts the ability of temporarily by-passing a ts block in the G1 phase of the cell cycle and (2) the myb product, under these conditions, regulates the mRNA levels of PCNA and histone H3 either directly or indirectly by a post-transcriptional mechanism.

Animals↗
PubMed1991

Gene-targeted specific inhibition of chronic myeloid leukemia cell growth by BCR-ABL antisense oligodeoxynucleotides.

Philadelphia-chromosome positive chronic myeloid leukemia cells in chronic phase (CML-CP) or blast crisis (CML-BC) and normal bone marrow cells (NBMC) were incubated in vitro with antisense oligonucleotide specific against the BCR/ABL breakpoint junction to examine the possibility of selective inhibition of leukemia growth. Growth capability was determined in vitro by colony assay in semisolid medium in the presence of interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF). The 18-mer antisense directed against the specific BCR/ABL mRNA breakpoint region diminished the colony formation by CML-CP and CML-BC cells, but not by NBMC. Scrambled oligomer did not affect significantly the growth of leukemic and normal cells. If CML-BC cells were mixed with NMBC and incubated with specific BCR/ABL antisense oligomer, leukemic colonies were selectively inhibited, as was shown by reverse, transcriptase-polymerase chain reaction (RT-PCR) performed to detect BCR/ABL mRNA in single colonies. These results confirm the possibility of selective inhibition of leukemia cells by antisense treatment.

Base Sequence↗
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 regulation of human megakaryocyte development.

Extracellular regulators of human megakaryocyte development are becoming better defined. How these regulators function at the subcellular and, in particular, the molecular levels remains almost completely unknown. The recent development of molecular micromethodologies such as in situ hybridization, the polymerase chain reaction, and the use of antisense oligodeoxynucleotides now make such studies possible in normal cells. We therefore examined the effect of several recombinant human hematopoietic growth factors and the maturation agonist phorbol myristate acetate on the expression of selected growth-regulated and maturation/function-related genes. We also examined the role of the c-myb proto-oncogene in regulating megakaryocyte proliferative activity and ploidy development. Our results demonstrate that growth factors have complex time and concentration effects on gene expression in morphologically recognizable human megakaryocytes. They also suggest that a more complete understanding of normal megakaryocyte development at the molecular level will soon be possible.

Actins↗
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

Inhibition of T-cell proliferation by a MYB antisense oligomer is accompanied by selective down-regulation of DNA polymerase alpha expression.

We recently found that inhibition of MYB protein synthesis in human peripheral blood mononuclear cells (PBMC) exposed to human c-myb (designated MYB) antisense oligodeoxynucleotides prevents entry into S phase and cell proliferation. To determine the mechanism(s) by which down-regulation of human c-myb protein (MYB) synthesis interferes with DNA synthesis, we analyzed mRNA levels of DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), transcripts of two genes required for DNA synthesis, in normal and leukemic T lymphocytes exposed to MYB antisense oligodeoxynucleotides. Expression of DNA polymerase alpha was inhibited both in normal T lymphocytes progressing from G0 to S phase and in exponentially growing CCRF-CEM leukemic cells, whereas expression of PCNA was inhibited only in mitogen-stimulated PBMC and remained essentially unaffected in the leukemia T-cell line. The functional link between expression of MYB and DNA polymerase alpha mRNAs was further demonstrated by analyzing DNA polymerase alpha mRNA levels in a temperature-sensitive (ts) fibroblast cell line (TK-ts13; TK is thymidine kinase) constitutively expressing human MYB mRNA driven by the simian virus 40 (SV40) promoter. In the MYB-expressing TK-ts13 cells, DNA polymerase alpha mRNA levels were unaffected following shift to the nonpermissive temperature of 39.6 degrees C, whereas in the parental line, DNA polymerase alpha mRNA levels were readily down-regulated. These findings indicate that the expression of MYB is related to that of DNA polymerase alpha in cells expressing MYB at high levels and suggest that there is a functional link between c-myb and DNA polymerase alpha mRNA expression during cell cycle progression of normal T lymphocytes.

Cell Division↗
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

Altered expression of growth-regulated protooncogenes in human malignant plasma cells.

The expression of three growth-regulated protooncogenes, c-myc, c-myb, and p53, and the S-phase-specific histone H3 gene, was compared in bone marrow cells from multiple myeloma patients and normal controls by measuring the amount of specific RNA by Northern blot analysis. Four samples contained at least 72% of myeloma cells, one sample 43%, and one 11%. Expression of the protooncogenes was similar in normal and myeloma bone marrow cells, whereas that of histone H3 gene was significantly reduced (between 10 and 15 times) in samples containing at least 43% of malignant plasma cells and not detectable in those containing more than 72% of neoplastic cells. Protooncogene levels of expression were compared to those of the H3 gene to distinguish the increased expression of a growth-regulated gene due to a true deregulation from overexpression reflecting solely an increase in the fraction of cycling cells. The ratios of expression of protooncogenes to histone H3 were markedly increased in multiple myeloma cells; the highest ratios were found in the patients with the highest number of malignant plasma cells. These results suggest that the expression of three growth-regulated oncogenes (c-myc, c-myb, p53) is altered in myelomatous plasma cells.

Actins↗
PubMed1989

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

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

Cell Division↗