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Biomedical subjects

B Calabretta

Publications and source records attributed to B Calabretta.

At least 109 records · Page 6Linked to original sources
PubMed1994

Lack of correlation between N-myc and MAX expression in neuroblastoma tumors and in cell lines: implication for N-myc-MAX complex formation.

Detectable levels of MAX messenger RNA were found in a set of human neuroblastoma tumors and established cell lines. MAX mRNA levels were independent of tumor stage and N-myc genomic amplification. By contrast, N-myc mRNA transcripts were detectable only in tumors with amplification of N-myc gene and in cell lines. Analysis by reverse transcriptase polymerase chain reaction and hybridization to specific oligodeoxynucleotide probes revealed approximately equal amounts of two MAX transcripts in all cases analyzed. Immunoprecipitations with a specific antibody to MAX detected two proteins of M(r) 21,000 and 22,000 in approximately equal amounts in all neuroblastoma lines regardless of N-myc amplification and/or expression. On the other hand, protein binding to the myc DNA consensus sequence correlated with N-myc expression in neuroblastoma cells. Thus, N-myc expression might be a limiting factor in the formation of the N-myc-MAX heterodimer in neuroblastomas.

Amino Acid Sequence↗
PubMed1994

Correlation between E2F-1 requirement in the S phase and E2F-1 transactivation of cell cycle-related genes in human cells.

The mammalian nuclear protein E2F-1 has recently been cloned based on its ability to bind the retinoblastoma protein. To determine whether E2F-1 plays a role in the control of the cell proliferation, we introduced an inducible construct expressing an E2F-1 antisense RNA into the human glioblastoma T98G cell line and assessed DNA synthesis during the cell cycle. Expression of the antisense transcripts during the G1-S transition resulted in a marked delay in the completion of DNA synthesis. Band-shift analysis of bacterially produced E2F-1 showed that this protein bound to the promoters of human DNA polymerase-alpha, cyclin D1, and c-myb but not to the cdc2 gene promoter. E2F-1 also transactivated the bound promoters in transient transfection assays. These results suggest a major role for E2F-1 in the control of cell cycle progression via transcriptional regulation of proliferation-associated genes.

Base Sequence↗
PubMed1994

Regulation of the expression of the hematopoietic stem cell antigen CD34: role of c-myb.

The CD34 antigen defines a subset of hematopoietic progenitor cells with self-renewal capacity and the ability to reconstitute hematopoiesis in irradiated primates and marrow-ablated humans, but its function remains unknown. The c-myb protooncogene plays a fundamental role in hematopoiesis, most likely via its transcriptional regulator function. We report that c-myb protein transactivates the CD34 promoter via specific interaction with multiple Myb binding sites in the 5' flanking region of the gene and induces expression of the endogenous CD34 mRNA in rodent fibroblasts. Also, constitutive expression of c-myb in CD34-negative human glioblastoma cells induces expression of CD34 mRNA and synthesis of the surface membrane antigen. These data directly demonstrate that c-myb regulates the expression of the hematopoietic stem cell antigen CD34 and raise the possibility that c-myb regulates hematopoiesis inducing a cascade of differentiation-related events.

Animals↗
PubMed1994

Oncogene-targeted antisense oligodeoxynucleotides combined with chemotherapy or immunotherapy: a new approach for tumor treatment?

Synthetic oligodeoxynucleotides (antisenses) complementary to bcr/abl breakpoint junction transcript on Philadelphia chromosome, or c-myb protooncogene inhibit partially the proliferation of Philadelphia positive leukemic cells (antisenses against bcr/abl and c-myb) and other tumor cells (antisenses against c-myb). This phenomenon is accompanied by specific downregulation of mRNA level of the particular gene. To develop a more effective procedure of tumor treatment the combination of low dose of cytostatic and bcr/abl or c-myb antisenses against Philadelphia chromosome positive cell line BV173, and the combination of anti-tumor cytotoxic T lymphocytes (CTL) and c-myb antisenses against melanoma cell line MM-28, were tested in vitro. Our results indicate that the combinations of conventional chemotherapeutic agent and antisense against bcr/abl or c-myb or tumor specific CTL and antisense against c-myb, are highly effective in killing of tumor cells and sparing normal cells. This creates the possibility to develop a more selective and effective treatment of neoplasia.

Bone Marrow Cells↗
PubMed1994

Inhibition of leukaemia cell proliferation by folic acid-polylysine-mediated introduction of c-myb antisense oligodeoxynucleotides into HL-60 cells.

The inhibitory effect of c-myb antisense oligodeoxynucleotides (ODNs) conjugated to folic acid (FA) on HL-60 cell proliferation was examined. Folic acid was covalently linked to a polylysine chain and purified by gel chromatography. Sterile FA-polylysine was complexed with c-myb sense and antisense. Exposure of HL-60 cells to the FA-polylysine-c-myb antisense ODN complex resulted in a down-regulation of c-myb expression and a greater inhibition of proliferation than that obtained using free ODNs. Moreover, FA-polylysine conjugate alone or complexed to c-myb sense ODN was not toxic to cells. The antigenic properties and uptake of the vitamin were not affected by the polylysine chain. These data suggest that this strategy is potentially useful for the selective delivery of anti-oncogene-targeted ODNs into cancer cells.

Base Sequence↗
PubMed1994

c-myb down regulation is associated with apoptosis in human neuroblastoma cells.

The c-myb transcriptional regulator plays a crucial role in the control of several proliferative/differentiative processes in haematopoietic cells. Its expression and function is not lineage-restricted, since c-myb is also expressed in solid tumours such as neuroblastomas, where its transcription is decreased by retinoic acid. In response to retinoic acid, neuroblastomas differentiate either towards a neuronal phenotype or undergo apoptosis. The temporal relationship between reduction in c-myb mRNA levels and the differentiative/proliferative/apoptotic processes suggests that c-myb may play a key role in the control of growth of these neuroectodermal tumours. Transfection of neuroblastoma cells with expression vectors containing segments of human c-myb cDNA in antisense orientation yielded fewer transfectant clones, with a far slower proliferation rate, than transfection with the corresponding sense construct. The dramatic growth arrest and reduction in cell number in the antisense transfectants is due to the induction of apoptosis. Apoptosis in the c-myb antisense-transfected cells is further increased in reduced serum conditions.

Journal Article↗
PubMed1994

The role of c-Myc protooncogene in chronic myelogenous leukemia.

c-Myc transcriptional factor encoded by c-myc protooncogene plays an important role in the regulation of cell cycle. It was also established that c-Myc is important for the transformation of fibroblasts and murine bone marrow cells induced by BCR/ABL tyrosine kinase encoded by bcr/abl oncogene localized on Philadelphia-chromosome (Ph1). The role of c-Myc in the proliferation of the leukemic cells was not known. Therefore, we examined the effect of c-Myc protein downregulation, using antisense oligodeoxynucleotides, on the growth of the BCR/ABL- dependent cell line and chronic myelogenous leukemia (CML) patients cells. Downregulation of c-Myc expression caused complete inhibition of the proliferation of BCR/ABL-dependent BV173 cell line and 50-70% inhibition of the colony formation of CML cells. These results suggests that c-Myc cooperates with BCR/ABL and is necessary for the growth of Ph1-positive leukemias.

Base Sequence↗
PubMed1994

Antisense BCR-ABL oligonucleotides induce apoptosis in the Philadelphia chromosome-positive cell line BV173.

BCR-ABL antisense oligonucleotides can specifically reduce colony formation of early hematopoietic progenitor cells from chronic myeloid leukemia (CML) patients. Little is known about the mechanism of this inhibition. We studied the inhibition of the bcr-abl oncogene using fluorescein-labeled phosphorothioate oligonucleotides in the Philadelphia chromosome-positive cell line BV173. Oligonucleotide stability, uptake, bcr-abl mRNA degradation, inhibition of cell proliferation, and cell death were studied. The oligonucleotide uptake was directly dependent on the extracellular concentration and was constant over the first 18 h of incubation. After that the uptake rate decreased. We detected a decrease in bcr-abl mRNA after 3 days of treatment with antisense oligonucleotides, but much less in controls. The controls used in the experiments were the sense oligonucleotide, equimolar amounts of sense and antisense, and an untreated control. Antisense oligonucleotides completely inhibited cell growth of BV173 cells and did not inhibit growth of HL-60 cells, whereas control oligonucleotides had no such effect on either cell line. An oligonucleotide specific for the other CML breakpoint was also effective in reducing cell growth of BV173. By the use of a DNA double staining technique to discriminate between necrotic and apoptotic cells, we detected a large number of apoptotic cells in antisense treated BV173 cultures after 5 days of treatment as compared to controls. We conclude that antisense BCR-ABL oligonucleotides reduce bcr-abl mRNA expression in BV173 cells mainly in a sequence-specific manner and induce apoptosis.

Apoptosis↗
PubMed1993

p120 GAP requirement in normal and malignant human hematopoiesis.

There is evidence to suggest that the p120 GAP (GAP), originally described as an inhibitor of p21ras, may also serve as a downstream effector of ras-regulated signal transduction. To determine whether GAP expression is required for the growth of human normal and leukemic hematopoietic cells, we used GAP antisense oligodeoxynucleotides to inhibit it and analyzed the effects of this inhibition on the colony-forming ability of nonadherent, T lymphocyte-depleted mononuclear cells and of highly purified progenitors (CD34+ MNC) obtained from the bone marrow and peripheral blood of healthy volunteers or chronic myeloid leukemia (CML, bcr-abl-positive) patients. The acute myelogenous leukemia cell line MO7, the Philadelphia BV173 cell line, and the acute promyelocytic leukemia NB4 and HL-60 cell lines were similarly examined. GAP antisense treatment inhibited colony formation from normal myelo-, erythro-, and megakaryopoietic progenitor cells as well as from CML progenitor cells. Proliferation of MO7 (growth factor-dependent) and BV173 (bcr-abl-dependent) cells, but not that of NB4 and HL-60 (growth factor-independent) cells, was also inhibited, even though a specific downregulation of GAP was observed in each cell line, as analyzed by either or both mRNA and protein expression. Stimulation of MO7 cells with hematopoietic growth factors increased the expression of GAP as well as the levels of active GTP-bound p21ras. Stimulation of GAP expression was inhibited upon GAP antisense treatment. These data indicate that p120 GAP is involved in human normal and leukemic hemopoiesis and strongly suggest that GAP is not only a p21ras inhibitor (signal terminator), but also a positive signal transducer.

Bone Marrow Cells↗
PubMed1993

Regulation of proliferation and cytokine expression of bone marrow fibroblasts: role of c-myb.

The c-myb protooncogene plays a major role in regulating the process of in vitro and in vivo hematopoiesis via its activity as transcriptional regulator in hematopoietic progenitor cells. Since the bone marrow microenvironment appears to regulate in vivo hematopoiesis by maintaining the growth of multipotent progenitors via secretion of specific cytokines, we asked whether c-myb is also required for the proliferation of and/or cytokine production by stromal cells that generate fibroblast-like colonies (fibroblast colony-forming units [CFU-F]). Using the reverse transcriptase polymerase chain reaction technique, we detected low levels of c-myb mRNA transcripts in human normal bone marrow fibroblasts. Treatment of these cells with c-myb antisense oligodeoxynucleotides caused downregulation of c-myb expression, decreased in the number of marrow CFU-F colonies (approximately 54% inhibition) and in the cell number within residual colonies (approximately 80%), and downregulation of granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) mRNA expression. Transfection of T98G glioblastoma cells, in which expression of c-myb, GM-CSF, and SCF mRNAs is undetectable or barely detectable, with a plasmid containing a full-length c-myb cDNA under the control of the SV40 promoter induced the expression of biologically active SCF and GM-CSF in these cells. Regulation of GM-CSF expression by c-myb was due in part to transactivation of the GM-CSF promoter. These results indicate that, in addition to regulating hematopoietic cell proliferation, c-myb is also required for proliferation of and cytokines synthesis by bone marrow fibroblasts.

Base Sequence↗
PubMed1993

c-myb transactivates cdc2 expression via Myb binding sites in the 5'-flanking region of the human cdc2 gene.

The c-myb protooncogene is preferentially expressed in hematopoietic cells and is required for cell cycle progression at the G1/S boundary. Because c-myb encodes a transcriptional activator that functions via DNA binding, it is likely that c-myb exerts its biological activity by regulating the transcription of genes required for DNA synthesis and cell cycle progression. One such gene, cdc2, encodes a 34-kDa serine-threonine kinase that appears to be required for G1/S transition in normal human T-lymphocytes. To determine whether c-myb is a transcriptional regulator of cdc2 expression, we subcloned a segment of a cdc2 human genomic clone containing extensive 5'-flanking sequences and part of the first exon. Sequence analysis revealed the presence of two closely spaced Myb binding sites that interact with bacterially synthesized Myb protein within a region extending from nucleotides -410 to -392 upstream of the transcription initiation site. A 465-base pair segment of 5'-flanking sequence containing these sites was linked to the CAT gene and had promoter activity in rodent fibroblasts. Cotransfection of this construct with a full-length human c-myb cDNA driven by the early simian virus 40 promoter resulted in a 6-8-fold enhancement of CAT activity that was abrogated by mutations in the Myb binding sites. These data suggest that c-myb participates in the regulation of cell cycle progression by activating the expression of the cdc2 gene.

Animals↗
PubMed1993

Wild-type p53 differentially affects tumorigenic and metastatic potential of murine metastatic cell variants.

The structure and the function of the p53 gene were studied in two metastatic cell variants derived from Lewis lung carcinoma. Single missense mutation at codon 334 was detected in the p53 gene of both cell variants. In spite of the identical mutation, the in vitro and in vivo growth rates of the two cell variants were differentially affected by the constitutive expression of exogenous wild-type (wt) p53 gene. In fact, only the more malignant cell line (C87) was severely affected by the wt-p53 gene introduction. However, the in vivo effects on this cell line were transient because during serial in vivo passages, cell populations lacking the wt-p53 gene were selected. Genetic mechanisms responsible for the resistance of the less metastatic cell variant (BC215) to the wt-p53 expression, were investigated. Intrinsic ability to mutate exogenous cDNA sequences was tested. We report that BC215 cells continued to express exogenous wt-p53 sequences after several in vitro passages. The expression of mdm2 gene was evaluated. The data demonstrated that BC215 cells constitutively express higher levels of mdm2 gene than C87 cells. We conclude that the overexpression of this gene might be responsible for the resistance of BC215 cells to exogenous wt-p53 gene expression.

Animals↗
PubMed1993

Highly efficient elimination of Philadelphia leukemic cells by exposure to bcr/abl antisense oligodeoxynucleotides combined with mafosfamide.

Synthetic oligodeoxynucleotides complementary to the break-point junction of bcr-abl transcripts selectively inhibit the proliferation of Philadelphia-positive leukemic cells, but residual leukemic cells persist in antisense oligodeoxynucleotides-treated cultures. Cyclophosphamide derivatives such as mafosfamide and 4-hydroperoxycyclophosphamide are used at high doses for purging of Philadelphia leukemic cells from marrows but such treatment can be associated with delayed engraftment and prolonged cytopenias. To develop a more effective procedure that might optimize the killing of leukemia cells and the sparing of normal hematopoietic progenitor cells, a 1:1 mixture of Philadelphia leukemic cells and normal bone marrow cells was exposed to a combination of a low dose of mafosfamide and bcr-abl antisense oligodeoxynucleotides and assayed for growth ability in clonogenic assays and in immunodeficient mice. Bcr-abl transcripts were not detected in residual colonies, and cytogenetic analysis of individual colonies revealed a normal karyotype. Normal but not leukemic hematopoietic colonies of human origin were also detected in marrows of immunodeficient mice 1 mo after injection of the treated cells. Our results indicate that a combination of a conventional chemotherapeutic agent and a tumor-specific antisense oligodeoxynucleotide is highly effective in killing leukemic cells and in sparing a much higher number of normal progenitor cells as compared with high-dose mafosfamide treatment. This offers the prospect of a novel and more selective ex vivo treatment of chronic myelogenous leukemia.

Animals↗
PubMed1993

Successful mafosfamide purging of bone marrow from chronic myelogenous leukemia (CML) cells.

The in vitro sensitivity of human chronic myeloid leukemia-blast crisis and chronic phase (CML-BC and CML-CP, respectively) cells as well as adherent cell-depleted, T lymphocyte-depleted normal bone marrow cells (A-T-NBMC) to various concentrations of mafosfamide (ASTA Z7654), was examined by colony formation assay in the presence of IL-3 and GM-CSF, to test the possibility of purging of BMC from CML cells. Colony formation by CML cells was inhibited more efficiently than by NBMC. After the incubation with 50 micrograms/ml or 100 micrograms/ml of mafosfamide, the growth of leukemic CFU-GM was totally abrogated in 2/11 or 9/11 cases of CML-BC and in 1/7 or 6/7 cases of CML-CP, respectively. At the same time the CFU-GM arising from normal BMC were not inhibited totally with 50 or 100 micrograms/ml of the drug in any of five experiments. CML cells were still unable to form secondary colonies, while normal BMC were capable of regrowth. The CD34+ cells isolated form CML-BC and CML-CP patients were also more susceptible to mafosfamide cytotoxicity in comparison to CD34+ cells derived from NBMC. To confirm the possibility of purging, CML-BC cells were mixed with NBMC (1:1) and incubated with mafosfamide. Finally, the growing colonies were examined for the presence of bcr/abl hybrid gene by reverse transcriptase-Taq polymerase chain reaction (RT-PCR) and specific hybridization. The bcr/abl gene was not detected in the colonies growing after 100 micrograms/ml, and the signal was diminished after incubation with 50 micrograms/ml of mafosfamide, as compared to control. These results strongly suggest that high concentrations of mafosfamide may be useful for the purging of autologous BMC from CML cells.

Antineoplastic Agents↗
PubMed1993

Elimination of clonogenic Philadelphia-positive cells using BCR-ABL antisense oligodeoxynucleotides.

Synthetic BCR-ABL antisense oligodeoxynucleotides are known to suppress in vitro clonogenic growth of primary cells from patients with CML. To evaluate the use of BCR-ABL antisense oligodeoxynucleotides as in vitro purging agents before autografting, we studied their effect on the clonogenic BV-173 cell line. At a concentration of 80 micrograms/ml, antisense oligodeoxynucleotides suppressed 85%, 95.5% and 95% of leukaemic cell growth after 24, 48 and 72 h of incubation, respectively. A correlation was observed between the concentration of oligomers and cell inhibition; however, concentrations higher than 80 micrograms/ml did not produce significant increase of BV-173 cell elimination. When the antisense treatment was performed on a wide range of target cell concentrations (from 1 x 10(5) to 5 x 10(6)/ml), the efficacy of purging was similar in all the groups. Addition of antisense oligodeoxynucleotides every 24 h produced, after 3 days, a cell growth inhibition superior to any single treatment; in particular, three treatments for 24 h at a concentration of 80 micrograms/ml were more effective than a single treatment at the same concentration or a single treatment at a concentration of 160 micrograms/ml. Incubation of normal human BM cells with the same doses which were very effective at inhibiting BV-173 cell proliferation, did not affect the growth of normal CFU-GM and of more immature progenitors.

Base Sequence↗
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↗