Biomedical subjects
B Calabretta
Publications and source records attributed to B Calabretta.
Blastic transformation of p53-deficient bone marrow cells by p210bcr/abl tyrosine kinase.
Blastic transformation of chronic myelogenous leukemia (CML) is characterized by the presence of nonrandom, secondary genetic abnormalities in the majority of Philadelphia1 clones, and loss of p53 tumor suppressor gene function is a consistent finding in 25-30% of CML blast crisis patients. To test whether the functional loss of p53 plays a direct role in the transition of chronic phase to blast crisis, bone marrow cells from p53+/+ or p53-/- mice were infected with a retrovirus carrying either the wild-type BCR/ABL or the inactive kinase-deficient mutant, and were assessed for colony-forming ability. Infection of p53-/- marrow cells with wild-type BCR/ABL, but not with the kinase-deficient mutant, enhanced formation of hematopoietic colonies and induced growth factor independence at high frequency, as compared with p53+/+ marrow cells. These effects were suppressed when p53-/- marrow cells were coinfected with BCR/ ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow cells had a proliferative advantage, as reflected by an increase in the fraction of S+G2 phase cells and a decrease in the number of apoptotic cells. Immunophenotyping and morphological analysis revealed that BCR/ABL-positive p53-/- cells were much less differentiated than their BCR/ABL-positive p53+/+ counterparts. Injection of immunodeficient mice with BCR/ABL-positive p53-/- cells produced a transplantable, highly aggressive, poorly differentiated acute myelogenous leukemia. In marked contrast, the disease process in mice injected with BCR/ABL-positive p53+/+ marrow cells was characterized by cell infiltrates with a more differentiated phenotype and was significantly retarded, as indicated by a much longer survival of leukemic mice. Together, these findings directly demonstrate that loss of p53 function plays an important role in blast transformation in CML.
A cell proliferation-dependent multiprotein complex NC-3A positively regulates the CD34 promoter via a TCATTT-containing element.
The CD34 cell surface antigen is a glycoprotein expressed by hematopoietic stem and progenitor cells and also by certain nonhematopoietic cell-types. Because CD34 expression is regulated both at the transcriptional and the posttranscriptional level, we attempted to identify factors that, by interacting with the 5' flanking region of the human CD34 gene, may regulate its promoter activity in proliferating hematopoietic cells. By electrophoretic mobility shift assay, UV cross-linking and DNase I footprinting analyses, we identified a multiprotein complex, designated NC-3A, that specifically interacts with the CD34 promoter region from nucleotides -375 to -351. Sequence analysis of this region revealed the presence of a distinct motif, TCATTT. Chloramphenicol acetyl-transferase assays used to assess promoter activity in transiently transfected cells showed that this TCATTT-containing element, which is conserved in both the human and the murine CD34 genes, mediates positive regulatory activity in hematopoietic and nonhematopoietic cells, and acts as an enhancer when placed upstream of a heterologous promoter. Moreover, loss of CD34 promoter activity was caused by mutation of the TCATTT motif. In addition, the interaction of the nuclear multiprotein complex NC-3A with this enhancer element is proliferation-dependent. These data indicate that, although not cell-type specific, the formation of a multiprotein complex NC-3A interacting with the region from nucleotides -375 to 351 plays an important role in controlling CD34 promoter activity in proliferating hematopoietic cells.
In vitro and in vivo reversal of multidrug resistance in a human leukemia-resistant cell line by mdr1 antisense oligodeoxynucleotides.
A major obstacle to successful cancer chemotherapy is the development of multidrug resistance (MDR) by tumor cells. Overexpression of the mdrl gene product P-glycoprotein (P-170) is characteristic of such cells. In this study, in vitro and in vivo reversion of MDR was attempted in a human leukemia cell line resistant to vincristine (HL-60/Vinc) using an 18-mer mdr1 antisense phosphorothioate oligodeoxynucleotide ([S]ODN) in combination with vincristine. As control of sequence specificity, both sense and scrambled [S]ODNs were used. The ability of these [S]ODNs to reverse MDR was studied in vitro and in severe combined immunodeficient (SCID) mice. In vitro treatment with antisense [S]ODNs restored vincristine sensitivity of HL-60/Vinc cells, whereas no changes in drug sensitivity were observed upon treatment with the sense or scrambled sequence. The in vitro effects correlated with inhibition of P-170 expression in HL-60/Vinc cells exposed to the mdr1 antisense [S]ODNs. In vivo reversal of MDR was obtained in SCID mice given injections of HL-60/Vinc cells and systemically treated with [S]ODNs plus vincristine, as indicated by a significantly prolonged survival of SCID mice that received the combination therapy of mdr1 antisense [S]ODNs + vincristine. Treatments with mdr1 antisense or scrambled [S]ODNs, vincristine, or scrambled [S]ODNs + vincristine had no effect on survival. These results suggest that the use of mdr1 antisense ODNs in combination with standard antineoplastic drugs might be useful in reversing MDR in vitro and in vivo.
Antisense oligodeoxynucleotide combination therapy of primary chronic myelogenous leukemia blast crisis in SCID mice.
The proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC. To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c-myc antisense phosphorothioate oligodeoxynucleotides ([S]ODNs), individually or in combination. Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense [S]ODNs resulted in a synergistic antiproliferative effect. Colony formation of normal bone marrow cells was not affected by either treatment. To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense [S]ODNs or with a combination of both antisense [S]ODNs. Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both [S]ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions. These effects correlated with a markedly increased survival of leukemic mice treated with both antisense [S]ODNs. Leukemic cells harvested from antisense [S]ODN-treated mice were sensitive to the effects of antisense [S]ODNs in vitro, suggesting that the treatment can be successfully repeated. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.
The transcription factors c-myb and GATA-2 act independently in the regulation of normal hematopoiesis.
The transcription factors c-myb and GATA-2 are both required for blood cell development in vivo and in vitro. However, very little is known on their mechanism(s) of action and whether they impact on complementary or overlapping pathways of hematopoietic proliferation and differentiation. We report here that embryonic stem (ES) cells transfected with c-myb or GATA-2 cDNAs, individually or in combination, underwent hematopoietic commitment and differentiation in the absence of added hematopoietic growth factors but that stimulation with c-kit and flt-3 ligands enhanced colony formation only in the c-myb transfectants. This enhancement correlated with c-kit and flt-3 surface receptor up-regulation in c-myb-(but not GATA-2-) transfected ES cells. Transfection of ES cells with either a c-myb or a GATA-2 antisense construct abrogated erythromyeloid colony-forming ability in methyl cellulose; however, introduction of a full-length GATA-2 or c-myb cDNA, respectively, rescued the hematopoiesis-deficient phenotype, although only c-myb-rescued ES cells expressed c-kit and flt-3 surface receptors and formed increased numbers of hematopoietic colonies upon stimulation with the cognate ligands. These results are in agreement with previous studies indicating a fundamental role of c-myb and GATA-2 in hematopoiesis. Of greater importance, our studies suggest that GATA-2 and c-myb exert their roles in hematopoietic gene regulation through distinct mechanisms of action in nonoverlapping pathways.
B-myb promotes S phase and is a downstream target of the negative regulator p107 in human cells.
The retinoblastoma protein family has been implicated in growth control and modulation of the activity of genes involved in cell proliferation, such as B-myb. Recent evidence indicates that the product of the B-myb gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression, B-myb induces deregulated cell growth of certain cell lines. Here we show that B-myb overexpression is able to induce DNA synthesis in p107 growth-arrested human osteosarcoma cells (SAOS2). p107 might exert its growth-suppressive activity by regulating B-myb gene transcription. Indeed, p107 down-modulated B-myb promoter activity and drastically decreased E2F-mediated transactivation. Finally, B-myb was able to stimulate DNA synthesis of both stably and transiently transfected human glioblastoma cells (T98G). Altogether, these data provide definitive evidence that the human B-myb protein is involved in growth control of human cells, and that p107 has a significant role in regulating B-myb gene activity.
Oligonucleotide N3'-->P5' phosphoramidates as antisense agents.
Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where every 3'-oxygen is replaced by a 3'-amino group, were synthesized. These compounds have very high affinity to single-stranded RNAs and thus have potential utility as antisense agents. As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake venom phosphodiesterase, to nuclease activity in a HeLa cell nuclear extract, or to nuclease activity in 50% human plasma, where no significant hydrolysis was observed after 8 h. These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-myb antisense phosphoramidates at 5 microM caused sequence and dose-dependent inhibition of HL-60 cell proliferation and a 75% reduction in c-myb protein and RNA levels, as determined by Western blot and RT-PCR analysis. Analogous results were observed for anti-c-myc phosphoramidates, where a complete cytostatic effect for HL-60 cells was observed at 1 microM concentration for fully complementary, but not for mismatched compounds, which were indistinguishable from untreated controls. This was correlated with a 93% reduction in c-myc protein level. Moreover, colony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleotides, respectively, in a sequence- and dose-dependent manner within a 0.5 nM-5 microM dose range. At these concentrations the colony-forming ability of normal bone marrow cells was not affected. The presented in vitro data indicate that oligonucleotide N3'-->P5' phosphoramidates could be used as specific and efficient antisense agents.
Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotides on human melanoma cells in vitro and and in mice.
BACKGROUND: Phosphorothioate oligodeoxynucleotides ([S]ODNs) contain a modified internucleoside phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used with some therapeutic success in animal models human leukemia; however, the potential for antisense [S]ODN treatment of solid tumors has only recently been explored. PURPOSE: We evaluated the effects of antisense [S]ODNs targeted to the c-myc oncogene on the proliferation of human melanoma cells in vitro and on the growth of human melanoma xenografts in CD-1 nude (nu/nu) mice, METHODS: The effects of 15-mer [S]ODNs containing c-myc sense, c-myc antisense, and two different scrambled sequences on the proliferation and viability of cultures of three established human melanoma cell lines (M14, JR8, and PLF2) were determined by measuring cell numbers and use of the trypan blue exclusion test. The induction of apoptosis in these cells following treatment with [S]ODNs was evaluated by fluorescence-activated cell sorter (FACS) analysis. FACS analysis was also used to determine the effects of [S]ODN treatment on the proliferation of primary cultures of a human melanoma explant (NG cells). The expression of c-Myc protein in cultured NG cells after treatment with [S]ODNs was examined by western blot analysis. The antitumor activity and the toxic effects of several [S]ODN treatment regimens were monitored by measuring differences in tumor weight (percent tumor weight inhibition), tumor growth rate (tumor growth inhibition), animal lifespan (percent increase in lifespan), the number of toxic deaths and the median number of long metastases in treated and control mice bearing NG xenografts. c-Myc protein expression in NG tumor cells following [S]ODN treatment was evaluated by FACS analysis, and the extent of apoptosis in these cells was determined by FACS analysis and morphologic examination. RESULTS: Treatment with antisense [S]ODNs, but not the others, inhibited the growth of all tested melanoma cultures in vitro; FACS analysis revealed that growth inhibition was associated with the induction of apoptosis. Antisense [S]ODN treatment also led to reduced celluLar levels of c-Myc protein. In vivo, [S]ODN antitumor activity and toxicity were dose and schedule dependent; however, only antisense [S]ODNs exhibited antitumor activity. Mice bearing NG xenografts treated with antisense [S]ODNs showed a marked inhibition of tumor growth, a reduction in the number of long metastases, and an increase in life span. Reduced levels of c-Myc protein and increased levels of apoptosis were also observed in NG tumor cells following antisense [S]ODN treatment. CONCLUSIONS: treatment of human melanoma cells and solid tumors with antisense [S]ODNs targeted to c-Myc inhibits their growth and is associated with the induction of apoptosis.
Induction of hematopoietic commitment and erythromyeloid differentiation in embryonal stem cells constitutively expressing c-myb.
To provide insight into the mechanisms by which c-myb regulates hematopoiesis, we analyzed the expression of markers for multiple hematopoietic lineages in differentiating parental embryonic stem (ES) cells and in ES cells transfected with c-myb or with a mutant c-myb deficient in DNA binding and assessed the ability of these cells to undergo hematopoietic commitment and colony formation. Undifferentiated ES cells transfected with intact c-myb, but not cells transfected with mutant c-myb, expressed CD34, c-kit, GATA1, and flt3 mRNA as well as surface CD34, c-kit, and flt3 product. In contrast, the kinetics of GATA-2 mRNA expression was identical in parental and Myb-transfected ES cells. Transient expression assays suggested transactivation of gene expression dependent on interaction with Myb binding sites in the CD34 and GATA1 5' flanking regions. Undifferentiated parental and c-myb mutant-transfected ES cells were not clonogenic, whereas c-myb transfectants formed erythromyeloid colonies in methylcellulose cultures in the absence of added hematopoietic growth factors and, at higher frequency, in the presence of kit and flt-3 ligands. Colony formation was suppressed by treatment with antisense oligodeoxynucleotides specifically downregulating c-kit and flt-3 expression. These findings indicate that c-myb regulates hematopoietic commitment and progenitor cell proliferation and differentiation through the activation of certain genes that define the stem/progenitor cell compartment.
Double knockout of the ALL-1 gene blocks hematopoietic differentiation in vitro.
The ALL-1 gene is involved in translocations with many partner genes in different types of the acute leukemias, but it is not clear whether it acts as an oncogene or whether the fusion proteins resulting from the translocations have dominant negative effects. To distinguish between these two possibilities, we analyzed the ability of wild-type AB2.1 embryonal stem (ES) cells and of single or double ALL-1 gene knockout cells derived from them to differentiate along hematopoietic lineages after withdrawal of leukemia inhibitory factor, using in vitro colony formation assays. All-1 double knockout ES cells formed a significantly greater number of colonies with faster kinetics than wild-type and ALL-1 single knockout ES cells. Parental ES cells formed lineage-restricted colonies, whereas single and double knockout ES cells developed, at high frequency, immature and/or "biphenotypic" colonies, mimicking the aberrant hematopoiesis typical of leukemic patients. These data are consistent with the possibility that loss of function of the ALL-1 gene is important in leukemogenesis.
B-myb transcriptional regulation and mRNA stability during differentiation of neuroblastoma cells.
B-myb and c-myb expression is high in neuroblastoma cells and declines during retinoic acid-induced differentiation. We show here that B-myb down-regulation during retinoic acid-induced differentiation of LAN-5 neuroblastoma cells occurs at the transcriptional level. In addition, we measured B-myb and c-myb messenger RNA half-lives, and found that, unlike c-myb, B-myb messenger RNA was remarkably stable (> 10 h). Inhibition of protein synthesis by treatment with cycloheximide increased B-myb messenger RNA levels, suggesting that one or more labile proteins act as repressors of B-myb transcription. In the same cell line, blocking protein synthesis decreased the level of c-myb mRNA under both normal and differentiative conditions. Thus, B-myb and c-myb undergo similar transcriptional regulation, but there are specific differences in the stability of their messenger RNAs and in the mechanisms through which their transcription is controlled. These differences could reflect different functional roles played by c-myb and B-myb in neuroblastoma cells.
Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo.
We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.
Effect of basic (FGF-2) and acidic (FGF-1) fibroblast growth factors on early haemopoietic cell development.
Basic fibroblast growth factor (FGF-2) and acidic fibroblast growth factor (FGF-1) are mitogens for a variety of cell types. Many reports suggest that haemopoietic cells are among these. Nevertheless, when we examined the effect of recombinant human FGF-1 or 2 on normal human marrow cell proliferation in vitro, only minimal stimulatory activity could be detected. In this regard, the addition of either growth factor to cultures of ancillary cell depleted marrow mononuclear cells (MNC), or to highly enriched CD34+ MNC, failed to enhance haemopoietic colony number and induced only a slight increase in colony size. Perturbation of FGF receptor (FGF-R) expression on CD34+ MNC with antisense (AS) oligodeoxynucleotides (ODN) was also without apparent effect on cell growth. Neither could we demonstrate any effect of FGF-1 or 2 on survival of early progenitor cells in serum-free culture. To explain these findings, we examined progenitor cells for expression of the FGF-R at the mRNA and protein level using RT-PCR and flow cytometry. Primitive CD34+/KIT+ MNC had no detectable FGF-R (FGF-R1, 2, 3 or 4) mRNA or protein expression. In fact, direct immunofluorescence labelling of MNC for CD34 antigen and FGF-R1 demonstrated that expression of these markers was mutually exclusive in the populations examined. FGF-R1 expression was detected on subpopulations of MNC and on cells derived from day-6 CFU-GM and BFU-E colonies. Accordingly, FGF-R1 is either absent, or present at very low levels, on primitive haemopoietic cells. This fact, combined with our in vitro culture data, suggest that receptors are unlikely to play a significant role in the development of these early cells. Nevertheless, the development of mature cells may be influenced by the FGFs since the FGF-Rs are expressed on more mature cells.
BCR/ABL regulation of PI-3 kinase activity.
The ability of BCR-ABL oncoproteins to induce leukemic transformation of hematopoietic cells depends on their tyrosine kinase activity, which is essential for recruitment and activation of multiple pathways that transduce oncogenic signals. Although it is unknown yet whether activation of PI 3-kinase is required for transformation, the colony-forming ability of Philadelphia cells is dependent on PI 3-kinase activity, as indicated by the results of studies using a number of strategies to interfere with the synthesis and/or the function of the regulatory and catalytic subunits of this kinase. In particular, wortmannin, a specific PI 3-kinase inhibitor, preferentially affected colony formation of Philadelphia cells over that of normal marrow hematopoietic progenitors. The mechanism(s) of such effects are unknown, but PI 3-kinase inhibitors may represent a novel class of therapeutic agents for the ex vivo and/or in vivo treatment of Philadelphia leukemias.
Antisense strategies in the treatment of leukemias.
Oligodeoxynucleotides are short nucleotide sequences of DNA synthesized as reverse complements of the desired mRNA target's nucleotide sequence. On formation of the RNA-DNA duplex, gene expression is prevented. Delivery of oligodeoxynucleotides targeting oncogene-encoded mRNAs to human cells in culture is associated with inhibition of cell proliferation and, in some circumstances, cell death. Oligodeoxynucleotides chemically modified to survive nuclease attack have been used systemically in murine models of human hematologic malignancies. In some studies, a measurable antileukemia effect has been observed. On the basis of these preclinical investigations, phase 1 clinical trials involving ex vivo and systemic administration of such compounds are now in progress at different institutions. Despite the remarkable progress of the past few years, much remains to be addressed on uptake, cellular distribution, mechanism(s) of action, and metabolism of such compounds. Furthermore, the "antisense effects" of the oligodeoxynucleotides might also be associated with nonspecific effects. In conclusion, time, a great deal of effort, and patience will tell whether such compounds will have a role as novel antileukemia therapeutic agents.
The influence of phosphorothioate oligodeoxynucleotides on various organs in vivo.
To characterize the distribution and toxicity of phosphorothioate antisense oligodeoxynucleotides ([S]ODNs) in vivo, the mice, previously injected with BV173 leukemic cells (Philadelphia chromosome-positive chronic myeloid leukemia blast-crisis), received intravenously 26-mer BCR-ABL antisense oligodeoxynucleotides (1 mg/mouse/day) for 9 consecutive days. Our investigation revealed that [S]ODNs were distributed to almost all organs except the brain with the highest level in the liver, spleen and kidneys. They were also detected in CD10+ leukemic cells isolated from spleen and bone marrow. Intracellular distribution assay showed the presence of [S]ODNs most prominently in nuclear and cytoplasmic fractions. Our data demonstrated no significant toxicity of [S]ODNs except the increase in spleen weight.
Inhibition of in vitro proliferation of chronic myelogenous leukemia progenitor cells by c-myb antisense oligodeoxynucleotides.
Normal hematopoietic progenitors and acute myelogenous leukemia cells show a differential requirement for the encoded product of c-myb proto-oncogene for proliferation. To determine whether c-myb is also differentially required for the proliferation of hematopoietic progenitors of chronic myelogenous leukemia (CML), mononuclear cells derived from both chronic phase and blast crisis were exposed to c-myb antisense oligodeoxynucleotides and assayed for colony-forming ability. Exposure of CML-BC cells from 12 patients to c-myb antisense oligodeoxynucleotides resulted in significant (p<001) inhibition of leukemia colony formation (average inhibition 63%) and was accompanied by down-regulation of c-myb expression. Colonies derived from CML chronic phase progenitors were virtually unaffected in 10 cases, but down-regulation of c-myb expression was not detected. However, in studies conducted with CD34+ leukemia cells, a subset highly enriched for hematopoietic progenitors, colony formation was inhibited at both disease stages, whereas CFU-GM colony formation derived from normal CD34+ cells was not affected by exposure to c-myb antisense oligodeoxynucleotides. These data suggest that CML chronic phase and blast crisis progenitors are both sensitive to the inhibitory effects of c-myb antisense oligomers, and that the lack of inhibition in partially purified CML-chronic phase progenitors is probably due to inefficient penetration of oligodeoxynucleotides into the clonogenic cells. The preferential effect of c-myb antisense oligodeoxynucleotides on colonies arising from the compartment that includes CML-CD34+ progenitors likely reflects the expansion of a cell population with high proliferative potential and elevated c-myb mRNA levels.