PubMed Health⌕ Search

Biomedical subjects

B Calabretta

Publications and source records attributed to B Calabretta.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1989

Structure of the human gene for the proliferating cell nuclear antigen.

The proliferating cell nuclear antigen (PCNA, cyclin) was originally defined as a nuclear protein whose appearance correlated with the proliferative state of the cell. It is now known to be a co-factor of DNA polymerase delta and to be necessary for DNA synthesis and cell cycle progression. cDNA clones of human PCNA have been isolated and, using one of these cDNA, we have now obtained from a lambda phage library a clone containing the entire human PCNA gene and flanking sequences. The human PCNA gene is a unique copy gene and has 6 exons. It spans, from the cap site to the poly(A) signal 4961 base pairs. We have identified, in the 5'-flanking sequence, a region with promoter activity, a well as other structural elements common to other promoters. An interesting feature of the PCNA gene is the presence of extensive sequence similarities among introns and between introns and exons.

Base Sequence↗
PubMed1989

Human gene for proliferating cell nuclear antigen has pseudogenes and localizes to chromosome 20.

We have isolated from a human genomic library a pseudogene of the proliferating cell nuclear antigen (PCNA) gene. Its sequence shows a 78% similarity with the human PCNA/cDNA. The PCNA gene is located on human chromosome 20, while the pseudogene maps to chromosome region Xpter in equilibrium Xq13. An additional locus detected by the full-length PCNA cDNA, but not by intron probes, segregates concordantly with chromosome region 6p12 in equilibrium 6pter and probably represents a second pseudogene.

Amino Acid Sequence↗
PubMed1989

An oligomer complementary to c-myb-encoded mRNA inhibits proliferation of human myeloid leukemia cell lines.

To study the role of the protooncogene c-myb in regulating myeloid leukemia cell proliferation and differentiation, we exposed cells of the human leukemia lines HL-60, ML-3, KG-1, and KG-1a to an oligodeoxynucleotide complementary to an 18-base-pair (bp) sequence of c-myb-encoded mRNA. This treatment resulted in a significant decrease in cell proliferation in all of the lines, which was most marked in HL-60 cells. After 5 days in culture, in several separate experiments with different oligomer preparations, 75% growth inhibition was observed in c-myb antisense treated cells in comparison to untreated HL-60 cells. Two c-myb antisense oligomers of identical length with either 2- or 4-bp mismatches had no effect on cell growth nor did an 18-bp c-myb sense or myeloperoxidase antisense oligomer. The effect of a c-myc antisense oligomer (18 bp) on the growth of HL-60, KG-1, and KG-1a cells was also studied. This oligomer had much less inhibitory effect on cell proliferation than did the c-myb antisense sequence. Interestingly, although c-myc antisense treatment induced maturation of HL-60 cells while it inhibited cell proliferation, such an effect was not noted in c-myb antisense treated cells. These studies indicate that the nuclear protein encoded by the c-myb protooncogene is required for maintenance of proliferation in certain leukemia cell lines. In compared to c-myc protein suggest that, at least in HL-60 cells, c-myc amplification or N-ras activation may not be sufficient to maintain the leukemic growth in the absence of c-myb protein. These findings support the hypothesis that development and maintenance of a malignant phenotype requires a multiplicity of interrelated genetic events.

Cell Differentiation↗
PubMed1989

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis.

Amino Acid Sequence↗
PubMed1989

Expression of the myeloperoxidase gene in acute and chronic myeloid leukemias: relationship to the expression of cell cycle-related genes.

The expression of the myeloperoxidase (MPO) gene was studied, by means of Northern blot analysis in 14 cases of acute myeloid leukemia (AML), 11 cases of chronic myeloid leukemia (CML), and 6 cases of CML blast crisis, and in HL60 cells before and after induction of terminal differentiation with retinoic acid (RA), phorbol esters (TPA), or vitamin D. The expression of a panel of cell cycle-related genes, namely C-MYC, histone H3, ornithine decarboxylase, P53, vimentin, and calcyclin, was also studied in the same cell populations. Our results indicate that: (a) MPO gene expression (steady state mRNA levels) is strictly confined to the first stages of myeloid differentiation, reaching its peak at the promyelocyte stage and becoming undetectable in mature granulocytes and monocytes; (b) cells devoid of any detectable MPO enzymatic activity such as leukemic basophils have a high content of MPO mRNA; and (c) MPO gene expression is not related to the growth activity of the cell population. Finally, our results show that the pattern of expression of growth-regulated genes in the neoplastic myeloid disorders AML, CML, and CML blast crisis is remarkably different.

Blast Crisis↗
PubMed1988

A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis in vitro.

The nuclear protein encoded by the proto-oncogene c-myb has been hypothesized to play an important role in the process of hematopoiesis, but direct proof of this function has been lacking. To address this issue, normal human bone marrow mononuclear cells were exposed to c-myb sense and antisense synthetic oligodeoxynucleotides, and the effects on hematopoietic colony formation and maturation were examined. Exposure of these cells to c-myb antisense, oligodeoxynucleotides resulted in a decrease in both colony size and number, without apparent effect on the maturation of residual colony cells. Exposure to c-myb sense, or irrelevant antisense, oligonucleotides had no such effect. These results show that (i) c-myb plays a critical role in regulating normal human hematopoiesis and (ii) the combined use of antisense oligodeoxynucleotides and hematopoietic cell culture techniques will provide a powerful tool for studying the role of proteins encoded by proto-oncogenes, or other specific genes, in normal human hematopoiesis.

Cell Differentiation↗
PubMed1988

Regulation of the proliferating cell nuclear antigen cyclin and thymidine kinase mRNA levels by growth factors.

The enzymes of the DNA synthesizing machinery constitute a group of gene products that are generally expressed co-ordinately at the G1/S boundary of the cell cycle. We have investigated how growth factors regulate the steady-state mRNA levels of two of these genes, the PCNA (proliferating cell nuclear antigen)/cyclin and the thymidine kinase genes. To detect the PCNA/cyclin mRNA, we isolated a cDNA clone from a human library. Two different cell lines were used for these studies: BALB/c3T3 cells, which are exquisitely sensitive to growth factors, and ts13 cells, a temperature-sensitive (ts) mutant of the cell cycle, which arrests in G1 at the restrictive temperature. The steady-state levels of the RNAs for these two genes under different growth conditions were also compared with the levels of histone H3 RNA which are good indicators of the fraction of cells in S phase. Both PCNA/cyclin and thymidine kinase genes share two fundamental characteristics, i.e. they are not inducible in a G1-specific ts mutant of the cell cycle at the restrictive temperature and their expression is inhibited by cycloheximide, indicating that unlike early growth-regulated genes, they require the previous expression of other growth-regulated genes. However, the two genes also show differences, the most notable being that PCNA/cyclin is inducible by epidermal growth factor alone, while thymidine kinase is not.

Animals↗
PubMed1988

Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin.

The proliferating cell nuclear antigen (PCNA or cyclin) is a nuclear protein recently identified as a cofactor of DNA polymerase delta. When exponentially growing Balb/c3T3 cells are exposed to antisense oligodeoxynucleotides to PCNA, both DNA synthesis and mitosis are completely suppressed. A corresponding sense oligodeoxynucleotide has no inhibitory effects. These experiments indicate that PCNA (cyclin) is important in cellular DNA synthesis and in cell cycle progression.

Animals↗
PubMed1988

Expression of c-myc and induction of DNA synthesis by platelet-poor plasma in human diploid fibroblasts.

When WI-38 human diploid fibroblasts become confluent, they stop synthesizing DNA and dividing. Addition of serum causes the quiescent cell to reenter the cell cycle. Prolonged quiescence after confluence decreases and delays the response to serum. For a few days after reaching confluence, WI-38 cells also respond to platelet-poor plasma. During this period, although not cycling, WI-38 cells still express c-myc and other growth-regulated genes, as measured by steady-state RNA levels. If the quiescence is prolonged further, c-myc expression (and that of two other growth-regulated genes) is no longer detectable, and its disappearance coincides with a loss of response to platelet-poor plasma. These results suggest that, also under physiological conditions, the expression of c-myc and other growth-regulated genes can cooperate with platelet-poor plasma in inducing cellular DNA synthesis in human diploid fibroblasts.

Blood Platelets↗
PubMed1988

Prognostic significance of "short-term" effects of chemotherapy on MYC and histone H3 mRNA levels in acute leukemia patients.

We have found that administration of chemotherapy alters expression of growth-regulated genes in leukemia blast cells. To determine if such changes might be correlated with therapeutic outcome, we studied steady-state mRNA levels of MYC and histone H3 in the leukemic blasts of patients just prior to and 24 hr after the administration of the first doses of antileukemic drug therapy. Among nine patients with acute myelogenous leukemia, mRNA levels of MYC and histone H3 were reduced in five patients, and hematologic remission was achieved in three of these individuals. No remission was obtained in the four patients without reduction in MYC and histone H3 mRNA. Among acute lymphocytic leukemia patients, the mRNA levels of MYC and/or histone H3 were reduced by the therapy in seven of nine patients. A complete hematologic remission was obtained in five of them, and a partial remission was obtained in the other two. No remission was obtained in the patients in which MYC and H3 mRNA levels were unaffected by the therapy. These studies are of interest because they suggest that a decrease in the mRNA levels of MYC and histone H3 24 hr after a single dose of antineoplastic drugs may predict which patients will achieve complete remission; lack of reduction in these mRNAs correlates with failure to achieve remission. In addition, these studies also provide further proof of the heterogeneity of altered growth regulation among human leukemias.

Antineoplastic Agents↗
PubMed1988

Expression of oncogenes and cell cycle related genes in acute and chronic leukemias.

The authors have assayed the level of expression of several cell-cycle related genes in several populations of circulating myeloid leukemic blast cells. The genes explored included oncogenes such as c-myc, c-myb, p53, and cell-cycle-related genes such as vimentin, calcyclin, ornithine decarboxylase (ODC) and histone H3. Particular attention was given to analysis of the relationship existing between the mRNA levels of the histone H3 gene, which is expressed specifically in the S phase of the cell cycle, and the levels of other genes that are expressed in different stages of the G1 phase. Remarkable differences were observed among the different cases indicating that a differential expression of cell-cycle-related genes characterizes many acute leukemias. This differential expression is reflected in an altered ratio among G1-related genes and the H3 histone gene. The large fraction of leukemic cells which does not express histone H3 and therefore is functionally noncycling, shows a heterogeneous pattern of G1-related gene expression. This reflects the inability of most leukemic cells to progress through the G1 phase into the S phase of the cell cycle. This inability represents an abnormality of the cell cycle. It is concluded that the study of the expression of cell-cycle genes and protooncogenes in in understanding how leukemic cells enter a state of proliferation arrest, which appears to occur in a large fraction of leukemic cells.

Cell Cycle↗
PubMed1987

Expression of c-myb protooncogene and other cell cycle-related genes in normal and neoplastic human colonic mucosa.

The expression of c-myb, c-myc, histone H3, and ornithine decarboxylase genes was examined by Northern blot analysis in the normal and neoplastic mucosa of ten subjects affected by colon cancer. The mRNA levels of c-myb protooncogene were detected at low levels in all normal samples but were increased in the neoplastic mucosa of six cases in comparison to the normal counterpart. In five of these six cases the mRNA levels of c-myc, histone H3, and ornithine decarboxylase mRNAs were also increased, suggesting that there is a relation between the high expression of c-myb and the fraction of cycling neoplastic cells.

Cell Cycle↗
PubMed1987

TPA-induction of c-fos mRNA during the cell cycle of WI-38 human fibroblasts.

A brief exposure of quiescent (Go) WI-38 human fibroblasts to the tumor promoter TPA results in an increase in the mRNA levels of c-fos protooncogene. The same effect is produced by exposing to TPA human diploid fibroblasts WI38 synchronized in S phase by treatment with 2.5 mM hydroxyurea. Induction of c-fos mRNA in response to TPA occurs also during the progression of synchronized WI38 throughout the second and third cell cycle, but it is not associated with measurable changes in the cell cycle progression of these cells. These findings suggest that TPA induction of c-fos mRNA levels in proliferating cells is a stimulus specific rather than a function specific event.

Cell Cycle↗