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Biomedical subjects

A D Friedman

Publications and source records attributed to A D Friedman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Identification of transcriptional activation and repression domains in human CCAAT/enhancer-binding protein epsilon.

Human CCAAT/enhancer-binding protein epsilon (C/EBPepsilon), a new member of the C/EBP family, significantly up-regulates both the mim-1 and human myeloperoxidase promoters, suggesting an important role for C/EBPepsilon in the transcriptional regulation of a subset of myeloid-specific genes. To elucidate the structure and function of C/EBPepsilon in transcriptional activation, amino acid residues 1-115, 147-249, or 1-249 of C/EBPepsilon were fused to the yeast GAL4 DNA binding domain. These expression vectors were cotransfected with a chloramphenicol acetyltransferase reporter gene and, in all cell lines tested, only the GAL-C/EBPepsilon-(1-115) fusion protein significantly activated expression from the chloramphenicol acetyltransferase reporter gene. Sixteen deletion mutants of C/EBPepsilon mapped the transactivation domain to amino acids 1-18 at the N terminus and revealed the presence of a transcription repression element between amino acid residues 116 and 162. Expression vectors containing the repression domain of C/EBPepsilon strongly inhibited gene transcription from TK, SV40, and adenoviral major late promoters bearing GAL4 binding sites. Fusion of this repression domain to the VP16 activation domain inhibited the transactivation function of VP16. Deletion of this repression domain increased gene transcription from a neutrophil elastase promoter-luciferase reporter. Taken together, these data suggest that C/EBPepsilon regulates transcription by utilizing both activation and repression functions.

CCAAT-Enhancer-Binding Proteins↗
PubMed1998

Frequent detection of tumor cells in hematopoietic grafts in neuroblastoma and Ewing's sarcoma.

Many poor-risk neuroblastomas and tumours of the Ewing's sarcoma family (ET) recur despite autologous transplants. Recurrence may be due to tumor cells contained in the BM harvests or PBSC harvests. The objectives of this prospective study were to: (1) determine the incidence and degree of tumor cell contamination in paired BM and PBSC harvests; and (2) determine the efficacy of tumor cell purging by immunomagnetic CD34+ cell selection. 198 samples from 11 consecutive patients with neuroblastoma or Ewing's sarcoma were analyzed. We assayed tumor contamination by RT-PCR assay for PGP 9.5, plus immunohistochemistry for neuroblastoma-specific antigens (the latter in neuroblastoma only). None of these patients had tumor cells detected in their BM by clinical histology immediately before BM or PBSC harvests. However, 82% of PBSC and 89% of backup BM harvests were contaminated with tumor by RT-PCR and/or immunocytochemistry assays. Unselected PBSC and BM harvests contained similar quantities of tumor cells (median, approximately 200000 cells). Cyclophosphamide plus G-CSF mobilization did not affect the incidence or level of contamination in PBSC harvests, as compared to blood obtained before mobilization. Immunomagnetic CD34+ cell selection depleted tumor cells by a median of 3.0 logs for PBSC, and 2.6 logs for BM harvests.

Adolescent↗
PubMed1998

Multiple functional domains of AML1: PU.1 and C/EBPalpha synergize with different regions of AML1.

Control elements of many genes are regulated by multiple activators working in concert to confer the maximal level of expression, but the mechanism of such synergy is not completely understood. The promoter of the human macrophage colony-stimulating factor (M-CSF) receptor presents an excellent model with which we can study synergistic, tissue-specific activation for two reasons. First, myeloid-specific expression of the M-CSF receptor is regulated transcriptionally by three factors which are crucial for normal hematopoiesis: PU.1, AML1, and C/EBPalpha. Second, these proteins interact in such a way as to demonstrate at least two examples of synergistic activation. We have shown that AML1 and C/EBPalpha activate the M-CSF receptor promoter in a synergistic manner. As we report here, AML1 also synergizes, and interacts physically, with PU. 1. Detailed analysis of the physical and functional interaction of AML1 with PU.1 and C/EBPalpha has revealed that the proteins contact one another through their DNA-binding domains and that AML1 exhibits cooperative DNA binding with C/EBPalpha but not with PU.1. This difference in DNA-binding abilities may explain, in part, the differences observed in synergistic activation. Furthermore, the activation domains of all three factors are required for synergistic activation, and the region of AML1 required for synergy with PU.1 is distinct from that required for synergy with C/EBPalpha. These observations present the possibility that synergistic activation is mediated by secondary proteins contacted through the activation domains of AML1, C/EBPalpha, and PU.1.

Animals↗
PubMed1997

The myeloperoxidase gene proximal enhancer directs hematopoietic-specific expression in transgenic mice.

The myeloperoxidase (MPO) gene is expressed specifically in immature myeloid cells. The MPO gene includes a promoter proximal enhancer which is coincident with DNaseI hypersensitive chromatin sites and is specifically active in myeloid cell lines. We developed transgenic murine lines in which 1.3 kb of murine MPO proximal 5' flanking region DNA was linked to a TATAA homology and RNA initiation site derived from the HSV-TK promoter and to a luciferase reporter (MPOTKLUC). In each of six founder lines, high-level luciferase activity was evident in marrow, thymus and spleen. Modest- to high-level luciferase expression was also evident in brain and in the heart in several of the lines, and luciferase activity was at or near background levels in lung, liver, kidney, stomach, colon, bladder, skeletal muscle, skin and small intestine in all of the MPOTKLUC transgenic mice. Within marrow cells, luciferase activity was evident in myeloid (GR-1+), B lymphoid (B220+) and T-lymphoid (CD4+) cells. Additional regulatory regions, thus, may be required to further restrict MPO gene expression to immature myeloid cells.

Animals↗
PubMed1997

GABP cooperates with c-Myb and C/EBP to activate the neutrophil elastase promoter.

Neutrophil elastase (NE) is a serine protease that is transcriptionally regulated during early myeloid differentiation. The murine NE (mNE) promoter contains functionally important c-Myb, C/EBP, and ets binding sites. Deletion of the ets site reduced promoter activity by 90%. Although the ets transcription factor, PU.1, bound to this ets site, it only modestly activated the mNE promoter. Here, we show that a second transcription factor from myeloid cells-GABP-binds to the mNE ets site but strongly activates the mNE promoter. GABP is a heteromeric transcription factor complex that consists of GABP alpha, an ets factor, and GABP beta, a Notch-related protein. GABP alpha bound to the mNE ets site and, in turn, recruited GABP beta to form a transcriptionally active complex. GABP alpha and PU.1 competed with each other for binding to the mNE ets site. GABP increased the activity of the mNE promoter sevenfold in U937 myeloid cells. GABP cooperated with c-Myb and C/EBP alpha to activate the mNE promoter more than 85-fold in otherwise nonpermissive, nonhematopoietic NIH 3T3 cells. Thus, GABP binds to the crucial mNE promoter ets site and powerfully activates its expression alone and in cooperation with the transcription factors c-Myb and C/EBP.

Animals↗
PubMed1997

CBF beta-SMMHC, expressed in M4Eo AML, reduced CBF DNA-binding and inhibited the G1 to S cell cycle transition at the restriction point in myeloid and lymphoid cells.

CBF beta-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBF beta-SMMHC or AML1-ETO oncoproteins failed to develop definitive hematopoiesis. To investigate these effects on hematopoiesis, we expressed CBF beta-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBF beta-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBF beta. Indirect immunofluorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and diffuse nuclear staining in 32D cl3 cells. CBF beta-SMMHC reduced endogenous CBF DNA-binding fivefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBF beta-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBF beta-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor alpha or beta subunit levels. Neither apoptosis nor 32D differentiation was induced by zinc in IL-3 in these lines. Induction of CBF beta-SMMHC in 32D cl3 cells did not inhibit their differentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of differentiation by CBF beta-SMMHC by allowing its increased expression.

Animals↗
PubMed1997

Systemic ehrlichiosis presenting as progressive hepatosplenomegaly.

A 42-year-old white man had headache, fever, chills, abdominal pain, nausea and vomiting, night sweats, and dark urine for 3 days before admission; he had history of a tick bite 6 weeks earlier. Progressive systemic deterioration, heralded by progressive hepatosplenomegaly and pancytopenia, occurred despite doxycycline therapy. Subsequent recovery was preceded by progressive resolution of hepatosplenomegaly. Progressive hepatosplenomegaly has not been previously reported in association with systemic monocytic ehrlichiosis.

Adult↗
PubMed1997

Core binding factor cannot synergistically activate the myeloperoxidase proximal enhancer in immature myeloid cells without c-Myb.

The myeloperoxidase (MPO) gene is transcribed specifically in immature myeloid cells and is regulated in part by a 414-bp proximal enhancer. Mutation of a core binding factor (CBF)-binding site at -288 decreased enhancer activity 30-fold in 32D cl3 myeloid cells cultured in granulocyte colony-stimulating factor (G-CSF). A novel functional analysis, linking the CBF-binding site to an enhancer deletion series, located at -147 an evolutionarily conserved c-Myb-binding site which was required for optimal enhancer activity and synergy with CBF in 32D cells. These sites cooperated in isolation and independent of a precise spacing. Deletional analysis carried out in the absence of the c-Myb-binding site at -147 located at -301 a second c-Myb-binding site which also synergized with CBF to activate the enhancer. A GA-rich region at -162 contributed to cooperation with CBF when the adjacent c-Myb-binding site was intact. Mutation of both c-Myb-binding sites in the context of the entire enhancer greatly impaired activation by endogenous CBF in 32D cells. Similarly, activation by c-Myb was impaired in constructs lacking the CBF-binding site. CBF and c-Myb were required for induction of MPO proximal enhancer activity when 32D cells differentiated in response to G-CSF. A fusion protein containing the Gal4 DNA-binding domain and the AML-1B activation domain, amino acids 216 to 480, activated transcription alone and cooperatively with c-Myb in nonmyeloid CV-1 cells. Determining how CBF and c-Myb synergize in myeloid cells might contribute to our understanding of leukemogenesis by the AML1-ETO, AML1-MDS1, CBFbeta-SMMHC, and v-Myb oncoproteins.

Animals↗
PubMed1997

Pediatric morning report: an appraisal.

We examined and contrasted morning reports at two hospitals, university and community, that have a pediatric residency program. Patient diagnoses assigned at morning report were compared with final diagnoses to assess disease categories discussed and the value of including outpatient follow-up in this educational forum. Data were obtained during morning reports for 6 months by chief residents at university and private community hospitals. Pertinent history, physical examination, and laboratory and radiologic findings were recorded and were assigned a tentative morning report diagnosis based on morning report discussion. Cases were then reviewed at discharge and at 6 months to determine final diagnoses. At the university hospital, 58% of the cases were undiagnosed before presentation at morning report. Of those cases, 23% were assigned a diagnosis at morning report that differed from the final diagnosis. Similarly, at the private community hospital, 28% of cases were undiagnosed before presentation at morning report. Of those cases, 73% were assigned a diagnosis that differed from the final diagnosis. We conclude that the provision of follow-up at morning report is important for maximizing resident education.

Child↗
PubMed1997

An analysis of morning report at a pediatric hospital.

The purpose of this study was to determine the types of cases residents select for morning report discussion and the educational value of postdischarge follow-up of unknown cases. Between April and December of 1994, at Cardinal Glennon Children's Hospital in St. Louis, Missouri, random, resident, and group-selected patients listed at morning report were followed up throughout hospitalization. Patients were categorized based upon whether or not their morning report and discharge diagnoses were the same or different. Patients discharged without a diagnosis were followed up by chart review at 6 months to determine whether a diagnosis had been made. Data were analyzed by Chi-square analysis with Bonfferoni adjustment factor for multiple comparisons. Residents were more than two times more likely to select cases for discussion in which the diagnosis changed during hospitalization (P < 0.01). The 6-month follow-up yielded new diagnoses in only 21% of previously unknown cases. We concluded that residents do an exceptional job of selecting difficult diagnostic cases for discussion at morning report. Postdischarge follow up of unknown cases adds little new information for discussion at morning report.

Child↗
PubMed1996

GADD153/CHOP, a DNA damage-inducible protein, reduced CAAT/enhancer binding protein activities and increased apoptosis in 32D c13 myeloid cells.

GADD153/CHOP is a DNA damage-inducible, nuclear leucine zipper protein that is capable of producing a G1-S arrest in fibroblastic cells and of dimerizing with and inhibiting CAAT/enhancer binding protein (C/EBP) activities. CHOP was induced in 32D cl3 myeloid cells exposed to methylmethane sulfonate (MMS), a DNA alkylating agent. The degree of induction was dependent upon the dose of MMS to which the cells were exposed. CHOP was not expressed, at least at similar levels, during 32D cl3 cell granulocytic differentiation or during their apoptosis upon growth factor withdrawal. High-level expression of exogenous CHOP in 32D cl3 cells markedly inhibited the trans-activation activities of endogenous C/EBPs. These cells proliferated in IL-3, although low-level ongoing apoptosis not observed with control cells was detected. When these cultures were transferred to granulocyte colony-stimulating factor (G-CSF), the majority of the cells underwent apoptosis, although the levels of CHOP did not increase. Similarly, 32D cl3 cells treated with doses of MMS sufficient to induce endogenous CHOP underwent apoptosis more rapidly when placed in G-CSF-containing, compared with interleukin 3 (IL-3)-containing, medium. However, induction of CHOP by MMS was similar in IL-3 and in G-CSF. The heightened sensitivity of 32D l13 cells to CHOP in G-CSF could result either from the loss of IL-3-specific signals or from increased expression of C/EBPs. Because myeloid leukemias express C/EBPalpha, induction of CHOP might contribute to their chemotherapy-induced apoptosis, and alterations in CHOP expression could contribute to their development of chemotherapy resistance.

Animals↗
PubMed1996

The murine myeloperoxidase gene contains a bipartite distal enhancer, including a novel region regulated by PEBP2/CBF.

The myeloperoxidase (MPO) gene is expressed specifically in immature myeloid cells. DNA (27 kb) derived from the murine MPO gene was subcloned into nine segments, and each examined for its ability to enhance transcription in 32D cl3 myeloid cells. Only one segment, besides the known proximal enhancer, was active. Deletional analysis of this region delimited two functional regions. One is located 3.4 kb upstream of the proximal enhancer and was also recently identified in a different cell system. The second novel region is a 133 bp segment located 1.1 kb further upstream. This region, like the proximal enhancer, binds and is regulated by PEBP2/CBF. The PEBP2/CBF binding-site in the distal enhancer, AACCAAT, differs from the PEBP2/CBF consensus, PuACCPuCA.

Animals↗
PubMed1996

C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter.

The murine neutrophil elastase (NE) gene is expressed specifically in immature myeloid cells. A 91-bp NE promoter region contains three cis elements which are conserved evolutionarily and are essential for activation of the promoter in differentiating 32D cl3 myeloid cells. These elements bound c-Myb (at -49), C/EBPalpha (at -57), and PU.1 (at -82). In NIH 3T3 cells, the NE promoter was activated by c-Myb, C/EBPalpha, and PU.1, via their respective binding sites. Cooperative activation was seen by any combination of c-Myb, C/EBPalpha, and PU.1, including all three together, again via their DNA-binding sites. In CV-1 cells, but not in NIH 3T3 cells, cooperation between Myb and C/EBPalpha depended on the integrity of the PU.1-binding site. In addition to C/EBPalpha, C/EBPdelta strongly activated the NE promoter, alone or with c-Myb, but C/EBPbeta was less active. Either of C/EBPalpha's two transactivation domains cooperatively activated the promoter with c-Myb, in both NIH 3T3 and 32D c13 cells. Synergistic binding to DNA in a gel shift assay between C/EBPalpha, c-Myb, and PU.1 could not be demonstrated. Also, separation of the C/EBP- and c-Myb-binding sites by 5 or 10 bp did not prevent cooperativity. These results suggest that a coactivator protein mediates cooperative activation of the NE promoter by a C/EBP and c-Myb. These factors, together with PU.1, direct restricted expression of the NE promoter to immature myeloid cells.

3T3 Cells↗
PubMed1996

A shunt infection caused by Francisella tularensis.

We report the first case of a child with a shunt infection caused by Francisella tularensis, the causative agent of tularemia. This patient is also unique in that the disease was limited to the central nervous system.

Child, Preschool↗
PubMed1996

Hematologic manifestations of viral infections.

Many infections have an impact on the hematologic system. There also are many noninfectious processes associated with abnormalities of the blood and iatrogenic causes such as pharmacologic therapy that also may affect the marrow or circulating cellular elements. Viral disease may impact on one or more components of blood, and familiarity with these associations can assist in anticipating the course of disease and reducing the need to seek out other etiologies for the usually transient hematologic abnormalities that often occur in patients with viral infections.

Child↗
PubMed1996

DNA-binding domain of AML1, expressed in t(8;21) and t(3;21) myeloid leukemias, inhibits PEBP2/CBF DNA-binding but is not sufficient to transform 32D cl3 myeloid cells.

Truncated AML1 proteins are predicted to be expressed from out-of-frame AML1 transcripts present in myeloid leukemia cells harboring t(8;21) and t(3;21). To test whether these proteins, consisting of almost exclusively an N-terminal AML1 DNA-binding domain, interfere with myeloid differentiation we expressed a similar truncated AML1 protein in 32D cl3 myeloid cells. In all clones examined, the ectopically expressed truncated AML1 protein prevented binding of endogenous PEBP2/CBFs to DNA, possibly by interacting with all available CBF beta subunits. However, compared to control clones, the 32D cl3 clones expressing truncated AML1 remained IL-3 dependent for survival, proliferated similarly in low and high concentrations of IL-3, and differentiated similarly upon transfer to G-CSF. Thus, truncated AML1 proteins may contribute to myeloid leukemogeneis by inhibiting PEBP2/CBF activities, although contributions from other oncoproteins are likely required as well.

Animals↗