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R Hromas

Publications and source records attributed to R Hromas.

At least 55 records · Page 3Linked to original sources
PubMed1995

The HEM proteins: a novel family of tissue-specific transmembrane proteins expressed from invertebrates through mammals with an essential function in oogenesis.

We report the identification of a new family of proteins, termed the HEM family, which show distinct expression patterns in blood cells and the central nervous system. Through the isolation and characterization of the corresponding brain-specific Drosophila (dhem-2) and rat orthologues (Hem-2), and through the detection of the Caenorhabditis elegans Hem-2 orthologue in the database, we show that this family is conserved throughout evolution. HEM proteins show a conserved length ranging from 1118 to 1126 amino acid residues. Moreover, they are at least 35% identical with each other and harbour several conserved membrane-spanning domains, indicative for their location on the cell surface. One of the members, the Drosophila orthologue dhem-2, was analysed in detail for its spatial expression pattern during development and for its mutant phenotype. dhem-2 is expressed maternally in the oocyte and shows uniform expression during the first half of embryogenesis, but becomes restricted to the brain and the nervous system during late embryogenesis, consistent with the expression of its vertebrate orthologue in the brain. One P-element insertion, located 39 base-pairs downstream from the dhem-2 transcription start site, causes female sterility, due to the fact that developmental processes in the oocyte are disturbed. Of the vertebrate HEM family members, the mammalian Hem-1 gene is expressed only in cells of hematopoietic origin, while Hem-2 is preferentially expressed in brain, heart, liver and testis.

Adaptor Proteins, Signal Transducing↗
PubMed1995

The hepatocyte nuclear factor-3/forkhead transcription regulatory family in development, inflammation, and neoplasia.

HNF-3/FKH genes are a large family of transcriptional activators. They are expressed in specific developmental and tissue patterns. Indeed, several of them are known to be essential for normal development (e.g. Dfkh and slp-1,2). Mutation within one of these genes produces mutant fruitfly embryos that are unable to survive. This family shares conserved DNA binding and transcriptional activation domains. The DNA binding domain has been crystallized, and its structure determined. Although it has resemblance to helices of homeodomains and H5 histones, it represents a new DNA binding motif, which has been called the 'winged helix,' because it contains additional interactive peptide regions called termed wings. Subtle amino acid variations in a region adjacent to the DNA recognition helix influence the recognition specificity of each HNF-3/FKH protein and therefore confer selectivity in promoter regulation. Members of this family are important in regulating the inflammatory response of the liver (the three HNF-3 genes). In addition, several members may be important in blood cell development (H3 and 5-3). Finally, two of these genes have been found to produce neoplasia (qin and FKHR). As investigation progresses, the mechanism by which these genes regulate development, inflammation and neoplasia will become more clear.

Animals↗
PubMed1995

Tandem autotransplantation for the treatment of metastatic breast cancer.

PURPOSE: To investigate the tolerability and impact on progression-free and overall survival of two consecutive cycles of high-dose chemotherapy (HDC) with autologous bone marrow transplantation (ABMT) in patients with previously untreated metastatic breast cancer. PATIENTS AND METHODS: Twenty-eight patients received conventional-dose induction therapy (ITx) followed by a planned two cycles of HDC with ABMT. Median age was 45 years (range, 34 to 60 years). Sites of disease were bone (seven patients), visceral (three), soft tissue (11), multiple (six), and CNS (one). The ITx regimens of cyclophosphamide, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), methotrexate, fluorouracil, prednisone, and tamoxifen (CAMFTP) (three patients); fluorouracil, doxorubicin, and cyclophosphamide (FAC; 11 patients); cyclophosphamide, methotrexate, and fluorouracil (CMF; four patients); or doxorubicin or mitoxantrone/cyclophosphamide (10 patients) were given to maximum response (three to five cycles). HDC was cyclophosphamide 6 g/m2, carboplatin 2 g/m2, and etoposide 625 mg/m2 with ABMT. RESULTS: Of 28 patients, 24 received two (86%) cycles of HDC. Four received only one cycle due to persistent toxicity from course 1 (one patient), no response to course 1 (two), and death on course 1 (one). Grade 3 to 4 nonhematologic toxicities included mucositis (in one or both cycles in 21 of 28 patients; 75%), diarrhea, nausea, and vomiting. Reversible peripheral neuropathy was seen in 15 of 28 patients and was severe in one. Documented infections were seen in 19 of 52 cycles. There was one transplant-related death. Six patients were converted from partial remission (PR) to complete remission (CR) with HDC; two of 24 patients (8%) were converted from PR to CR with the second cycle of HDC. Progression-free survival rate is nine of 28 patients (32%) with median follow-up of 23 months (range, 13 to 36+ months). Eighteen of 28 patients (64%) have progressed at 1 to 17 months from ABMT. CONCLUSION: Two cycles of HDC with ABMT was well tolerated with a high response rate in patients with metastatic breast cancer. The importance of the second cycle of HDC in this population is unclear.

Adult↗
PubMed1995

Dose escalation study of high-dose carboplatin and etoposide with autologous bone marrow support in patients with recurrent and refractory germ cell tumors.

Thirty-three patients with germ cell cancer (GCT) recurrent after two cisplatin-based regimens or cisplatin refractory (progression within 4 weeks of the last dose of cisplatin) were enrolled in a trial to establish the maximum tolerated doses (MTD) of carboplatin and etoposide given in combination with ABMT for two cycles. BM harvest of > or = 2 x 10(8) nucleated cells/kg preceded two cycles of therapy. Each agent was dose escalated, carboplatin from 1650 mg/m2 to 2100 mg/m2 and etoposide from 1200 mg/m2 to 2250 mg/m2 per cycle in successive cohorts. Twenty patients completed two cycles, 13 underwent only one due to: early death (4), toxicity (2), and progressive disease (6). There were four CR, three of whom achieved NED status with surgery, 14 PR, of whom eight have progressed. Four patients with stable disease and seven PD have died with a median survival of 6 months. There were six treatment-related deaths, four on course 1 and two on course 2. Causes of death on course 1 were: CNS hemorrhage (1), multiorgan failure (3); and on course 2: sepsis (1) and sudden death (1). Severe but reversible mucositis, transaminase and creatinine elevations were observed at the highest dose level. Three of five patients treated at this dose level had severe neurologic toxicity, manifested by both peripheral neuropathy and ototoxicity. The MTD in this patient population was carboplatin 2100 mg/m2 and etoposide 2250 mg/m2 on each of two cycles of therapy. Neurologic and mucosal toxicity were dose limiting.

Adolescent↗
PubMed1994

PE-1, a novel ETS oncogene family member, localizes to chromosome 1q21-q23.

The v-ets oncogene family shares a conserved peptide motif called the ETS domain that mediates sequence-specific DNA binding. This motif is unique among transcription factor families. Using partially degenerate oligonucleotides from conserved regions of the ETS domain and the polymerase chain reaction, we isolated a new member of the v-ets family designated PE-1 from HL60 cells. PE-1 was expressed as an approximately 7.5-kb transcript in most cell lines tested. In the hairy cell leukemia line Eskol, there was an additional 1.8-kb transcript observed. PE-1 was the most common ETS domain gene found in CD34+HLA-DR- hematopoietic progenitors. PE-1 was localized to human chromosome 1q21-q23 using both in situ chromosomal hybridization and human-hamster hybrids.

Amino Acid Sequence↗
PubMed1994

Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core.

The myeloid zinc finger gene 1, MZF1, encodes a transcription factor which is expressed in hematopoietic progenitor cells that are committed to myeloid lineage differentiation. MZF1 contains 13 C2H2 zinc fingers arranged in two domains which are separated by a short glycine- and proline-rich sequence. The first domain consists of zinc fingers 1 to 4, and the second domain is formed by zinc fingers 5 to 13. We have determined that both sets of zinc finger domains bind DNA. Purified, recombinant MZF1 proteins containing either the first set of zinc fingers or the second set were prepared and used to affinity select DNA sequences from a library of degenerate oligonucleotides by using successive rounds of gel shift followed by PCR amplification. Surprisingly, both DNA-binding domains of MZF1 selected similar DNA-binding consensus sequences containing a core of four or five guanine residues, reminiscent of an NF-kappa B half-site: 1-4, 5'-AGTGGGGA-3'; 5-13, 5'-CGGGnGAGGGGGAA-3'. The full-length MZF1 protein containing both sets of zinc finger DNA-binding domains recognizes synthetic oligonucleotides containing either the 1-4 or 5-13 consensus binding sites in gel shift assays. Thus, we have identified the core DNA consensus binding sites for each of the two DNA-binding domains of a myeloid-specific zinc finger transcription factor. Identification of these DNA-binding sites will allow us to identify target genes regulated by MZF1 and to assess the role of MZF1 as a transcriptional regulator of hematopoiesis.

Amino Acid Sequence↗
PubMed1994

Drosophila Forkhead homologues are expressed in CD34+/HLA-DR- primitive human hematopoietic progenitors.

The Forkhead gene (FKH) regulates morphogenesis in Drosophila. It is the prototype of a new family of transcriptional activators. We used the polymerase chain reaction (PCR) to analyze the expression pattern of this new transcriptional regulatory gene family in primitive hematopoeitic progenitors. Partially degenerate oligonucleotides to two conserved amino acid sequences of this family were used to prime a PCR amplification of cDNA synthesized from CD34+/HLA-DR- hematopoietic cells. Known and novel FKH genes were found to be expressed in these cells.

Animals↗
PubMed1994

The ETS oncogene family in development, proliferation and neoplasia.

V-ets was found as an oncogenic sequence in the E26 avian retrovirus, which produced leukemias in chickens. There are a large number of mammalian homologues of v-ets, all sharing a common sequence called the ETS domain. These mammalian homologues have been found to be transcriptional activators, and the ETS domain is the DNA binding portion of the gene. This family has been found to be important in regulating embryonic development and the response to growth stimuli. In addition, PU.1 and FLI-1 are dysregulated by Friend leukemia virus insertion in mice resulting in erythroleukemia. Significantly, the DNA binding portion of FLI-1 is the 3' part of an oncogenic fusion transcript (termed EWS-FLI) in human Ewing's sarcoma and neuroepithelioma.

Amino Acid Sequence↗
PubMed1993

Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1.

The ETS oncogene family member PU.1 is a transcriptional activator that is dysregulated by Friend erythroleukemia virus insertion. Northern analysis found that PU.1 is highly expressed in cells of myeloid and B-lymphoid origin, but not expressed at all in a number of nonhematopoietic tissues. Interferon-gamma and retinoic acid downregulated PU.1 expression in marrow macrophages. In situ immunohistochemistry found that PU.1 is expressed only in early granulocytic and erythroid cells and megakaryocytes, but not in mature erythroid cells, mature granulocytes, endothelial cells, or osteocytes. Thus, its expression pattern makes PU.1 a candidate for a genetic determinant of lineage commitment and stage progression in blood cell development. It also lends insight into how PU.1 might play a role in Friend virus erythroleukemia.

Animals↗
PubMed1993

PCR cloning of an orphan homeobox gene (PRH) preferentially expressed in myeloid and liver cells.

Homeobox genes are members of a family of transcription factors that regulate tissue development in many different organisms. We set out to identify homeobox genes that might play a role in hematopoiesis. Using degenerate oligonucleotide primers corresponding to conserved homeobox sequences in the polymerase chain reaction (PCR), we cloned from the HL60 promyelocytic cell line a homeobox gene we now designate PRH. We mapped this gene, which was not homologous to any of the previously described homeobox genes, to chromosome 10, a region outside of the known genomic homeobox clusters. Northern blot hybridization indicates that PRH is preferentially expressed in myeloid and liver cells. The structure, chromosomal location, and pattern of expression of PRH indicate that it represents an orphan homeobox gene that may regulate the lineage development of certain hematopoietic cells.

Amino Acid Sequence↗
PubMed1993

Drosophila forkhead homologues are expressed in a lineage-restricted manner in human hematopoietic cells.

The forkhead gene (FKH) regulates morphogenesis in Drosophila. It is the prototype of a new family of transcriptional activators. Partially degenerate oligonucleotides to two conserved amino acid sequences of this family were used to prime a polymerase chain reaction (PCR) amplification of HEL cell cDNA. Two unique clones, designated H3 and H8, were isolated that contained homologies to FKH. A third novel clone, 5-3, was isolated by low stringency screening of a chronic myelogenous leukemia cDNA library using H8 as a probe. H3 and 5-3 are preferentially expressed in restricted hematopoietic lineages, while the expression of H8 was ubiquitous. Southern analysis showed that FKH 5-3 is conserved through yeast, which is rare among tissue-specific transcription factors. The H3 and 5-3 clones provide evidence that FKH family members are present in a tissue-restricted manner in humans.

Amino Acid Sequence↗
PubMed1993

Characterization of the ets oncogene family member, fli-1.

The recently cloned fli-1 gene is a member of the ets oncogene family that is preferentially expressed in hematopoietic cells. It is a target of dysregulation by Friend leukemia virus insertion and translocation in Ewing's sarcoma and neuroepithelioma. In this report, we have studied the function and regulation of both murine and human fli-1. Analysis of the human and mouse fli-1 proteins showed that fli-1 binds to specific DNA sequences highly related to m-ets-2 binding sites. Methylation protection experiments showed that fli-1 and m-ets-2 contacted the same nucleotides in two different binding sites. The fli-1 protein was shown to be a transcriptional activator in co-transfection studies. Stimulation of murine bone marrow macrophages by mediators of inflammation, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, interleukin-1, and interferon-gamma resulted in the reduced expression of fli-1 mRNA. fli-1 was only expressed in a defined subset of human erythroleukemia cell lines.

Animals↗
PubMed1993

Human FLI-1 localizes to chromosome 11Q24 and has an aberrant transcript in neuroepithelioma.

The v-ets oncogene family shares a conserved motif, termed the ETS-domain, that mediates sequence-specific DNA binding. This motif is unique among transcription factor families. Using partially degenerate oligonucleotides to highly conserved amino acids in this motif as primers for the polymerase chain reaction, a novel ETS-domain cDNA fragment was generated. This fragment was subsequently used to clone both mouse and human full length cDNAs for this gene. The amino acid sequence of the longest open reading frame showed that this gene was homologous to the mouse FLI-I gene, an ETS family gene activated by Friend erythroleukemia virus insertion. The gene is normally expressed only in hematopoietic cells. The gene was localized to chromosome 11q24, a region of aberrations in Ewing's sarcoma and neuroepithelioma. In the neuroepithelioma cell line TC-32 the FLI-1 transcript is present but has an aberrant structure, indicating that it may be rearranged in neuroepithelioma.

Amino Acid Sequence↗
PubMed1993

Mutant H-ras over-expression inhibits a random apoptotic nuclease in myeloid leukemia cells.

Cell suicide, or apoptosis, is now recognized as an essential regulatory step in such diverse developmental processes as embryogenesis, thymocyte restriction, and hematopoiesis. One of the major features of apoptosis is the activation of an endogenous nuclease that cleaves DNA into nucleosomal fragments. Little is known about the activation or specificity of the apoptotic endonuclease. In this study, we investigated signalling pathways and the specificity of the apoptotic nuclease. We found that forced over-expression of activated H-ras inhibited activation of the apoptotic endonuclease. Since a high percentage of myelodysplasias and leukemias have mutations that activate ras, this finding lends insight into how ras might be leukemogenic. In addition, the phorbol ester TPA and a cyclic AMP analogue also slowed activation of this endonuclease. Interestingly, protein synthesis inhibition stimulated the endonuclease activity. In addition, by cloning and sequencing apoptotic fragments we found that the apoptotic nuclease has no sequence specificity. Thus, the apoptotic nuclease inhibited by H-ras over-expression was random in nature.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1.

EWS/FLI-1 is a chimeric protein formed by a tumor-specific 11;22 translocation found in both Ewing's sarcoma and primitive neuroectodermal tumor of childhood. EWS/FLI-1 has been shown to be a potent transforming gene, suggesting that it plays an important role in the genesis of these human tumors. We now demonstrate that EWS/FLI-1 has the characteristics of an aberrant transcription factor. Subcellular fractionation experiments localized the EWS/FLI-1 protein to the nucleus of primitive neuroectodermal tumor cells. EWS/FLI-1 specifically bound in vitro an ets-2 consensus sequence similarly to normal FLI-1. When coupled to a GAL4 DNA-binding domain, the amino-terminal EWS/FLI-1 region was a much more potent transcriptional activator than the corresponding amino-terminal domain of FLI-1. Finally, EWS/FLI-1 efficiently transformed NIH 3T3 cells, but FLI-1 did not. These data suggest that EWS/FLI-1, functioning as a transcription factor, leads to a phenotype dramatically different from that of cells expressing FLI-1. EWS/FLI-1 could disrupt normal growth and differentiation either by more efficiently activating FLI-1 target genes or by inappropriately modulating genes normally not responsive to FLI-1.

3T3 Cells↗