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E V Prochownik

Publications and source records attributed to E V Prochownik.

At least 55 records · Page 3Linked to original sources
PubMed1990

Regulation of megakaryocyte phenotype in human erythroleukemia cells.

Induction of human erythroleukemia (HEL) cells with nanomolar tumor-promoting phorbol myristate acetate (PMA) diesters results in the synchronous acquisition of multiple markers of the megakaryocyte phenotype. Induced cells markedly increase their content of cytoplasm and show features of morphological maturation. At the ultrastructural level, PMA-treated cells show increases in cytoplasm, nuclear lobulation and nucleolar content, and free ribosomes. Limited numbers of cells also express alpha-granules and nascent demarcation membrane systems. Functionally, PMA-stimulated HEL cells express increased amounts of the megakaryocyte/platelet proteins: glycoprotein IIb/IIIa, platelet factor 4, von Willebrand factor, glycoprotein Ib, and thrombospondin. No changes are observed in antigenic markers of the erythroid (glycophorin A) or macrophage lineages (MO-1 or MO-2). The increases in antigenic expression are rapid, reaching maximum levels within 3-4 d under serum-free conditions. Treatment with PMA also abruptly (within 1-2 d) inhibits cellular division in these cells. Washout studies indicate that phorbols exert their effect within 18-24 h, the approximate cell cycle time for these cells. Consistent with proliferative arrest, c-myc proto-oncogene transcripts begin to decline within 8 h of PMA treatment, although transcripts of c-myb are unaffected. Importantly, megakaryocyte differentiation is associated with endomitotic DNA synthesis (i.e., continued DNA synthesis in the absence of mitosis and cytokinesis), with HEL cells reaching a DNA content of 3-12 times that of unstimulated cells. Endomitosis is coordinately regulated with changes in antigenic expression and cell size such that those cells having the highest DNA content are the largest and also express the greatest levels of antigen.

Antigens↗
PubMed1989

The antimitogenic action of tumor necrosis factor is associated with increased AP-1/c-jun proto-oncogene transcription.

Tumor necrosis factor alpha (TNF) mediates its in vivo antineoplastic effect primarily through its action on the endothelial surfaces of tumor vessels. Among other changes, endothelial cells increase the expression of surface procoagulant molecules which allow for the genesis of microthrombi and the eventual anoxic killing of the tumor tissue. TNF is also a potent antimitotic factor for endothelial cells. We investigated the molecular basis for these diverse actions of TNF by differential screening of a cDNA library prepared from TNF and cycloheximide-treated human umbilical vein endothelial (HUVE) cells. One of the cDNA clones identified encodes the transcription factor and proto-oncogene AP-1/c-jun. The expression of AP-1/c-jun following TNF treatment is mediated largely at the transcriptional level. Neither c-myc nor c-fos transcripts were induced by TNF. Since AP-1/c-jun has been previously identified as one of the earliest transcripts expressed in quiescent cells that have been stimulated by growth factors, our results suggest that mitogenic and antimitogenic stimuli evoke distinct yet overlapping sets of molecular responses and that the coordinate expression of AP-1/c-jun and c-fos is not mandatory. AP-1/c-jun transcript induction is thus one of the initial events mediated by TNF treatment of HUVE cells.

Blotting, Northern↗
PubMed1989

Molecular genetics of inherited antithrombin III deficiencies.

The cloning of antithrombin III (ATIII) complementary deoxyribonucleic acids and the determination of the ATIII gene structure have permitted a systematic evaluation of the molecular basis for inherited ATIII deficiencies. Sixteen kindreds with the most common form of the deficiency, in which plasma ATIII antigen levels and activity are proportionately reduced, were studied. Two polymorphic deoxyribonucleic acid markers were used to resolve parental ATIII alleles and to trace their inheritance patterns. In 15 of 16 cases, the structure of the affected ATIII allele was indistinguishable from normal, suggesting that relatively small mutations, resulting in gene inactivation, are responsible for the lower ATIII levels in these affected families. In the remaining kindred, complete deletion of one ATIII allele was seen. Also investigated was the molecular basis for a qualitative form of ATIII deficiency in a French-Canadian family with normal levels of immunoreactive protein but only half the expected levels of serine protease inhibitor activity. Using polymorphic markers, the abnormal allele was identified, cloned, and partially sequenced from the propositus. A single G----A transition was seen in the first base of codon 382, resulting in an alanine----threonine substitution in the defective protein. This mutation, together with others in this vicinity, defines a minimal length for a fully functional thrombin-binding domain.

Antithrombin III↗
PubMed1989

c-myb but not c-myc suppresses the hemin-induced nonterminal expression of hemoglobin by murine erythroleukemia cells.

Clonal lines of Friend murine erythroleukemia (F-MEL) cells have been generated following transfection with c-myc or c-myb expression plasmids. These clones produce high levels of abnormally regulated proto-oncogene transcripts and fail to terminally differentiate in the presence of dimethyl sulfoxide. To determine the relative levels at which the two proto-oncogenes might exert their inhibitory effects, we asked whether these clones could express differentiated functions in the absence of terminal differentiation. It was found that exposure of c-myc-transfected cells to hemin allows for the induction of hemoglobin, whereas c-myb-transfected cells were refractory to hemin induction. It thus appears that c-myb exerts a more globally suppressive effect on F-MEL-differentiated functions than does c-myc and may prevent the expression of those events that can otherwise be dissociated from the terminally differentiated state.

Animals↗
PubMed1989

Expression and analysis of COOH-terminal deletions of the human thrombospondin molecule.

Thrombospondin (TSP) is a homotrimeric extracellular glycoprotein with a subunit molecular mass of 140 kD. The subunits have a modular or domain-like structure and are held together by interchain disulphide bonds. A number of domains have been identified including those for the binding of collagen, fibrinogen, and heparin. Due to the trimeric form of the TSP molecule, the various domains are trivalent in nature and this contributes to the ability of TSP to mediate cell-substrate interactions. Indeed, TSP has recently been shown not only to promote cell adhesion but also to be intimately involved in cell growth and migration. The adhesive function of TSP is attributable to the "solid-phase" or matrix-bound form of the molecule. There is some evidence that the heparin-binding domain mediates incorporation of soluble TSP into the insoluble matrix form. The heparin-binding domain of TSP is a compact globular amino-terminal moiety that contains two clusters of basic amino acids and a single intrachain disulphide bond. To delineate the role of the heparin-binding domain in matrix assembly and to define further the precise region of interchain disulphide bonding that results in trimer formation, we have expressed deleted forms of the cDNA encoding TSP in SV-40-transformed. African green monkey kidney cells. The proteins synthesized from the various deleted TSP cDNAs were examined for (a) secretion into the culture medium and incorporation into the extracellular matrix; (b) binding to heparin-Sepharose; (c) immunoprecipitability by a conformation-specific monoclonal antibody; and (d) ability to form trimers. This analysis allowed us to draw the following conclusions. (a) A 218 amino acid NH2-terminal protein that preserves the intrachain disulphide bridge of the heparin-binding domain is capable of binding to heparin-Sepharose and incorporating into the extracellular matrix. (b) A shorter 164 amino acid NH2-terminal peptide that does not contain the intrachain disulphide bridge of the heparin-binding domain is neither able to bind to heparin-Sepharose nor able to incorporate into the extracellular matrix. (c) The region of interchain disulphide bridging necessary for trimer assembly resides within a cluster of seven cysteine residues immediately adjacent to the heparin-binding domain.

Animals↗
PubMed1988

Constitutive expression of a c-myb cDNA blocks Friend murine erythroleukemia cell differentiation.

A full-length human c-myb cDNA clone has been isolated from a CCRF-CEM leukemia cell cDNA library. The plasmid vector contains simian virus 40-derived promotor, splice, and polyadenylation sequences as well as a transcription unit for a dihydrofolate reductase cDNA. We have introduced this construct into Friend erythroleukemia (F-MEL) cells and have isolated a number of clones which contain intact and transcriptionally active human c-myb sequences. F-MEL clones expressing the highest levels of the human c-myb mRNA differentiate poorly in response to dimethyl sulfoxide. Two clones which initially expressed low levels of human c-myb transcripts and which differentiated normally were subsequently inhibited in their ability to differentiate when grown in successively higher concentrations of methotrexate, due to amplification and enhanced expression of plasmid sequences. The inhibitory effect on F-MEL differentiation appeared to be independent of the early decline in c-myc transcripts which were normally regulated in all cases examined. Our results indicate that constitutive expression of a nontruncated human c-myb cDNA can exert profound effects on erythroid differentiation and argue for a causal role of c-myb in the F-MEL differentiation process.

Animals↗
PubMed1988

c-myc antisense transcripts accelerate differentiation and inhibit G1 progression in murine erythroleukemia cells.

Friend murine erythroleukemia (F-MEL) cells were transfected with a plasmid bearing tandemly arranged mouse c-myc antisense and dihydrofolate reductase transcription units. Sixteen clones were isolated, each containing unrearranged c-myc sequences and expressing high levels of antisense transcripts. All antisense clones examined contained reduced amounts of cytoplasmic endogenous c-myc transcripts. The kinetics of reaccumulation of endogenous c-myc mRNA during a 24-h exposure to dimethyl sulfoxide (DMSO) were also retarded and the ultimate transcript levels attained were less than in control cells. Antisense clones grew as well as control F-MEL cells in medium containing 10% fetal calf serum but at only a half and a quarter of the control rates in media containing 5 and 2% serum, respectively. Antisense clones differentiated faster and to a greater degree than control cells following DMSO exposure. myc antisense transcript expression was increased by growing cells in methotrexate, which resulted in an enhanced response to DMSO. Fluorescence-activated cell sorter (FACS) analysis of cellular DNA content indicated that a greater fraction of antisense nuclei contained a G0/G1 2n DNA content following a 24-h exposure to DMSO. When density-arrested antisense clones were diluted into fresh medium to allow reentry into the cell cycle, they incorporated less [3H]thymidine than control cells. FACS analysis showed that this was because only a portion of the cell population was entering S phase. Whereas control cells did not increase in size following release from density arrested antisense cells contained a subpopulation which were initially smaller and which eventually attained the same size as control cells. Quiescent antisense cells thus comprise two populations, each arrested at a different point in G1. Dilutional replating allowed both populations to reenter the cell cycle. We propose a model which postulates that certain minimal myc levels are necessary for cells to traverse G1. Those with insufficient levels, due, for example, to antisense inhibition, are unable to completely traverse G1 during density arrest and synchronize at an earlier point than do control cells. This earlier point may be along the differentiation pathway and may account for the greater responsiveness of antisense cells to DMSO induction. This model postulates that F-MEL cells overexpressing myc fail to differentiate because myc levels are never sufficiently low enough to allow cells to enter the differentiation pathway.

Animals↗
PubMed1988

Antithrombin-III-Hamilton: a gene with a point mutation (guanine to adenine) in codon 382 causing impaired serine protease reactivity.

Antithrombin-III-Hamilton is a structural mutant of antithrombin III with defective serine protease reactivity, demonstrable in three members of a French Canadian family. The propositus, a 54-year-old man with a history of recurrent thromboembolic events, and his two asymptomatic grown children are heterozygous for the mutant antithrombin III gene. In all three individuals, the immunoreactive antithrombin III level is normal, while the antithrombin and antifactor Xa activity is approximately 50% of the control value. Two dimensional immunoelectrophoresis of antithrombin-III-Hamilton in the presence of heparin is normal. Purified antithrombin-III-Hamilton did not form thrombin-antithrombin III complex when incubated with thrombin for up to 30 minutes. The normal and mutant antithrombin III alleles of the propositus could be distinguished by linkage to Pstl restriction fragment length polymorphisms (RFLP). Genomic DNA from the propositus was cloned into EMBL 3 phage vectors and two clones containing nearly complete copies of the antithrombin-III-Hamilton allele were identified. Exon 6 of both clones was subcloned into M13 phage vector and sequenced, revealing a G----A point mutation in the first base of codon 382. Codon 382 codes for alanine in the normal allele and for threonine in the antithrombin-III-Hamilton allele. Alanine-382, 12 residues from the reactive center, is a highly conserved amino acid in the family of serine protease inhibitors known as the serpins. We postulate that, as a result of the substitution of threonine for alanine in antithrombin-III-Hamilton, either the tertiary structure or the hydrophobicity of the thrombin-binding region is altered, causing aberrant conformation of the Arg-393-Ser-394 bond at the reactive center impairing the interaction between antithrombin-III-Hamilton and the activated serine proteases.

Alleles↗
PubMed1987

Two regions downstream of AATAAA in the human antithrombin III gene are important for cleavage-polyadenylation.

We have investigated the sequence requirements for the cleavage-polyadenylation reaction in the human antithrombin III (ATIII) gene. A series of 5'-3' and 3'-5' deletions were produced around the AATAAA site using Bal31 nuclease. Ligation of appropriate pairs of such mutations resulted in the generation of varying sized deletions or duplications of sequences either upstream of, downstream of, or within the region encompassing the poly(A) site. Whereas a large deletion 3' to the AATAAA signal abolished cleavage and polyadenylation of ATIII transcripts, smaller deletions, all of which were subsets of the large one, did not. This indicated that the ATIII gene contains at least two independently acting poly(A)-cleavage signals 3' of AATAAA. When one of these signals was eliminated and the other was partially deleted at its 3'-end, we were able to disrupt the normal spacing between AATAAA and the cleavage site without substantially affecting the efficiency of the cleavage reaction. This suggested that the distance between AATAAA and the cleavage site is determined by the same sequence which, along with AATAAA, specifies cleavage and polyadenylation. The duplication of regions either upstream or downstream of AATAAA affected neither the efficiency nor the site of cleavage of the ATIII transcript. When a duplication included a large region containing AATAAA as well as downstream sequences, both sites were chosen for cleavage reactions. With a more delimited duplication, which included AATAAA but not the downstream cleavage signals, both sites were again used. However, two new cleavage sites were now detected. These results suggested that the distance between AATAAA sites and critical downstream cleavage-spacing sequences may also be important in determining the site of cleavage. This may in part stem from spatial constraints imposed by RNA-protein complexes which have been postulated to be critical in catalyzing the cleavage-polyadenylation reaction.

Antithrombin III↗
PubMed1987

Changes in protein kinase C activity are associated with the differentiation of Friend erythroleukemia cells.

We investigated the activity and cellular distribution of protein kinase C during the dimethylsulfoxide (DMSO) and hypoxanthine-induced differentiation of Friend murine erythroleukemia cells. Most of the cellular protein kinase C activity was found in the soluble fraction of unstimulated Friend cells. Within 15 min of the addition of DMSO or hypoxanthine, protein kinase C underwent a dramatic and prolonged reversal of this distribution which was accompanied by a gradual decline in total cellular protein kinase C activity over the ensuing 5 days. The loss of total activity was found to be dose dependent although maximal translocation from soluble to insoluble components occurred at even lower concentrations of the inducers tested. Two clones of Friend cells, selected for their failure to differentiate in response to DMSO, showed alterations in protein kinase C activity and/or distribution following DMSO addition when compared to wild-type Friend cells. These data show that different inducers of Friend cell differentiation have similar effects on cellular protein kinase C, that the protein kinase C changes accompanying this process are immediate but prolonged, and that changes in protein kinase C activity and distribution are associated with Friend cell differentiation.

Animals↗
PubMed1987

Molecular genetic survey of 16 kindreds with hereditary antithrombin III deficiency.

Molecular genetic techniques were utilized to examine antithrombin III (ATIII) gene status in 16 independently ascertained kindreds with hereditary ATIII deficiency. In one of these families antithrombin III deficiency is caused by hemizygosity of the ATIII locus. In the remaining 15 kindreds, two copies of the ATIII gene are present and appear to be grossly normal at the level of whole genome Southern blotting, suggesting that small deletions, insertions or limited nucleotide substitution(s) in the antithrombin III gene, or "trans-acting" defects at other loci involved in the processing, modification, and secretion of biologically active ATIII are responsible for the observed anticoagulant disorders.

Antithrombin III↗
PubMed1985

Intron structure of the human antithrombin III gene differs from that of other members of the serine protease inhibitor superfamily.

Antithrombin III (ATIII) plays an integral role in the coagulation system by inhibiting thrombin and several other activated clotting factors. Inherited deficiency of ATIII is quite common and can result in life-threatening thrombotic complications. In order to understand the basis of ATIII deficiency, we have isolated and characterized the normal human ATIII gene from a recombinant Charon 4A bacteriophage genomic library. The ATIII gene contains six exons and five introns distributed over approximately 19 kilobases of DNA. The positions of introns in the ATIII gene were compared with other members of the serine protease inhibitor family which share 17-31% amino acid homology. When aligned to achieve maximal protein homology, only one of the ATIII introns corresponded to the four introns of rat angiotensinogen or human alpha 1-antitrypsin. Similarly, only one ATIII intron was homologous to the seven introns of chicken ovalbumin. We present two testable models to explain the discrepancy in intron positions among members of the serine protease inhibitor superfamily of genes.

Amino Acid Sequence↗
PubMed1984

In vivo transcription of a human antithrombin III "minigene".

A 2.2-kilobase human antithrombin III (ATIII) "minigene" has been constructed which preserves many of the features of the 16-kilobase parental gene. These include approximately 300 base pairs of the 5'-flanking region containing the "TATA"-less ATIII promoter, the entire protein-coding sequence including the signal peptide, a single intervening sequence with intact donor and acceptor splice sites, and a polyadenylation signal. Unique PstI termini allow the minigene to be cloned in many commonly used plasmid vectors. The minigene has been reintroduced into heterologous cells by calcium phosphate precipitation. RNA is transcribed with heterogeneous 5'-ends and is processed by splicing either to the normal acceptor site or to an alternate site within the intervening sequence. The alternately spliced mRNA is also found in liver where it accounts for 20-40% of ATIII-specific transcripts. The alternate splicing event introduces a 42-nucleotide segment between codons -19 and -18 of the signal peptide. This segment contains an in-frame ochre termination codon such that the predicted protein product encoded by the alternately spliced mRNA would be only 19 amino acids long. The minigene is transcribed to high levels in mouse fibroblasts when present as an episome in a bovine papilloma virus-derived vector although no protein product is detected.

Amino Acid Sequence↗
PubMed1983

Isolation of a cDNA clone for human antithrombin III.

Antithrombin III (ATIII) is an important plasma protease inhibitor with a central role in the coagulation system. On the basis of its protein sequence, ATIII is one member of a "super family" of protease inhibitors that includes alpha 1-antitrypsin and chicken ovalbumin. An increased risk of thromboembolism is associated with inherited ATIII deficiency. To study the structure and expression of the human ATIII gene, we have isolated complementary (cDNA) clones for ATIII from human liver mRNA. ATIII cDNA clones were identified by hybridization to a mixture of synthetic oligodeoxynucleotides encoding amino acids 251-256 of the ATIII protein sequence. The largest cDNA clone (1.4 kilobases) included the coding region of ATIII mRNA from codon 10 through a 3'-untranslated region. Comparison of ATIII cDNA clones from two different sources revealed a sequence polymorphism at an internal PstI restriction site. Analysis of both total genomic DNAs and an ATIII gene cloned in a bacteriophage Charon 4A showed that the ATIII gene is present once per haploid genome and is distributed over 10-16 kilobases of DNA. Computer-assisted comparison of the cDNA sequence with those for baboon alpha 1-antitrypsin and chicken ovalbumin revealed homologies consistent with their inclusion in the protease inhibitor superfamily.

Amino Acid Sequence↗
PubMed1983

Molecular heterogeneity of inherited antithrombin III deficiency.

Inherited antithrombin III deficiency is associated with an increased risk of thromboembolism. Using recombinant-DNA techniques, we isolated a molecular probe for the antithrombin III structural gene and identified a common DNA polymorphism within the gene. We found that there is genetic heterogeneity in this disorder. In one family, the antithrombin III gene was deleted in affected members, whereas in another no deletion occurred. Use of the DNA polymorphism should allow identification and further characterization of abnormal antithrombin III genes.

Adolescent↗
PubMed1979

Type C virus induced by iododeoxyuridine in the human embryonic cell strain HEL-12.

The induction of a type C virus from a strain of human embryonic lung cells (HEL-12) by iododeoxyuridine (IdUrd) was examined at various times during in vitro propagation. IdUrd did not elicit type C virus production immediately following initiation of cultures from frozen primary HEL-12 cells. After overnight treatment with 30 microgram/ml IdUrd the cells expressed viral antigens and produced a type C virus between the 25th and 80th day of in vitro growth. Production of the induced type C virus was transient. Single-cell clones of the parental HEL-12 strain were likewise susceptible to type C virus production by IdUrd. The ability of IdUrd to induce virus terminated with the onset of spontaneous type C virus production from HEL-12 cells at between 80 and 120 days of in vitro growth.

Antigens, Viral↗
PubMed1977

Nucleic acid sequences of primate type C viruses in normal and neoplastic human tissues.

Recent reports on the isolation of type C viruses from human cells have generated considerable controversy because of the close relationship of the viruses to the type C viruses of subhuman primates. These findings have been interpreted as evidence for contamination despite the repeated isolation of some type C viruses from separate clinical specimens or from separate frozen primary cell stocks of the same donor. Our laboratory has described a type C virus which is released from the HEL-12 strain of normal human embryonic lung fibroblasts. The HEL-12 virus is related immunologically to simian sarcoma (woolly monkey fibrosarcoma) virus (SiSV) and the endogenous type C viruses of baboons and domestic cats. To demonstrate that the HEL-12 virus is not an adventitious contaminant, fresh human tissues were examined for antigenic cross-reactivity with HEL-12 virus antigens. Some patients with myelogenous leukemia contain circulating immunoglobulins which specifically inhibit the reverse transcriptases of HEL-12 virus and primate type C viruses. In addition, the glomerular immune complexes in patients with systemic lupus erythematosus contain antigens related to HEL-12 virus proteins. Using the techniques of molecular hybridisation, we demonstrate here that HEL-12 cells contain proviral DNA sequences before antigen expression or spontaneous virus release can be detected. We also present evidence for the existence of nucleic acid sequences homologous to HEL-12 viral RNA in the DNAs from certain cancer patients.

Animals↗