Arja Kallio

Difluoromethylornithine-induced amplification of ornithine decarboxylase genes in Ehrlich ascites carcinoma cells.

Stepwise increments of the concentration of 2-difluoromethylornithine, a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (EC 4.1.1.17), resulted in a selection of cultured Ehrlich ascites carcinoma cells capable of growing in the presence of up to 50 mM difluoromethylornithine. Dialyzed extracts of drug-resistant tumor cells exhibited a very high ornithine decarboxylase activity and contained large excess of immunoreactive ornithine decarboxylase protein. Hybridization analyses with cloned complementary DNA revealed that the difluoromethylornithine-resistant tumor cells also expressed mRNA of the enzyme at greatly enhanced rate. The overproduction of ornithine decarboxylase by the tumor cells grown under the pressure of difluoromethylornithine was at least partly attributable to a 10 to 20-fold increase in the total gene dosage of ornithine decarboxylase involving an amplification of several genes of the gene family. The gene amplification developed appeared to be stable, as the gene dosage only slowly (during a period of several months) returned towards the normal level upon the removal of difluoromethylornithine. The overproduction of ornithine decarboxylase was accompanied by an enhanced resistance of the enzyme towards difluoromethylornithine in vitro.

Inhibition of prereplicative polyamine accumulation in regenerating rat liver

Abstract Injections of 1,3-diaminopropane, an inhibitor of mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, during the first 10 h after partial hepatectomy markedly delayed the stimulation of liver DNA synthesis from [3H]thymidine normally occurring at the beginning of second day of liver regeneration. Inhibition of ornithine decarboxylase by repeated injections (every 2 h) of diaminopropane for 10 h after partial hepatectomy abolished the enhancement in DNA synthesis at 20 h postoperatively, where shorter periods of postoperative diaminopropane treatment resulted in less complete prevention of the synthesis of DNA. Under these experimental conditions the rate of liver DNA synthesis in vivo from [3H]thymidine showed a highly significant positive correlation with the concentration of tissue spermidine but not with that of putrescine or spermine. It was also possible to prevent the accumulation of spermidine and to depress liver DNA synthesis at 24 h postoperatively by starting the treatment with diaminopropane after varying lag periods following partial hepatectomy. Treatment with diaminopropane started as late as at 12 h after partial hepatectomy still prevented any accumulation of liver spermidine and resulted in a profound inhibition (about 85%) of DNA synthesis at 24 h postoperatively. The inhibition of DNA synthesis was gradually subsiding when the commencement of the amine treatment was moved more far from the time of the operation.

Regulation of ornithine decarboxylase by diamines in regenerating rat liver.

1. Introduction Unlike the inducible enzymes in bacteria, many of the mammalian enzymes, which regulate the flow of substrates through metabolic pathways rapidly chang ing their activity in response to a variety of stimuli, are labile proteins with short molecular half-lives [

An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences

The construction of a mammalian cell expression vector using human cytomegalovirus immediate early gene enhancer to initiate transcription of inserted coding sequences is described. The vector also carries Epstein-Barr virus EBNA-1 nuclear antigen gene, ori-P sequences and hygromycin B resistance gene hph from E. coli. The expression capacity of this construct was tested by inserting the chloramphenicol acetyltransferase (CAT) gene into the vector. The EBV-CAT construct was transfected into various cell lines and high levels of CAT activity were obtained in human and monkey cells. In these cells, the vector DNA also replicates as an extrachromosomal element having 1 to 20 copies per cell. In most cases, the vector copy number and the expression level of inserted gene was in positive correlation in different cell clones.

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo.

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activity in rat kidney. Administration of diaminopropane 60 min before partial hepatectomy only marginally inhibited ornithine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant. An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed S-adenosyl-L-methionine decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy. Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals. Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life. Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [14C]methionine 4 h after partial hepatectomy or after administration of porcine growth hormone. Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornithine decarboxylase activity and spermidine synthesis.

Human ornithine decarboxylase(ODC)-encoding gene; cloning and expression in ODC-deficient CHO cells

We have cloned a full-length human ornithine decarboxylase (ODC)-encoding gene from a genomic library of human myeloma cells which overproduce ODC due to a selective gene amplification. Correct expression of the cloned gene was assessed by transfecting it into a Chinese hamster ovary (CHO) cell mutant devoid of ODC activity. Transfection with a 10-kb BamHI DNA fragment of the genomic clone, conferred ODC activity to the recipient cells and relieved them of dependence on exogenous polyamines for growth. A set of 40 transformants was isolated, eight of which were further characterized. The transfected ODC gene appeared to be hypomethylated at the cytosine residues in the sequence CpG. The transfectants were all responsive to serum stimulation, but showed different levels of ODC expression depending on both copy number and integration site of the transfected ODC gene. ODC serum induction in the transfectants was sensitive to cycloheximide and polyamine additions, and the half-life of the enzyme was very short, like that in normal CHO cells. These results suggest that the human ODC gene we transfected contains all the elements needed for normal control of ODC expression.

A solution hybridization method for quantification of mRNAs: Determining the amount and stability of oncogene mRNA

A solution hybridization method for the quantification of specific mRNAs is described. This assay utilizes complementary RNA probes prepared by in vitro transcription, sandwich hybridization in solution, and affinity-based hybrid collection. The possibility of using this method for crude biological samples without purifying mRNAs makes it ideal when accurate quantification of multiple samples is needed. Human N-myc oncogene transcript was used as a model and as little as 0.24 pg (2 X 10(5) molecules) of N-myc mRNA could be detected. Using this assay it was shown that human neuroblastoma IMR-32 cells contain approximately 500 N-myc mRNA molecules per cell having a half-life of approximately 35 min.

Efficient synthesis of influenza virus hemagglutinin in mammalian cells with an extrachromosomal Epstein-Barr virus vector

The capability of an Epstein-Barr virus hybrid vector (EBV-CMV), containing the cytomegalovirus (CMV) immediate early enhancer and simian virus 40 promoter, to produce large amounts of authentic mammalian proteins was studied. The cDNA of influenza virus hemagglutinin (HA), a cell surface glycoprotein, was inserted into this vector and the EBV-CMV-HA plasmid was transfected into two human and two monkey cell lines. Southern-blot analysis revealed that the EBV-CMV-HA plasmid was maintained in extrachromosomal state and the recombinant cell clones contained on the average three copies (range 1-24) of the transfected DNA. The recombinant HA polypeptides from different cell clones, selected either randomly or by fluorescence-activated cell sorter, were analysed using immunological techniques. Three of the four cell lines expressed recombinant HA on the cell surface in glycosylated form. The highest production levels, 11.5 micrograms/10(6) cells, were obtained in HeLa cells containing only two copies of EBV-CMV-HA DNA per cell. The protein levels correlated with the mRNA levels in Northern-blot analysis. A corresponding vector, containing the same regulatory signals for HA expression, but lacking the EBV sequences, yielded clones with significantly lower expression levels. The results confirm that the extrachromosomal EBV-CMV vector is very useful in the production of apparently authentic mammalian glycoproteins.

Comparison of mammalian cell expression vectors with and without an EBV-replicon.

SummaryWe have characterized the properties of an Epstein-Barr virus vector (EBV-CMV) and compared its expression potential with a respective integrating vector (CMV). These vectors were used to express chloramphenicol acetyltransferase (CAT) gene in human HeLa, 293, monkey CV-1, dog MDCK, and hamster R 1610 cells. The EBV-CMV-cat DNA replicates extrachromosomally in HeLa, 293 and CV-1 cells, where also high expression of CAT gene was observed. The EBV-CMV vector integrated in MDCK and R 1610 cells and the CMV vector integrated in all cells tested. Integration yielded mostly clones with low CAT expression. In all cell lines, except HeLa cells, the existence of the extrachromosomal but not the integrated vector DNA is strictly dependent on the Hygromycin B selection pressure. The extrachromosomal state of the EBV vector is a prerequisite for good expression particularly in human and monkey cells.

Structure, Amplification and Methylation of Ornithine Decarboxylase Genes in Human Malignant Cells

Ornithine decarboxylase (ODC; EC 4.1.1.17) belongs to those enzymes which in response to metabolic stress are overproduced by exposed tumor cells. Mouse tumor cells easily acquire resistance to α-difluoromethylornithine (DFMO), a mechanism based irreversible inhibitor of ODC by overproducing the enzyme through amplification of transcriptionally active genes (1, 2, 3) or as a results of more efficient transcription or translation at normal gene copy number (4, 5).