Biomedical subjects

F G Berger

Publications and source records attributed to F G Berger.

At least 55 records · Page 3Linked to original sources

PubMed1989

Evolution of androgen-regulated ornithine decarboxylase expression in mouse kidney.

The analysis of naturally occurring variations in hormone-regulated gene expression generates insights into the mechanisms governing evolutionary changes in hormone response. In the mouse (genus Mus), kidney ornithine decarboxylase (ODC) expression, which is regulated by androgens, has been modified during evolution. This has resulted in intra- and interspecies variations that have accumulated over a 10-15 million year time period. We have examined ODC expression and its response to androgens in eight Mus species. Induced ODC levels were found to be similar in six of these species. Two species (M. cookii, M. pahari) contain diminished enzyme levels that are the result of different mechanisms. In M. cookii, the low ODC levels reflect reduced ODC mRNA induction in response to hormone. In M. pahari, on the other hand, the low ODC levels are not derived from altered mRNA concentrations, but appear to be due to translational and/or posttranslational effects. Nuclear run-on assays indicate that ODC mRNA induction is associated with increases in rates of ODC gene transcription. The low ODC mRNA level in induced M. cookii reflects a low rate of ODC gene transcription. Thus, both transcriptional and posttranscriptional mechanisms have contributed to modification of the ODC expression phenotype during evolution. The significance of these findings to the evolution of androgen-regulated ODC expression is discussed.

Androgens↗

PubMed1988

Tissue- and species-specific regulation of murine alpha 1-antitrypsin gene transcription.

Previous studies from our laboratories have shown that the tissue specificity of alpha 1-antitrypsin (AT) expression differs among closely related mouse species. In laboratory mice (Mus domesticus), AT mRNA is found almost exclusively in the liver. In the wild-derived species Mus caroli, the mRNA is expressed not only in the liver but also in the kidney, where it is regulated by androgens during post-natal development. We presently show that the tissue specificity, the species specificity, and the developmental regulation of AT mRNA levels correlate with the transcription rate of the AT gene, as measured by nuclear run-on assays. During the course of these experiments, we found that some AT-specific probes are complementary to constitutively synthesized RNAs that do not accumulate and that are unrelated to functional AT mRNA expression. These RNAs, which result from both sense and anti-sense transcription, may derive from aberrant initiation events within certain regions of the AT gene.

Aging↗

PubMed1988

The role of thymidylate synthase in the response to fluoropyrimidine-folinic acid combinations.

A panel of human colorectal tumor cell lines has been examined to determine the role of TS in the response to fluoropyrimidine antimetabolites. Among these cell lines, the response to FdUrd does not correlate with the levels of TS. In cell lines HCT 116 and RCA, which are poorly responsive to FdUrd, structural alterations in TS have been identified. In HCT 116, two TS polypeptides are present: a common form, occurring in all the cell lines and a variant form. The variant TS polypeptide has a reduced affinity for the TS ligands, FdUMP and CH2H4PteGlu, relative to the common TS polypeptide. Clonal populations of HCT 116 that overproduce each form have been isolated. Clones that overproduce the variant polypeptide are 4-fold less responsive to TS-directed cytotoxic agents than those that overproduce the common; thus, the presence of the variant TS is associated with a reduced response to TS-directed cytotoxic agents. The response of cell line RCA to FdUrd is dependent upon the extracellular CF concentration: response increases as CF is increased. RCA contains a TS enzyme with reduced affinity for CH2H4PteGlu, relative to cell line C, which is sensitive to FdUrd at all CF concentrations. Both cells form high chain-length polyglutamates of CH2H4PteGlu at CF concentrations in which the response to FdUrd differs by 4-fold. In RCA, the TS structural gene is variant, relative to the other cell lines. This variation may underlie the altered enzyme affinity for CH2H4PteGlu and the sensitivity to modulation of FdUrd response by CF.

Cell Line↗

PubMed1988

Evolution of androgen-regulated mRNA expression in mouse kidney.

To gain information on the evolution of mammalian gene expression patterns, we studied the androgen-inducible expression of three kidney mRNAs in several mouse species (genus Mus). The RP2, ornithine decarboxylase, and beta-glucuronidase mRNAs have each evolved independently, in that the pattern of variation among species is unique for each. This suggests a role for gene-specific, cis-acting genetic elements. Relationships between the regulatory phenotypes and the species phylogeny suggest that the variations in hormone-inducible mRNA expression were generated by a series of independent mutations that occurred in specific lineages, resulting in modifications of the progenitor phenotype. Alternatively, the variations may have preexisted within the progenitor population as polymorphisms that were fixed during establishment of individual lineages. Thus, significant alterations in the androgen-regulated mRNA phenotype have occurred either prior to or during speciation within the Mus genus. These alterations are presumed to be in regulatory sequences that control the expression of the corresponding genes and their response to testosterone; as such, they should be useful in further studying the genetic determinants of gene expression and its evolution.

Androgens↗

PubMed1988

Glucocorticoid regulation of the genes encoding thymidine kinase, thymidylate synthase, and ornithine decarboxylase in P1798 cells.

The expression of a number of genes was measured in P1798 cells treated for various periods of time with 0.1 microM dexamethasone. Thymidine kinase (TK) activity decreased under these conditions with 50% inhibition achieved within approximately 8 h. Decreased TK activity was associated with reduced abundance of TK mRNA. Analysis of nuclear transcription indicated that this was attributable to a decrease in the number of RNA polymerase II molecules engaged in transcription of the TK gene. With respect to TK, there was an overall correlation between enzyme activity, mRNA, and nuclear transcription. The data are consistent with the hypothesis that glucocorticoid inhibition of expression of TK is primarily due to inhibition of transcription. Transcription of the TK gene was also reduced by greater than 90% after inhibition of protein synthesis for 6 h. This suggests that transcription of this gene requires a protein of short biological half-life. It is proposed that this hypothetical transcription factor is regulated by glucocorticoids. The amount of thymidylate synthase and dihydrofolate reductase remained constant for at least 24 h in dexamethasone-treated P1798 cells. Dihydrofolate reductase mRNA likewise remained constant. However, the mRNA encoding thymidylate synthase decreased 80-90% within 24 h. The mRNA encoding ornithine decarboxylase also decreased. In neither case did this appear to be primarily due to inhibition of transcription of the respective genes. The abundance of the mRNAs encoding hypozanthine-guanine phosphoribosyl transferase and phosphoglycerate kinase did not decrease in dexamethasone-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Thymidylate synthase as a determinant of 5-fluoro-2'-deoxyuridine response in human colonic tumor cell lines.

A panel of seven human colorectal cell lines of differing phenotype has been examined to elucidate the role of thymidylate synthase (TS) in the response to 5-fluoro-2'-deoxyuridine (FdUrd). Although TS is a major target of FdUrd, no consistent relationship was observed between the intracellular levels of TS and the response to FdUrd among the cell lines. Levels of thymidine kinase and dihydrofolate reductase, enzymes that are involved in generation of ligands that form the inhibitory ternary complex with TS, do not correlate with FdUrd response. Two cell lines that exhibit innate resistance to FdUrd, relative to the other cell lines, have variations in TS enzyme structure or gene structure. Cell line HCT 116 contains two forms of TS, as defined by isoelectric focusing. One form, which is unique to HCT 116, is more basic than the common form, which is present in all the cell lines. Cell line RCA contains a variation in the TS structural gene, as defined by restriction fragment-length analysis. These structural variations, which are associated with reduced response to FdUrd, may serve as markers for reduced clinical response to TS-directed chemotherapy.

Blotting, Northern↗

PubMed1988

A naturally occurring variation in thymidylate synthase structure is associated with a reduced response to 5-fluoro-2'-deoxyuridine in a human colon tumor cell line.

Inhibition of thymidylate synthase (TS) is an important mechanism of action of fluoropyrimidine antimetabolites. Thus, TS structure and expression are expected to be determinants of response to these agents. The role of TS in fluoropyrimidine response has been analyzed in a panel of human colonic tumor cell lines. Previous work has demonstrated that there is little correlation between TS concentration and sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd) among these cell lines, suggesting that parameters other than the TS levels are responsible for the variations in drug response. One such parameter has been identified in cell line HCT 116. This line, which is relatively resistant to FdUrd, produces two structural forms of TS, as determined by mobility of the enzyme in isoelectric focusing polyacrylamide gels. One form is common to all the cell lines, whereas a variant form, which is more basic and is encoded by a separate structural gene, is unique to HCT 116. Cells expressing one or the other TS form have been isolated and used to demonstrate that the variant form is associated with FdUrd resistance. Kinetic experiments indicate that the variant TS has reduced affinities for 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate, which are ligands involved in formation of a stable inhibitory complex with the enzyme. Thus, the innate resistance of cell line HCT 116 to FdUrd is derived, at least in part, from production of an altered structural form of TS having reduced affinity for ligands.

Blotting, Northern↗

PubMed1987

Developmental expression, cellular localization, and testosterone regulation of alpha 1-antitrypsin in Mus caroli kidney.

alpha 1-Antitrypsin (alpha 1-protease inhibitor), an essential plasma protein, is synthesized predominantly in the liver of all mammals. We have previously shown that Mus caroli, a Southeast Asian mouse species is exceptional in that it expresses abundantly alpha 1-antitrypsin mRNA and polypeptide, in the kidney as well as the liver (Berger, F.G., and Baumann, H. (1985) J. Biol. Chem. 260, 1160-1165) providing a unique model for examination of the evolution of genetic determinants of tissue-specific gene expression. In the present paper, we have further characterized alpha 1-antitrypsin expression in M. caroli. The extrahepatic expression of alpha 1-antitrypsin is limited to the kidney, specifically within a subset of the proximal tubule cells. The developmental pattern of alpha 1-antitrypsin mRNA expression in the kidney differs from that in the liver. In the kidney, alpha 1-antitrypsin mRNA is present at only 2-4% adult level at birth and increases very rapidly to adult level during puberty between 26 and 36 days of age. There are no significant changes in liver alpha 1-antitrypsin mRNA levels during this period. Testosterone, while having only modest affects on alpha 1-antitrypsin mRNA accumulation in the adult kidney, causes a 20-fold induction of the mRNA in the pre-pubertal kidney. This suggests that the increase in alpha 1-antitrypsin mRNA expression during puberty is testosterone mediated. Southern blot analyses of Mus domesticus and M. caroli genomic DNA and a cloned M. caroli alpha 1-antitrypsin genomic sequence, indicate that a single alpha 1-antitrypsin gene exists in M. caroli, whereas multiple copies exist in M. domesticus. These data show that the alteration in tissue specificity of alpha 1-antitrypsin mRNA accumulation that has occurred during Mus evolution is associated with distinctive developmental and hormonally regulated expression patterns.

Animals↗

PubMed1987

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.

Adenosylmethionine Decarboxylase↗

PubMed1987

Evolution of steroid-inducible RP2 mRNA expression in the mouse kidney.

We have examined the structure and expression of mRNAs encoded by the androgen-inducible RP2 gene in the kidneys of nine mouse species within the genus Mus. There is considerable interspecies variation in the lengths of the major RP2 transcripts; some of this variation is due to the presence or absence of a B1 repetitive element in the 3'-untranslated region of the gene. In addition, the extent of RP2 mRNA induction by testosterone differs among the species. Two species show 10-20-fold induction, while others display a reduced response or none at all. Analysis of an interspecific hybrid indicates that the inducibility phenotype is inherited in an additive fashion. A correlation between RP2 inducibility and the time of formation of lineages within the Mus genus suggests that induction evolved in a stepwise fashion, with the acquisition of a modest hormonal response being followed by the appearance of a greater response. The interspecies variations in RP2 mRNA structure and regulation provide a useful model for the identification and study of genetic elements that elicit evolutionary alterations in steroid-modulated gene expression.

Animals↗

PubMed1987

Thymidylate synthase gene amplification in a colon tumor resistant to fluoropyrimidine chemotherapy.

We have identified amplification of the thymidylate synthase gene in a colonic tumor that had developed resistance to 5-fluorouracil/leucovorin combination chemotherapy. The tumor had previously undergone a partial response to this combination but began to progress following a prolonged period of continuous therapy and relative disease stabilization. Since thymidylate synthase is a target enzyme for 5-fluorouracil, it is likely that the observed gene amplification is responsible for the resistance. Thus, gene amplification may be a relevant mechanism of acquired resistance to fluoropyrimidine chemotherapy in the clinic.

Colonic Neoplasms↗

PubMed1986

Putrescine does not mediate the androgen-response in mouse kidney.

We have tested the notion that polyamines, particularly putrescine, mediate the response of mouse kidney to androgens. Hormonal effects were measured in female mice maintained on the ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO), which results in a 85-90% reduction of ODC enzyme levels and a depletion of putrescine concentrations in kidney. These animals exhibited normal kidney cell hypertrophy in response to testosterone. In addition, androgen-inducibility of the RP2 gene was indistinguishable from that in normal mice. These results indicate that an increase in putrescine levels is not a prerequisite for androgen effects in mouse kidney and that putrescine does not mediate the hormonal response.

Animals↗

PubMed1986

Androgen induction of messenger RNA concentrations in mouse kidney is posttranscriptional.

The concentrations of several mRNAs in mouse kidney increase in response to testosterone. To determine if the increases are generated at the level of gene transcription, we have assayed transcription rates for several androgen-inducible mRNAs in kidney nuclei in vitro. No significant changes were found in the synthesis of three mRNAs whose concentrations increase 10-20-fold during testosterone treatment. Kinetic analysis of changes in transcript levels after testosterone administration and withdrawal suggests that mRNA stabilization is a major factor in the inductions. Thus, the androgen-mediated induction of these kidney mRNAs is generated predominantly at the posttranscriptional level.

Animals↗

PubMed1986

Rearrangement of genes located on homologous chromosomal segments in mouse and man: the location of genes for alpha- and beta-interferon, alpha-1 acid glycoprotein-1 and -2, and aminolevulinate dehydratase on mouse chromosome 4.

Gene mapping studies to determine the order of alpha- and beta-interferon (Ifa, Ifb), aminolevulinate dehydratase (Lv), and alpha-1 acid glycoprotein-1 and -2 (Orm-1, Orm-2) relative to each other and to the reference genes brown (b), B-cell maturation factor responsiveness (Bmfr-1), and major urinary protein-1 (Mup-1) are reported. The most likely order was Mup-1--Lv--b--Orm-1, Orm-2--Ifa, Ifb--Bmfr-1. This order suggested that two chromosomal segments located on chromosome 4 in the mouse and chromosome 9 in man have been conserved since divergence of lineages leading to man and mouse; these segments are marked by soluble aconitase-1 (Aco-1) and galactose-1 phosphate uridyl transferase (Galt) and by Lv and Orm-1. This order also demonstrated that, although genes located on opposite arms of chromosome 9 in man remain syntenic in the mouse, gene order has not been conserved; Ifa and Ifb are not located in their expected locations near Aco-1 and Galt. The position of Ifa and Ifb between Orm-1 and Bmfr-1 could not be determined with certainty because of apparent heterogeneity in recombination frequencies between crosses involving conventional laboratory strains of mice and crosses involving interspecific matings between laboratory mice and Mus spretus. This result suggests that caution must be exercised when using M. spretus in linkage crosses.

Animals↗

PubMed1985

An evolutionary switch in tissue-specific gene expression. Abundant expression of alpha 1-antitrypsin in the kidney of a wild mouse species.

alpha 1-Antitrypsin (AT), one of the major proteinase inhibitors in mammalian serum, is generally considered to be synthesized exclusively in the liver. We have found that a wild-derived Mus species, Mus caroli, expresses AT mRNA in kidney at levels approaching that in liver; no other mouse, inbred or wild-derived, exhibits this striking property. Liver and kidney mRNAs from M. caroli encode very similar AT polypeptides that are distinct from that encoded by Mus musculus liver mRNA. In vivo, liver AT is secreted into the bloodstream, while kidney AT, which is processed differently from the liver protein, is excreted into the urine. Analysis of RNA from a hybrid between M. musculus and M. caroli indicates that a cis-acting genetic element may be responsible for the difference in AT expression. Restriction enzyme digestion patterns of AT genomic sequences in M. caroli DNA are considerably different from those in M. musculus; in addition, these sequences are undermethylated in liver DNA from M. musculus and in liver and kidney DNA from M. caroli, reflecting the respective patterns of expression. Further studies of the altered tissue specificity of AT expression that is apparent in these two related species should lead to new insights into the nature and evolution of genetic determinants of tissue-specific phenotypes.

Animals↗

PubMed1985

Genetics and evolution of the acute phase proteins in mice.

In mammals, a number of liver-derived plasma proteins, termed acute phase reactants, are induced during an inflammation response. We have studied genetic variation in the structure and expression of several of these proteins in a variety of inbred and wild-derived mice. In a genetic cross, electrophoretic polymorphisms for the two alpha 1-acid glycoproteins, AGP-1 and AGP-2, co-segregated in 58 backcross progeny, indicating that either a single gene or two tightly-linked genes on chromosome 4 encode the AGPs. In the same backcross, segregation of variation in haptoglobin structure showed that the gene encoding this acute phase reactant is on chromosome 8. Structural variation in serum amyloid A correlated with restriction fragment length polymorphisms in the Saa gene determined by Taylor and Rowe (1984). Analysis of a number of highly diverged species of mice indicated that AGP expression has undergone considerable modification during evolution of the Mus genus; this is associated with alterations in Agp gene organization, which may include species-specific amplification and/or deletion events.

Animals↗

PubMed1985

Thymidylate synthase overproduction and gene amplification in fluorodeoxyuridine-resistant human cells.

Cytotoxicity to 5-fluoro-2'-deoxyuridine (FdUrd) derives from its conversion to 5-fluorodeoxyuridine-5'-monophosphate, which binds to and inhibits thymidylate synthase (TS) in the presence of the cofactor, 5,10-methylenetetrahydrofolate. We have selected FdUrd-resistant variants of the human cell line HEp-2 following adaptation to stepwise increases in drug concentration. In the initial selection, maximal drug resistance was associated with a 26-fold increase in the cellular level of TS. Greater TS overproduction (80-fold) was obtained by selection for FdUrd resistance in the presence of 10 microM folinic acid and 100 microM deoxyinosine. The latter agents were included to expand the folate pool to ensure adequate levels of cofactor during the selection process. Using cDNA plasmid pMTS-4, which is complementary to mouse TS mRNA, we show that TS overproduction in the HEp-2 variants is accompanied by a 100-fold increase in TS mRNA and a 100-fold amplification of the TS structural gene. Thus, TS overproduction and gene amplification is a mechanism of resistance to FdUrd in human cells.

Base Sequence↗

PubMed1984

Androgen-regulated ornithine decarboxylase mRNAs of mouse kidney.

Ornithine decarboxylase, the first enzyme of the polyamine biosynthetic pathway, is induced by androgens in the mouse kidney. We have isolated from a kidney cDNA clone bank two plasmids, pODC1440 and pODC934 , that contain different cDNA inserts corresponding to ornithine decarboxylase mRNA. Identification was based upon the ability of plasmid-specific mRNAs to encode a 53,000-dalton polypeptide that reacts with antibody to purified mouse kidney ornithine decarboxylase. Plasmid pODC1440 hybridizes predominantly to a mRNA that is 2.1 kilobases (kb) long and is induced about 20-fold in the kidneys of female mice treated with testosterone. Plasmid pODC934 hybridizes to mRNAs of lengths 2.2 and 2.6 kb, which are induced about 8-fold by testosterone. There are probably no more than 1-2 copies of the pODC1440 -specific sequence in the mouse genome, while there may be as many as 12 copies of the pODC934 -specific sequence. That the two plasmids correspond to different ornithine decarboxylase mRNAs is suggested by two observations. First, several mouse strains express normal levels of pODC934 -specific RNA and little or no pODC1440 -specific RNA; furthermore, pODC934 -specific RNA is expressed in several tissues while pODC1440 -specific RNA is kidney-specific. Thus, androgen-mediated stimulation of kidney ornithine decarboxylase activity levels involves alterations in the concentrations of at least two distinct mRNAs.

Androgens↗