The use of ureteral stents and suprapubic catheter in vesicoureteric reflux surgery.

The use of ureteric stents in reimplantation surgery is important. The younger the patient, the more important the stenting of ureters post reimplantation becomes, because even minimal oedema following surgery will produce ureteric obstruction unless stents are in place. JJ stents are now the preferred method of choice in ureteric reimplantation surgery, but in the past the patient required another admission to hospital and general anaesthetic to have the stents removed endoscopically. We describe a technique whereby the stents are attached to the suprapubic catheter and are therefore removed prior to the patient's discharge from hospital, thus obviating the need for a second admission and second anaesthetic for the stent removal. We have studied 23 patients with this technique and find that it is a reliable and safe method to use.

Activity of human trypanosome lytic factor in mice.

The inability of the cattle pathogen Trypanosoma brucei brucei to infect humans is due to an innate factor in human serum termed Trypanosome Lytic Factor (TLF). Human haptoglobin-related protein is the proposed toxin in TLF and can exist either as a component of a minor subclass of high-density lipoprotein (TLF-1) or as a lipid free, high molecular weight protein complex (TLF-2). The trypanolytic activity of both TLF-1 and TLF-2 has been studied in vitro but their relative contributions to protection against T. b. brucei infection in vivo has not been established. In the present studies we show that treatment of T. b. brucei infected mice with TLF-1 resulted in a dose dependent decrease in parasite numbers but did not affect parasite numbers in mice infected with Trypanosoma brucei rhodesiense, the causative agent of the human sleeping sickness. Similarly, pretreatment of mice with TLF-1 resulted in protection against a challenge by T. b. brucei but had no effect on T. b. rhodesiense challenge. Induction of the acute phase protein haptoglobin, a natural antagonist of TLF-1, diminished but did not abolish the protection against trypanosome challenge. In addition, haptoglobin knockout mice showed higher levels of TLF-1 mediated protection against a T. b. brucei challenge. These results suggest that while TLF-1 is active in vivo, even in the presence of elevated levels of haptoglobin, its activity is modulated in a dose dependent fashion by haptoglobin in the circulation.

Haptoglobin, inflammation, and tumorigenesis in the MIN mouse.

We have determined that the MIN mouse, which is predisposed to adenomas of the small and large intestine, exhibits chronic inflammation characterized by the induction of haptoglobin (HP), as well as other acute phase proteins. Inflammation is initiated at about 40-70 days of age, and is maintained throughout the life of the animal. Delayed onset of inflammation brought on by dietary means is associated with a reduction in tumor number and longer life-span. Knockout mice that lack haptoglobin exhibit an increase in tumor number, indicating that the acute phase reactant suppresses tumorigenesis, perhaps through inhibition of the inflammatory response. The MIN mouse is, therefore, a useful model for studying the roles of HP and inflammation in tumorigenesis in vivo.

Mechanisms of acquired resistance to thymidylate synthase inhibitors: the role of enzyme stability.

Inhibitors of the enzyme thymidylate synthase (TS), such as the fluoropyrimidines 5-fluorouracil and 5'-fluoro-2'-deoxyuridine (FdUrd) or the antifolates AG337, ZD1694, and BW1843U89, are widely used in the chemotherapy of cancer, particularly cancer of the colon and rectum. Numerous studies have shown that TS gene amplification, leading to mRNA and enzyme overproduction, is a major mechanism of resistance to these inhibitors. In the present work, we have isolated and characterized FdUrd-resistant derivatives of several human colon tumor cell lines. Although gene amplification was commonly observed, the increases in mRNA and enzyme were strikingly discordant. In one drug-resistant line, a deficiency of enzyme relative to mRNA was shown to be caused by expression of a metabolically unstable TS molecule. The reduced half-life of TS in this line was caused by a Pro-to-Leu substitution at residue 303 of the TS polypeptide. The mutant enzyme conferred resistance to FdUrd as well as antifolates in transfected cells. In another FdUrd-resistant line, which had an excess of enzyme relative to mRNA, the TS molecule was more stable than in the parent line. However, no amino acid substitutions were detected in the TS polypeptide from this line, which suggests that the stabilization must be caused by changes in one or more cellular factors that regulate TS degradation. The results indicate that changes in the stability of the TS polypeptide accompany, and even contribute to, acquired resistance to TS inhibitors in colon tumor cells.

Evolution of transcriptional regulatory elements within the promoter of a mammalian gene.

Transcription of the murine D7Rp2e gene is highly variable among species of the genus Mus, indicating that extensive modifications in the gene's regulatory elements have occurred during evolution. Since promoter regions are well known to harbor cis-acting information that controls gene transcription, we compared the sequence and function of the D7Rp2e promoter in several Mus species, with the goal of understanding the molecular mechanisms underlying the interspecies variations in expression. Three overlapping binding sites for nuclear factors (sites A, B, and C in proximal to distal order) were identified about 300 bp upstream of the transcriptional start site. The sequences of these sites differ between the species Mus domesticus and M. pahari, which exhibit distinct D7Rp2e expression phenotypes. Site A binds a factor called RPBF-I; sites B and C bind a distinct factor that is termed RPBF-II and is likely a member of the NF-I family of transcription factors. DNase I footprinting experiments with the M. domesticus promoter show that binding of RPBF-II at site B is very strong, while binding of RPBF-I and RPBF-II at sites A and C, respectively, is weak; in contrast, with the M. pahari promoter, factor binding at sites A and C is strong, while that at site B is weak. These differences in patterns of binding-site occupancy derive from changes in the affinities of individual sites for their cognate nuclear factors. Transient transfection experiments indicate that the M. pahari binding pattern is capable of repressing transcription of a linked reporter. Such repression may contribute to the differences in D7Rp2e expression between the two species. We suggest that the species-specific footprinting patterns represent the existence of a dynamic equilibrium between two states of nuclear factor binding, the nature of which can be modified during evolution to result in new patterns of gene transcription.

Expression of the alpha 1-proteinase inhibitor gene family during evolution of the genus Mus.

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1-PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.

Biphasic binding of 5-fluoro-2'-deoxyuridylate to human thymidylate synthase.

Thymidylate synthase (TS) is a homodimeric enzyme that catalyzes the reductive methylation of dUMP by N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, to form dTMP. Inhibition of TS by the dUMP analog 5-fluoro-dUMP (FdUMP) occurs through the formation of a covalent ternary complex containing the nucleotide analog, N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, and the enzyme; this complex is termed the inhibitory ternary complex (ITC). In the present report, the kinetics of FdUMP binding into an ITC with purified preparations of human TS were examined. Rapid chemical-quench techniques, as well as steady state binding methods, showed that the enzyme contains two distinct FdUMP binding sites with different affinities for the nucleotide analog. Binding to the first, or high affinity, site was rapid and reached a maximum stoichiometry of 1.0 mol of FdUMP/mol of dimer; binding to the second, or low affinity, site was much slower and reached a stoichiometry of 1.7 mol of FdUMP/mol of dimer. Rate constants for FdUMP binding to and dissociation from the ITC (kon and koff, respectively) were determined, as were equilibrium dissociation constants (Kd). A naturally occurring mutant form of TS, which contains a tyrosine to histidine substitution at residue 33 and renders cells relatively resistant to fluoropyrimidines, exhibited a lower affinity for FdUMP specifically at the second binding site, with little or no change at the first. Hill coefficients were < 1.0, with the His-33 enzyme having a significantly lower coefficient than the wild-type enzyme. The results, in total, indicate that the two FdUMP binding sites on the TS dimer are nonequivalent. We suggest that such nonequivalence may be due to negative cooperativity, where nucleotide binding to the first subunit elicits a conformational change that results in reduced affinity for ligand at the second subunit. This negative cooperativity may be stronger for the His-33 mutant. Thus, the relative fluoropyrimidine resistance conferred by the His-33 substitution may be due to enhanced negative cooperative effects on FdUMP binding into the ITC, thereby reducing the effectiveness of the pyrimidine analog as an inhibitor of thymidylate biosynthesis.

Functional effects of a naturally occurring amino acid substitution in human thymidylate synthase.

A major mechanism underlying the cytotoxicity of fluoropyrimidine analogs such as 5-fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) occurs via the formation of 5-fluoro-2'-deoxyuridylate (FdUMP), a tight-binding inhibitor of thymidylate synthase (TS). Genetic variation in the structure of the TS molecule is an important determinant of response to fluoropyrimidines, because such variation may affect the binding of FdUMP to the enzyme. Previous studies have shown that the colonic tumor cell line HCT116 expresses two structurally distinct TS polypeptides that differ by the presence of tyrosine or histidine at residue 33. Compared with the Tyr-33 form, the His-33 form confers a 3-4-fold level of FdUrd resistance to cells; this was postulated to be derived from the reduced affinity of the enzyme for FdUMP and N5,N10-methylenetetrahydrofolate, ligands required for the formation of a stable inhibitory complex. In the present study, the Tyr-33 and His-33 forms have been purified to homogeneity, and their properties have been compared in detail. The Km values for dUMP and N5,N10-methylenetetrahydrofolate in the TS reaction were not significantly different between the two enzymes. In contrast, the catalytic efficiency (kcat) was 8-fold lower for the His-33 form. Kinetic and equilibrium binding measurements demonstrated that the dissociation constant for FdUMP binding into the ternary complex was 3-4-fold higher for the His-33 form; this was shown to be due to both a decrease in the rate of FdUMP association with the enzyme and an increase in the rate of FdUMP dissociation from the ternary complex. A TS form containing phenylalanine at residue 33 was created by site-directed mutagenesis and was shown to be very similar to the Tyr-33 enzyme with regard to kcat, pH/activity profile, and effect on FdUrd response. Thus, it is the presence of histidine at residue 33, rather than the absence of tyrosine, that is responsible for the alterations in catalytic and ligand-binding functions exhibited by the His-33 form. Possible mechanisms by which the histidine residue perturbs the structure of the TS active site are discussed.

Genetic variation in thymidylate synthase confers resistance to 5-fluorodeoxyuridine.

The human colorectal tumor cell line HCT 116 was resident to the cytotoxic effects of 5-fluorodeoxyuridine (FdUrd). The response to FdUrd was increased only slightly by the presence of 10 microM folinic acid (CF). HCT 116 formed FdUMP and CH2H4PteGlu polyglutamates after exposure to FdUrd and CF. The sensitivity to FdUrd correlated well with the extent of TS inhibition. The role of TS in the resistance of the cells to FdUrd was examined. HCT 116 expresses two TS enzymes, which differ in pI. The more basic TS has been detected in only HCT 116 cells. The other TS is identical in pI to the enzymes detected in other human cells. The variant TS differs from the common by His replacement of Tyr at residue 33. The variant TS exhibited a 3-fold lower affinity for FdUMP than the common TS. The enzymes co-expressed in HCT 116 exhibited an FdUMP binding constant similar to that of the variant TS. TS-deficient cells were transfected with cDNAs encoding the two TS polypeptides. Transfectants expressing the variant TS were more resistant to FdUrd cytotoxicity than cells expressing the common TS. Thus, the structural variation in TS reduced enzyme affinity for FdUMP and conferred resistance to FdUrd.

A naturally occurring tyrosine to histidine replacement at residue 33 of human thymidylate synthase confers resistance to 5-fluoro-2'-deoxyuridine in mammalian and bacterial cells.

Structural changes in the macromolecular targets of pharmacological agents can result in alterations in the efficacy of these agents. In previous studies, we identified a variant structural form of thymidylate synthase (TS) that is associated with relative resistance to 5-fluoro-2'-deoxyuridine, in a human colonic tumor cell line. We now report on the use of DNA transfer techniques to examine directly the effects of each TS form on drug response. TS cDNA constructs, corresponding to the normal or variant TS mRNA, were expressed in Chinese hamster lung cells or in Escherichia coli, and response to 5-fluoro-2'-deoxyuridine was determined. We observed that expression of the variant TS, which differs from the normal form by a tyrosine to histidine substitution at residue 33, confers a 4-fold level of drug resistance in the mammalian cells, as well as in bacteria. The possible role of Tyr-33 in 5-fluoropyrimidine-mediated inhibition of TS is discussed.

Evolution of messenger RNA structure and regulation in the genus Mus: the androgen-inducible RP2 mRNAs.

The RP2 gene is one of several genes that are regulated by androgens in the mouse kidney. Previous studies have demonstrated that androgen inducibility of RP2 transcription varies among species within the genus Mus, indicating extensive evolutionary modification of the participating regulatory elements. Thus, while a five-fold induction of transcription occurs in M. domesticus, none is detectable in M. hortulanus or M. caroli. In the present paper, we have sequenced cDNAs representing the RP2 mRNAs of M. caroli and M. saxicola and have compared them with each other and with M. domesticus. Several findings from the sequence comparisons indicate that the encoded 41-kD polypeptide is physiologically functional. First, divergence within noncoding regions of the mRNAs exceeds that within coding regions. Second, the 357-codon open reading frame has been maintained among the species, with approximately 90% of the amino acid replacements being conservative. Finally, substitution rates at synonymous sites within the coding regions are from twofold to threefold greater than those at nonsynonymous sites. The genetic elements responsible for variations in RP2 inducibility among species were studied by cis/trans analyses of mice heterozygous for RP2 alleles, using a primer extension assay to measure expression of species-specific mRNAs. The results show that the absence of transcriptional induction in M. hortulanus is due to a cis-acting genetic element, while that in M. caroli is due to a trans-acting element. Thus, the androgen-resistant RP2 phenotypes of these two species derive from distinct genetic events.

Single amino acid substitution defines a naturally occurring genetic variant of human thymidylate synthase.

Previously, we identified an altered structural form of thymidylate synthase (TS) in a human colonic tumor cell line. This form, which is encoded by a variant structural gene, renders cells relatively resistant to 5-fluoro-2'-deoxyuridine as a result of the reduced affinity of the enzyme for the active metabolite 5-fluoro-2'-deoxyuridylic acid. We have isolated a cDNA clone specific to the altered TS and have determined its sequence. Two point mutations distinguish the normal from the altered TS mRNAs. One, a (A----G) change, is located within the 3'-untranslated region; the other, a T----C change within the amino acid-coding region, predicts replacement of tyrosine by histidine at residue 33 of the polypeptide. This sequence change was confirmed by direct analysis of cDNA amplified by the polymerase chain reaction and was further verified using allele-specific oligonucleotides as probes in Northern blots. These results, along with studies by other laboratories showing Tyr33 to be evolutionarily conserved, suggest that this residue plays an important role in TS function.

Molecular genetics of androgen-inducible RP2 gene transcription in the mouse kidney.

Androgen control of the RP2 gene in the mouse kidney has been modified during evolution. In inbred mice (Mus domesticus), the concentrations of mRNAs encoded by RP2 undergo a 10- to 12-fold induction in response to testosterone; in other Mus species (e.g., Mus hortulanus and Mus caroli), induction ranges from none to about two- to fourfold. In this communication, we show that androgens induced RP2 transcription in M. domesticus, although this induction may not have fully accounted for the increase in mRNA levels. Reduced mRNA inducibility in M. hortulanus and in several other species was associated with an absence of transcriptional induction. Analysis of an interspecies backcross population indicated that the difference in RP2 inducibility between M. domesticus and M. hortulanus was due to a single Mendelian locus tightly linked (0 of 47 recombinants) to RP2. The RP2 gene was found to contain at least two promoters, only one of which was highly sensitive to testosterone. These results indicate that induction of the RP2 mRNAs, as well as interspecies variations in RP2 inducibility, are primarily a consequence of effects on this promoter.

Transient activation of c-myc protooncogene expression in Leydig cells by human chorionic gonadotropin.

This study evaluated the effects of in vivo administration of human chorionic gonadotropin (hCG) on c-myc oncogene expression in Leydig cells. Sprague-Dawley rats (46-50 days old) were treated with hCG (10 units, i.p.), and purified Leydig cells were isolated 1-24 h later. HCG caused a transient elevation of c-myc mRNA in 4 h and returned to normal or lower than normal levels at 24 h. There was no change in c-fos or beta-actin mRNA levels. Our results suggest that the growth promoting effects of hCG on Leydig cells may be mediated by the transient expression of c-myc protooncogene.

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.

Biochemical and molecular characterization of the glucocorticoid receptor of lymphosarcoma P1798 variants.

Three phenotypically distinct isolates from lymphosarcoma P1798 have been compared with respect to properties of the glucocorticoid receptor. Wild type P1798 cells express functional receptors and glucocorticoid treatment of such cells causes cytolysis in vivo. Wild type cells do not undergo cytolysis in culture. Rather, such cells exhibit reversible inhibition of proliferation in the presence of dexamethasone. Two variant populations were selected from this background. One was selected for the ability to form tumors in mice receiving pharmacological doses of glucocorticoids. Cells from such tumors are resistant to the cytolytic effects of glucocorticoids in vivo, but are sensitive to the antiproliferative effects of the hormone in culture. Variants were also selected based upon their ability to proliferate in the presence of dexamethasone in culture. These variants were resistant to glucocorticoid-mediated cytolysis in vivo. Wild type P1798 cells express approximately 20,000 high affinity dexamethasone-binding sites per cell. Dexamethasone-mesylate labeling and immunoblotting experiments indicate that hormone binding is due to a polypeptide of Mr 90-100 K. This polypeptide is encoded in an mRNA species that resolved as a single entity of approximately 7000 nucleotides. Variants selected for resistance to cytolysis in vivo are indistinguishable in any of these respects from wild type cells. The receptors are fully functional, as evidenced by their ability to precipitate growth arrest of dexamethasone-treated cultures. Variants selected for resistance in culture harbor a receptor mutation. They express fewer than 500 dexamethasone-binding sites per cell. Such variants contain neither detectable dexamethasone-mesylate-binding protein nor any protein that is recognized by a receptor antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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.