RESUMEN

Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K(m) for dUMP and k(cat) were not substantially different among the variants and were comparable with the wild-type values, but K(m) for methylenetetrahydrofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) and k(off) for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i) values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40 degrees, and the side chain forms an ion pair with Arg-225.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Thymidylate synthase (TS) is indispensable in the de novo synthesis of dTMP. As such, it has been an important target at which anti-neoplastic drugs are directed. The fluoropyrimidines 5-fluorouracil and 5-fluoro-2'-deoxyuridine are cytotoxic as a consequence of inhibition of TS by the metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). This inhibition occurs through formation of a stable ternary complex among the enzyme, the nucleotide analog, and the co-substrate N5, N10-methylenetetrahydrofolate. Numerous studies have shown that cellular concentrations of TS undergo about a 2-4-fold induction following treatment with TS inhibitors. An extensive body of in vitro studies has led to the proposal that this induction occurs because of relief of the translational repression brought on by the binding of TS to its own mRNA. In the current study, we have tested several predictions of this autoregulatory translation model. In contrast to expectations, we find that fluoropyrimidines do not cause a change in the extent of ribosome binding to TS mRNA. Furthermore, mutations within the mRNA that abolish its ability to bind TS have no effect on the induction. Finally, enzyme turnover measurements show that the induction is associated with an increase in the stability of the TS polypeptide. Our results, in total, indicate that enzyme stabilization, rather than translational derepression, is the primary mechanism of TS induction by fluoropyrimidines and call into question the general applicability of the autoregulatory translation model.

Asunto(s)

RESUMEN

Circoviruses are a diverse group of animal and plant pathogens with undefined relationships to one another but for their non-geminate, non-enveloped capsids and circular, single-stranded DNA genomes. The sequences of the beak and feather disease virus and porcine circovirus genomic DNAs are presented and analyzed in the context of the other members of the family. Sequence comparisons, inferred phylogenies, and geographic occurrence suggest that the ambisense circoviruses, particularly the beak and feather disease virus, represent an evolutionary link between the geminiviruses and the plant circoviruses. We propose that the family members be reclassified into three groups: The family Circoviridae consists of the animal pathogens (beak and feather disease virus and porcine circovirus) that possess ambisense genomes with striking similarities to the geminiviruses. The BBTV-like viruses include the plant pathogens (coconut foliar decay virus, banana bunchy top virus, subterranean clover stunt virus) with a geminivirus-like stem-loop element in their DNAs, and single to multiple component genomes. The chicken anemia virus is an unassigned virus possessing unique characteristics bearing little similarity to the other ssDNA viruses.

Asunto(s)

RESUMEN

Fluorinated pyrimidines, such as 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd), are cytotoxic to cells as a consequence of generation of 5-fluoro-2'-deoxyuridylate (FdUMP), which is a mechanism-based inhibitor of the enzyme thymidylate synthase (TS). FdUMP inhibits TS via its binding into a stable inhibitory ternary complex (ITC) with the enzyme and the cosubstrate N5, N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4PteGlu). In previous studies, we identified a naturally occurring mutant form of human TS that contains a Tyr-->His substitution at residue 33 and confers relative resistance to FdUrd in both mammalian and bacterial cells. Kinetic studies indicated that the equilibrium dissociation constant (Kd) for binding of FdUMP into the ITC is altered in the mutant enzyme. In the current investigation, we have examined the kinetics of FdUMP binding into covalent binary complexes, i.e., in the absence of CH2H4PteGlu. Our results showed that although the rate constants for binary FdUMP binding (i.e., kon and koff) are altered by the Tyr-->His substitution, there is no measurable effect on the overall Kd. Analysis of a number of other amino acid substitutions at residue 33 indicated that maximal enzyme accumulation and function requires a bulky, hydrophobic side chain at this site.

Asunto(s)

RESUMEN

The growth of mouse L cell fibroblasts is inhibited by glucocorticoids, and we have selected spontaneous glucocorticoid-resistant L cells in culture. One cloned variant exhibits a stable phenotype in the absence of selective conditions. This variant contains no specific glucocorticoid-binding capacity, no immunoreactive glucocorticoid receptor protein, and no detectable glucocorticoid receptor messenger RNA. A glucocorticoid-dependent reporter gene requires exogenous glucocorticoid receptor cDNA and steroid in order to be expressed in this variant. Genomic DNA analysis of the variant cell line indicates that there has been no gross alteration in receptor gene structure. These results suggest that the variant may be deficient in transcription of the glucocorticoid receptor gene.

Asunto(s)

RESUMEN

Verapamil, the prototype calcium channel blocker, reversibly inhibits cell proliferation in many normal and tumour cell lines (Schmidt et al., Cancer Res., 48, 3617, 1988). We have found that two closely related cell lines - B16 murine melanoma cells and B10.BR normal murine melanocytes growing in culture - behave differently in the presence of verapamil, and we are now utilising these two related cell lines to help elucidate the molecular basis of verapamil's antiproliferative effect. In this study, we studied cell cycle phase distribution and c-myc gene expression in both cell lines in the absence of verapamil, during incubation with verapamil and after the cells were washed free of verapamil. Our studies show that 100 microM verapamil rapidly blocks DNA synthesis in melanocytes but not in B16 cells. Similarly, incubation with verapamil for 6-24 h results in a decreased c-myc signal in melanocytes, but a transient increase in c-myc expression in B16 cells. After verapamil is washed from the cells following a 24-h incubation with drug, c-myc expression increases in melanocytes as they begin again to proliferate, but decreases in B16 cells as they begin to die. Our disparate results with these cell lines suggest that c-myc gene expression, regardless of its known involvement in growth control, is not the immediate target for verapamil's inhibitory action.

Asunto(s)

RESUMEN

Glucocorticoids regulate proliferation of lymphosarcoma P1798 in culture. Treatment with dexamethasone caused a redistribution of cells with respect to the cell cycle. A decrease in cells in S and G2 + M phases was observed. This was accompanied by a corresponding increase in G1 cells. Growth arrest was preceded by a rapid and precipitous decrease in the expression of the cellular c-myc gene. Restriction analysis of the c-myc gene indicated that this locus was neither amplified nor grossly rearranged in P1798 cells. Glucocorticoids caused a decrease in the abundance of c-myc mRNA. After 24 h in 0.1 microM dexamethasone, c-myc mRNA levels declined to less than 5% of control. Fifty percent inhibition occurred within 90 min. The effects of dexamethasone were completely reversible. The amount of c-myc mRNA returned to control levels within 4 h after withdrawal of the hormone. Nuclear run-on transcription analysis indicated that glucocorticoids regulate transcription of the c-myc gene in P1798 cells. Transcription of exons I and II was inhibited to the same extent, suggesting that glucocorticoids inhibit initiation of transcription. Inhibition of transcription may account for decreased expression of c-myc which may, in turn, account for the antiproliferative effects of the hormone.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

An enzyme, galactosyltransferase, able to catalyze the formation of galactose polymers was detected in cell-free extracts of a wild type strain of Neurospora crassa. Enzyme activity was found in both the supernatant and the particle fractions after centrifugation at 100,000 X g. The enzyme assayed in the 100,000 X g supernatant showed a 4fold difference in specific activity as compared to that found in the particle fraction.

Asunto(s)