Ribonucleotide reductase M1 (RRM1) 2464G>A polymorphism shows an association with gemcitabine chemosensitivity in cancer cell lines.

OBJECTIVES: Significant variability in the efficacy and toxicity of an anticancer drug is observed in cancer patients. Currently, there are no standard tools for prediction of a patient's tumor response or his risk of adverse events to chemotherapy. METHODS: We investigate an association between polymorphisms of gemcitabine metabolism-related genes and its chemosensitivity in vitro using 62 human cancer cell lines of various origins. Polymorphisms of gemcitabine metabolism-related genes of deoxycytidine monophosphate deaminase (DCTD), deoxycytidine kinase (DCK) and ribonucleotide reductase M1 (RRM1) were evaluated using the CEQ8000 Genetic analysis system and GeneDoc software. Chemosensitivity of gemcitabine was expressed as an IC50 using MTT assay. RESULTS: The frequency of the polymorphisms was 21% in DCTD 315T>C, 45.2% in RRM1 1082C>A, 59.7% in RRM1 2455A>G, and 79% in RRM1 2464G>A. When examining the association between these polymorphisms and IC50, only the RRM1 2464G>A showed the tendency to be more chemosensitive to gemcitabine (P=0.011), and haplotypes containing 2464G>A polymorphism also showed the association with chemosensitivity when compared to wild-type RRM1 (G2464G). We could not see the significant differences of mRNA expression level with real-time PCR between cell lines according to G2464A polymorphism. In oligonucleotide microarray 73 GenBank Accession Number (69 genes) were selected which expressed differently between RRM1 wild-type and the G2464A polymorphism. CONCLUSIONS: RRM1 2464G>A polymorphism demonstrated an association with gemcitabine sensitivity, which needs functional studies with co-expressing genes and prospective clinical studies for the clinical application as a predictive bio-marker.

An increase in the expression of ribonucleotide reductase large subunit 1 is associated with gemcitabine resistance in non-small cell lung cancer cell lines.

The mechanisms of resistance to the antimetabolite gemcitabine in non-small cell lung cancer have not been extensively evaluated. In this study, we report the generation of two gemcitabine-selected non-small cell lung cancer cell lines, H358-G200 and H460-G400. Expression profiling results indicated that there was evidence for changes in the expression of 134 genes in H358-G200 cells compared with its parental line, whereas H460-G400 cells exhibited 233 genes that appeared to be under- or overexpressed compared with H460 cells. However, only the increased expression of ribonucleotide reductase subunit 1 (RRM1), which appeared in both resistant cell lines, met predefined analysis criteria for genes to investigate further. Quantitative PCR analysis demonstrated H358-G200 cells had a greater than 125-fold increase in RRM1 RNA expression. Western blot analysis confirmed high levels of RRM1 protein in this line compared with the gemcitabine-sensitive parent. No significant change in the expression of RRM2 was observed in either cell line, although both gemcitabine-resistant cell lines had an approximate 3-fold increase in p53R2 protein. A partial revertant of H358-G200 cells had reduced levels of RRM1 protein (compared with G200 cells), without observed changes in RRM2 or p53R2. In vitro analyses of ribonucleotide reductase activity demonstrated that despite high levels of RRM1 protein, ribonucleotide reductase activity was not increased in H358-G200 cells when compared with parental cells. The cDNA encoding RRM1 from H358-G200 cells was cloned and sequenced but did not reveal the presence of any mutations. The results from this study indicate that the level of RRM1 may affect gemcitabine response. Furthermore, RRM1 may serve as a biomarker for gemcitabine response.

Assessment of resistance induction to cytosine arabinoside following transfer and overexpression of the deoxycytidylate deaminase gene in vitro.

Recent attempts to protect hematopoietic progenitor cells from cytarabine (ara-C)-induced toxicity by transfer of the cytidine deaminase (CDD) gene resulted in efficient in vitro inducibility of ara-C resistance. Another enzyme involved in intracellular ara-CTP inactivation is the deoxycytidylate deaminase (dCMPD). We therefore transfected the human dCMPD cDNA gene into murine fibroblasts and investigated the relationship of forced dCMPD expression and resistance induction to ara-C. Several cell lines were established which demonstrated a 1.7-3.5-fold increase in cellular dCMPD activity and an up to 2-fold increase in the IC50 value of ara-C. However, increases in dCMPD activities did not show a positive linear correlation with the induction of ara-C resistance. In addition, CD34 + hematopoietic progenitor cells revealed the highest endogenous dCMPD enzyme levels among different human hematopoietic cells. Thus, despite the documented role for dCMPD in ara-CTP inactivation of certain cell types, these results suggest that the dCMPD gene may prove less useful than the CDD gene as a therapeutic target in attempts to attenuate ara-C-induced bone marrow toxicity.

Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein in Escherichia coli.

The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase. The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids with a calculated molecular mass of 19,985 daltons. The sequence of several cyanogen bromide-cleaved peptides derived from HeLa cell dCMP deaminase are all contained within the deduced amino acid sequence. A zinc binding region is present in the enzyme, similar to that reported for cytidine deaminase (Yang, E. C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). Northern blot analysis revealed a predominant messenger RNA species of 1.9 kilobases. Expression of the active protein to about 10% of Escherichia coli's total protein was achieved by subcloning the open reading frame into a high expression system using the polymerase chain reaction. Polyacrylamide gel electrophoresis revealed a prominent protein band which comigrated with affinity purified HeLa dCMP deaminase, while Western blot analysis yielded an immunoreactive band which comigrated with the single immunoreactive affinity column purified dCMP deaminase band. The enzyme which possesses a kcat of 1.02 x 10(3) s-1 was purified to homogeneity in over 60% yield. The overexpression of dCMP deaminase should permit more exacting studies on the regulation of this important allosteric enzyme which provides substrate for DNA synthesis.

Overcoming inclusion body formation in a high-level expression system.

Attempts at overexpressing T4-phage deoxycytidylate deaminase using the pET3c/BL21(DE3)/pLysS system resulted in this enzyme being part of an inactive inclusion-body complex. However, by employing an enriched growth medium it was found that the deaminase could be induced in a soluble active form to at least 20% of this organism's cellular protein. Insoluble inclusion bodies were obtained with less rich media. This procedure was employed successfully with other highly expressed proteins that formed inclusion bodies. The use of a rich growth medium during the course of protein induction may be a valuable adjunct to limiting inclusion body formation with this as well as other expression systems.

Effect of 5-fluoro-2'-deoxyuridine and hydroxyurea on the phytohemagglutinin-induced increase of thymidine kinase, replicative DNA polymerase, deoxycytidylate deaminase and CDP reductase activities in human lymphocytes.

The inhibitors of DNA synthesis, 5-fluoro-2'-deoxyuridine and hydroxyurea, caused an inhibition of thymidine kinase, replicative DNA polymerase and CDP reductase activities in stimulated lymphocytes when they were exposed to the inhibitors during the early transformation period (0-17 hr). However, the enzyme activities were unaffected when the inhibitors were added to cells stimulated for more than 17 hr. As opposed to these enzymes the deoxycytidylate deaminase activity was unaffected by the inhibitors during the entire transformation period (0-28 hr). This indicates a close regulatory mechanism in lymphocytes between DNA synthesis and induction of enzymes involved in DNA replication. The inhibitory mechanism exerted by the inhibitors is for the moment unknown. It might be independent of the well-known inhibition of the target enzymes, thymidylate synthetase and ribonucleoside diphosphate reductase, since there was no immediate apparent correlation in time between depletion of the pool sizes and the inhibition of the enzyme activities.

Allosteric transitions associated with the binding of substrate and effector ligands to T2 phage induced deoxycytidylate deaminase.

The binding characteristics of T2 phage induced deoxycytidylate deaminase were examined through the use of ultrafiltration and equilibrium dialysis. The positive effectors, 5-(hydroxymethyl)deoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate, were bound in a highly cooperative manner, which is consistent with the allosteric effects promoted by these compounds. Their respective S0.5 values were 8 and 2 microM. A similar degree of cooperativity was associated with the binding of such competitive inhibitors of deoxycytidylate deaminase as dGMP, 4-N-hydroxydeoxycytidine 5'-monophosphate, and tetrahydrodeoxyuridylate. The negative effector, dTTP, also inhibited the binding of dCTP in a pH-dependent manner, which is consistent with its previously demonstrated inhibition of catalysis [Maley, G. F., Guarino, D. U., & Maley, F. (1972) J. Biol. Chem. 247, 931-939]. The binding of dTTP could be demonstrated only at low phosphate concentrations and did not appear to be cooperative. The number of binding sites for the allosteric ligands, substrate, and substrate inhibitors was shown to be six, which coincides with the number of enzyme subunits. It was established by CD difference spectroscopy that dCTP, at concentrations normally employed to demonstrate enzyme activation, effects a dramatic conformation transition in the deaminase, as indicated by a sharp decrease in ellipticity at about 280 nm. The nature of this response suggests that the microenvironment of some of the enzyme's tyrosyl residues had been perturbed by the presence of this allosteric nucleotide.

Aanlysis of dCMP deaminase and CDP reductase levels in hamster cells infected by herpes simplex virus.

Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity.

Bromodeoxyuridine induction of deoxycytidine deaminase activity in a hamster cell line.

The Syrian hamster cell line, RPMI 3460, was found to express barely detectable levels of the enzyme deoxycytidine deaminase. In contrast, the cell lines B4 and HAB, which are derived from 3460 cells and have approx. 60 and 100% bromodeoxyuridine substitution in DNA, respectively, show an approx. 50-fold higher enzyme activity. Deoxycytidine deaminase activity can be "induced" in 3460 cells by growth in 10(-5) M bromodeoxyuridine, as well as by the other halogenated pyrimidines, iododeoxyuridine and chlorodeoxy-uridine. The time required for maximal enzyme activity to accrue (approx. 8 days) suggests that new genetic expression is required for enhanced deoxycytidine deaminase activity and inhibition of induction in the presence of Ara. C shows that bromodeoxyuridine must be incorporated into DNA. In addition, the extent of enhanced deoxycytidine deaminase activity is directly related to the level of bromodeoxyuridine substitution in DNA. Another hamster cell line, BHK21/C13, which shows no detectable deoxycytidine deaminase activity, cannot be induced by bromodeoxyuridine. These results are discussed with respect to a mechanism by which bromodeoxyuridine may alter gene expression due to an altered binding of both positive and negative regulatory proteins to DNA.

Effects of 6-thioguanine on macromolecular events in regenerating rat liver.

Regenerating rat liver was used as a semisynchronous system in which to investigate the effects of 6-thioguanine on biochemical processes occurring in discrete phases of the cell cycle. 6-Thioguanine inhibited the first wave of DNA biosynthesis in regenerating rat liver. This effect appeared to be the result of a decrease, caused by 6-thioguanine, in the induction of several enzyme activities (i.e., thymidine kinase, deoxycytidylate deaminase, cytidine diphosphate reductase, and DNA polymerase) necessary for the initiation of DNA replication in regenerating liver. There was a fairly short period during which 6-thioguanine could be given to rats to accomplish the inhibition of the appearance of the induced activities of these enzymes; this period corresponded to the time just before enzyme induction. The inhibition of the induced synthesis of this group of enzymes occurred in the presence of an intact translational apparatus and intact polysomes and in the absence of interference with the incorporation of radioactive leucine and tyrosine into total protein of liver. Synthesis of polyadenylate-containing RNA was depressed in 6-thioguanine-treated rats, whereas the synthesis of polyadenylate-lacking RNA was unaffected. It is suggested that the inhibition of the synthesis of polyadenylate-containing RNA by 6-thioguanine is at least in part responsible for the observed decrease in induced enzyme activities and the resulting interference with DNA replication.

Factors affecting deoxycytidylate deaminase activity in some transplantable rat hepatomas.

Dunning hepatoma has a low activity of deoxycytidylate deaminase, comparable to that of normal adult rat liver. This activity seems inconsistent with the rapid proliferation rate of the tumor. Factors which might affect the activity of deoxycytidylate deaminase in the Dunning hepatoma have been examined in it and compared to the Novikoff hepatoma which has high activity of this enzyme. The low activity in Dunning hepatoma does not appear to be the result of any inhibition or, possibly, proteolytic enzyme as judged by mixing experiments, nor does it appear to be due to in vivo differences in nucleotide concentrations especially deoxycytidine 5'-monophosphate, deoxycytidine 5'-triphosphate, or deoxyguanosine 5'-monophosphate which might either help stabilize the enzyme, allosterically increase its activity, or inhibit it. The Dunning hepatoma does not convert cytosine deoxyriboside to uridine deoxyriboside at a significant rate, and the formation of uridine deoxyriboside from deoxyuridine monophosphate is 1% or less during a 30-min incubation of high-speed supernatant fraction from the tumor in either the presence or absence of fluoride. It is concluded that the Dunning hepatoma probably has intrinsically low deoxycytidylate deaminase activity.

Level of specific prereplicative mRNA's during bacteriophage T4 regA-, 43- and T4 43- infection of Escherichia coli B.

The role of the T4 bacteriophage regA gene in stabilizing early mRNA was investigated by assaying the level of functional mRNA from eight prereplicative genes (56 [dCMP hydroxymethylase], cd [dCMP deaminase], 1 [deoxynucleotide kinase], rIIA, rIIB, 46 [DNA arrest], and 45) during extended infection of Escherichia coli B with T4 regA-, 43- and T4 43- bacteriophage. The above gene-specific transcripts in RNA isolated from infected cells were quantitated by translation with an E. coli B cell-free system. Conditions were chosen to insure that the amount of gene product formed in vitro, measured either as an enzyme activity or as a radioactive band in acrylamide gel, was directly proportional to the level of mRNA present. The failure of T4 regA-, 43- phage to terminate prereplicative synthesis (Wiberg et al., 1973) resulted in an enhanced production of many early gene products over those formed during T4 43- infection. This increase did not appear to be associated with an increment in mRNA levels, since in the present study gene-specific early mRNA's were found to be only marginally elevated and slightly more stable in T4 regA-, 43-- than in T4 43--infected cells. Of interest was the observation that significant quantities of all of the mRNA's studied; with the exception of those from genes 45 and 46, could be isolated from T4 43--infected cells after synthesis of the respective gene products had ceased. On termination of normal prereplicative synthesis during infection with T4 43- phage, polyribosomes were found to be dissociated completely, a finding which suggests that the residual mRNA present in these cells is free in the cytoplasm. The persistence in T4 43--infected cells of translatable mRNA for many prereplicative genes after product synthesis ceased indicates that the impairment in protein synthesis is not due solely to regA-mediated messenger degradation or modification. Rather, the results suggest that the regA gene product may act either by interfering with early mRNA polypeptide chain initiation or by promoting prereplicative polysome dissociation.