Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis. The enzyme consists of two subunits, M1 and M2. Hydroxyurea (HU) is an M2-specific inhibitor. It has been shown that a HU-resistant clone derived from stepwise exposure to HU overexpresses the M2 mRNA and the RR protein (Y. Yen et al., Cancer Res., 54: 3868-3691, 1994). In this study, we established stable clones by transfecting human KB cells with the cDNA of human wild-type RR in which each subunit was overexpressed by a SV40 promoter. The mammalian cell expression vector ph beta APr-1 was used for constructing M1, M2, and M1/M2 subunit cDNA. The transfected cells were selected with G418. The clones designated M2-D, M1-D, X-D, and KB-V represent transfectant clones which contain M2 cDNA, M1 cDNA, M1/M2 cDNA, and vector alone, respectively. The parental KB cells and clones containing vector plasmid KB-V express equally low amounts of M2 and M1 mRNA from the endogenous genes. The expression of M2 mRNA and M1 mRNA is elevated 2-3 fold in the X-D transfectants. M2-D clone demonstrated a 6-fold higher M2 mRNA level although the M1 mRNA expression remains the same as parental cells. M1-D transfectants have a 3-fold increase in M1 mRNA expression relative to parental cells, but reveal no alteration of M2 mRNA. Southern analysis of genomic DNA suggested the incorporation of the plasmid into the genome. The X-D clone revealed both integration of the M2 and M1 gene while the M2-D clone only showed M2 gene integration. The M1-D clone revealed M1 gene integration relative to the parental cells. The Western blot of M2 protein showed a 3-fold increase in the X-D and M2-D clones whereas the M2 protein level in M1-D was the same as it was in parental cells. The M1 protein was increased 3-fold in X-D and 1.5-fold in M1-D over that of parental cells. However, lower M1 protein levels were identified in the M2-D clone. The specific activity of the RR enzyme from each transfectant showed a 3-fold increase in both the X-D and M2-D clones and slightly increased in M1-D clone over that of parental cells. However, X-D and M2-D both demonstrated a 3-fold increase in resistance to HU as compared to M1-D which showed the same sensitivity as the parental enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Overexpression of ribonucleotide reductase in transfected human KB cells increases their resistance to hydroxyurea: M2 but not M1 is sufficient to increase resistance to hydroxyurea in transfected cells. 788 31

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis and repair. The enzyme consists of two dissimilar subunits, M1 and M2. It is known that the M2 subunit plays a role in tumorgenicity and metastasis. In this study, we transfected human oropharyngeal KB cancer cells with human RR M1 and M2 antisense cDNA expressed by an inducible vector system. The transfectants were double-selected with hygromycin and G418. The clones, designated KB-M1AS, KB-M2AS and KB-CAT, represented transfectant clones that contained M1 antisense cDNA, M2 antisense cDNA, and a CAT reporter gene, respectively. In a colony-forming assay, colony formation for the KB-M2AS clone decreased approximately 50% when M2 antisense mRNA expression was induced by isopropylthiogalactose (IPTG). However, the KB-M1AS clone revealed no significant inhibition under IPTG induction. RR enzyme activity, as measured by 14CDP reduction assay, revealed a 30% decrease in the IPTG-induced KB-M2AS clone relative to non-IPTG-induced samples at 144 hours. As shown by Northern blot, expression of the M2 antisense mRNA showed peaks at 48 hours and 144 hours after induction by IPTG. M2 antisense mRNA expression induced by IPTG was 33-fold greater than the uninduced control at 144 hours. Western blot analysis showed that the M2 subunit protein level decreased in the KB-M2AS clone beginning at 72 hours after induction and continued to decrease to 50% of the uninduced control at 144 hours, then showed a slight recovery at 168 hours. In conclusion, M2 antisense mRNA expression by an inducible system can effectively decrease RR M2 protein expression, reduce enzyme activity, and inhibit growth. Furthermore, this approach can be employed in future antisense investigations.
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PMID:Inhibition of human cancer cell growth by inducible expression of human ribonucleotide reductase antisense cDNA. 1080 62