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Title: The effect of cytotoxic drugs on the DNA metabolism and proliferation of lymphoblasts
Author: Fricker, Simon P.
ISNI:       0000 0001 3484 4875
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1981
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(A) The EBV transformed lymphoblastoid, Namalwa, cell line was used to investigate the effects of three cytotoxic agents on eukaryotic DNA replication. Cytosine arabinoside, an analogue of deoxycytidine, at a concentration of 5 x 10−9 M was shown to inhibit DNA synthesis by 50% in the Namalwa cell line as measured by the incorporation of 3H-thymidine into acid precipitable material. . A comparative study of the metabolism of cytosine arabinoside in transformed B and T lymphoblastoid cell lines and the protozoan, T. pyrifomis was also undertaken. AraC has to be phosphorylated to its active form, araCTP, before it can exert a cytotoxic effect. Alternatively it can be inactivated by deamination to araU. The metabolites of araC were identified in each test system by using thin layer chromatography. The levels of araC kinase and araC deaminase were assayed. Both types of lymphoblastoid cells possessed high levels of araC kinase and no araC deaminase. T. pyriformis, on the other hand, had a low level of araC kinase and a high level of araC deaminase. The latter enzyme was inhibited 100% by 10−4 M tetrahydro-uridine. The effect of cytosine arabinoside on DNA replication was investigated using sedimentation analysis of DNA on alkaline sucrose gradients. The results indicate that araC inhibits DNA chain elongation. A comparative study of the effect of aphidicolin on DNA synthesis showed that 10−7 M aphidicolin inhibited DNA synthesis in Namalwa cells by 50%. The inhibitory effect of aphidicolin on DNA chain elongation was demonstrated using DNA sedimentation analysis on alkaline sucrose gradients. The differing effects of cyclophosphamide, phosphoramide mustard and hydroperoxycyclophosphamide on DNA synthesis were demonstrated. DNA synthesis in Namalwa cells was inhibited 20% by 5 x 10−3 M cyclophosphamide, 65% by 5 x 10−3 M phosphoramide mustard and 90% by 5 x 10−3 M hydroperoxycyclophosphamide. Analysis of DNA on alkaline sucrose gradients indicated that the alkylating agents acted by cross-linking newly synthesised DNA giving unusual DNA intermediates but this hypothesis needs to be confirmed by further experiments. (B) The immunosuppressive effects of the glucocorticoids, clobetasol propionate, clobetasone butyrate and 6-α-methyl prednisolone, on the delayed-type hypersensitivity response to the contact sensitising agent oxazolone was demonstrated in CBA mice. The inhibitory effects of cytosine arabinoside, aphidicolin and 6-α-methyl prednisolone on DNA synthesis in PHA stimulated human peripheral lymphocytes were demonstrated, 60% inhibition was obtained with 10−6 M 6-α-methyl prednisolone, 10−7 M aphidicolin and 10−8 M cytosine arabinoside. Rat peripheral lymphocytes were stimulated to undergo blastogenesis with both PHA and an oxazolone-rat serum albumin conjugate. The DNA synthetic activity induced in both cases was inhibited by 6-α-methyl prednisolone. 34% inhibition was obtained with 10−4 M 6-α-methyl prednisolone in both the PHA and conjugate stimulated rat lymphocytes.
Supervisor: Not available Sponsor: Science Research Council ; Upjohn Limited
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry