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Title: Effects of 5-azacytidine on growth control mechanisms in PHA stimulated equine lymphocytes
Author: Zain, B. Sayeeda
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1971
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PHA stimulated lymphocytes offer a convenient system for the study of growth inhibition, cellular proliferation and cell differentiation. Because of the scarcity of human blood for experimental purposes, horse blood was utilised as a substitute. A standard method of culture and growth of equine lymphocytes invitro has been developed. In addition, studies connected with the isolation, purification and preservation of equine lymphocytes have been made. To understand the basic metabolic pattern of lymphocytes biochemical studies were carried out. It has been shown that on stimulation with PHA, horse lymphocytes, in many ways behave like human lymphocytes both morphologically and metabolically. On incubation with PHA, changes in the nuclear chromatin take place, and increase in the rate of proteins, RNA synthesis ensues. The quiscent cells start synthesizing DNA after 28-30h of incubation with PHA. The activities of the enzymes involved in the synthesis of RNA and DNA is increased. The uptake and phosphorylation of dThd and Urd is increased, which in turn reflects an increased thymidine kinase and uridine kinase activity. The DNA polymerase activity is increased by 50-150 fold. The DNA polymerase activity in the high speed supernatant fraction prefers the denatured DNA primer, while the nuclear enzyme has no specific requirements, The supernatant enzyme is less stable compared to the nuclear enzyme. Uridine has a stimulatory effect on RNA synthesis in PHA stimulated equine lymphocytes. An increased rate of RNA synthesis starts within 4h of PHA addition, and unlike in unstimulated cells rRNA accumulates. In an attempt to elucidate the mechanism of PHA stimulation cyclic AMP was used. Our results reveal the possibility of PHA acting as a hormone, utilizing cyclic AMP as the secondary messenger. In order to understand the growth control mechanisms in these cells 5-azaCytidine was used. It has been shown that 5-azaCyd inhibits the growth of these cells by virtue of getting incorporated into RNA and inhibiting the protein synthesis. If 5-azaCyd is added to the cultures for a short time and then removed, the growth does not resume after drug removal upto at least 50h. Addition of Cyd or Urd (at a concentration 10-100 fold higher than 5-azaCyd) simultaneously with or before 5-azaCyd treatment reverses the inhibitory affect of the drug; on the other hand addition of Cyd or Urd, 80 minutes after 5-azaCyd addition is of no use in reversing the growth inhibition. We have shown that the primary site of 5-azaCyd action is not the DNA metabolism, as DNA synthesis is inhibited slowly after a lag of an hour and is complete in 6-8 hours, The uptake and phosphorylation of radioactive dThd is not affected, for 2 hours, measured by looking at the intracellular pool of radioactive dThd and its phosphorylation products. To avoid the possibility of dThd kinase involvement, 32p[i] orthophosphate label was used but the same results were obtained, 5-azaCyd does not inhibit the enzyme DNA polymerase invitro but it does inhibit the induction of the enzyme invivo. Thus basing on this data, we believed that 5-azaCyd does not primarily affect DNA synthesis, but the DNA synthesis is inhibited because of inhibition of protein synthesis. Studies connected with RNA metabolism revealed that 2h treatment with 5-azaCyd inhibits the total RNA synthesis in equine lymphocytes. We have shown that the uptake and phosphorylation of Urd is not affected for 2-h, The enzyme RNA polymerase is not inhibited invitro, but total RNA synthesis is inhibited within 3-4h, Treatment of the cells with the drug simultaneously with PHA for 2 hours at the beginning of the culture, washing off the drug and reincubation of the cells with fresh medium containing PHA inhibits the expected induction of rRNA synthesis, Fractionation of nuclear .and cytoplasmic RNA from cells treated with the drug for 1, 2 and 4h on sucrose gradients reveal that neither the rRNA precursors, nor the rRNA itself is inhibited during the first 2h of treatment. By 4h all the species of RNA are inhibited. The cytoplasmic RNA sedimenting in the 4s region shows inhibition during the first two hourse of treatment, but there is no inhibition at 4th h. We have shown that 5-azaCyd also inhibits the protein synthesis in unstimulated lymphocytes. The mechanism of 5-azaCyd action on the inhibition of protein synthesis, after getting incorporated into RNA is discussed. The possibility of the involvement of tRNA rather than the mRNA is discussed in detail.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available