| dc.description.abstract | Among the various anticancer drugs available for clinical use, 5?fluorouracil (FUra) occupies a distinct position. It is used in the treatment of several cancers either singly or in combination with other anticancer agents. On one hand, it is converted to FdUMP, which causes an irreversible inhibition of the enzyme thymidylate synthetase, leading to inhibition of DNA synthesis. On the other hand, it readily gets incorporated into various classes of RNA, thereby affecting several vital cellular functions and causing cytotoxicity.
Fluorouracil is known to get incorporated into primer RNA, heterogeneous nuclear RNA, small nuclear RNA, ribosomal RNA, messenger RNA, and transfer RNA. The fluorouracil substitution into bacterial tRNA causes different degrees of inhibition of aminoacylation and changes in the base composition of uridine-derived bases. In higher eukaryotes like mice, FUra is known to inhibit uracil-modifying enzymes. Little is known about the altered functions of fluorouracil-substituted transfer RNA in eukaryotes, especially mammals. Hence, we chose to study the effect of FUra substitution on the altered functions of tRNA, taking rat liver as the model system.
In the present work we aimed at characterizing the FUra-substituted tRNA (FUra?tRNA or tRNApura) from rat liver. These studies included aminoacylation kinetics, requirement for Mg²?, ATP and K?, and thermal denaturation and renaturation studies in order to find out the properties of the FUra-substituted total tRNA and to study the altered function of a single species of tRNA during aminoacylation, taking initiator transfer RNA as a representative.
Total normal tRNA was isolated from rat liver. Four different protocols were devised for preparing tRNA in bulk quantities, taking the positive points from the existing methods of total tRNA isolation. Of the protocols devised, the DEAE?Sephacel chromatography was found to be good in obtaining total tRNA quantitatively and qualitatively of a better nature over the other three procedures, viz., modified Zubay’s method, three-step method, and polysomal elimination method.
Rat liver aminoacyl?tRNA synthetases (AARS) were isolated using 2–8% polyethylene glycol precipitation and DEAE?Sephacel chromatography, and were used in the aminoacylation of normal and FUra-substituted tRNAs. Total AARS were isolated from E. coli to identify the activity of initiator tRNA from rat liver.
The analogue 5?fluorouracil is acidic in nature compared to uracil and it tends to confer correspondingly more net negative charge to the tRNA molecule upon its incorporation than unsubstituted tRNA. When total tRNA from rat liver, isolated after injecting the rats with FUra (250 mg/kg body weight), was applied onto DEAE?Sephacel column and eluted with an upward concave buffer gradient of 0.325 M–0.6 M NaCl in 20 mM Tris?HCl (pH 8.9), the tRNApura eluted into a broad peak which was eluting at a slightly higher NaCl concentration than normal total tRNA. [³H]FUra?tRNA loaded on the same column also eluted in the latter fractions corresponding to cold FUra?tRNA, while normal total tRNA chromatographed on the same column eluted in slightly earlier fractions than FUra?tRNA. Thus the substituted tRNA could be separated from the unsubstituted normal total tRNA due to the shifting of FUra?tRNA more towards higher NaCl concentration on DEAE?Sephacel column chromatography.
FUra?tRNA showed inhibition of aminoacylation with [¹?C]leucine as well as [¹?C]methionine over total normal tRNA. There was slight difference in the requirement of Mg²?, KCl and ATP for normal and FUra?tRNA.
To study the effect of the dose of FUra on aminoacylation, total rat liver tRNA was isolated after injecting four groups of rats (four per group) with 0, 50, 250, 500 mg/kg body weight of FUra intraperitoneally. There was a decrease in the total yield of FUra?tRNA with increasing dosage of FUra, suggestive of metabolic toxicity to rats.
The aminoacylation of total tRNA was monitored with [¹?C]chlorella protein hydrolysate at varying concentrations of input tRNA. Unsubstituted tRNA served as the control. The FUra?tRNA isolated with the doses of 50, 250 and 500 mg/kg body weight of FUra were designated as tRNApura50, tRNApura250 and tRNApura500 respectively. The tRNApura50 showed increased amino acid acceptance with total amino acids, while tRNApura250 and tRNApura500 showed inhibition of amino acid acceptance. It was observed that injection of therapeutic dose (50 mg/kg body weight) of FUra resulted in an increased acceptance of several individual amino acids like methionine, lysine, aspartic acid, tryptophan, serine, and arginine. There was a decrease in the aminoacylation with increasing FUra dose for several of these individual amino acids indicating a common mechanism of action.
The presence of fluorouracil in tRNA brings about certain changes in the properties of tRNA. Firstly, it imparts more negative charge to tRNA. Heidelberger (1965) suggested that FUra could behave like cytosine in tRNA. So, instead of base pairing with adenine it could base pair with guanine with three hydrogen bonds. Since RNA molecules exhibit considerable double-strandedness forming stems and loops, the incorporation of fluorouracil in the place of uracil is expected to drastically alter the secondary structure of tRNA. Therefore, the number of paired bases in RNA and hence the number of hydrogen bonds would increase, which might affect the Tm of the tRNA.
Here, we examined the melting profiles of normal and FUra-substituted total tRNA in perchlorate?cacodylate?phosphate?EDTA buffer and calculated the Tm values. Normal total tRNA from rat liver showed a Tm of 63°C, whereas tRNA isolated after injecting 50, 250 and 500 mg/kg body weight FUra showed a Tm of 69°C, 71°C and 71°C respectively. The increase in the Tm implies a more rigid structure of the tRNA molecule due to more hydrogen bonds formed after FUra substitution.
In another experiment the effect of anti?tRNA antibodies was checked on the amino acid acceptance of total normal and FUra?tRNA. The total tRNA was preincubated with anti?tRNA antibodies followed by addition of total rat liver aminoacyl?tRNA synthetases. There was about 41% inhibition in the aminoacylation of tRNApura50. For tRNApura250, inhibition of aminoacylation was only 7%. The anti?tRNA antibodies failed to inhibit aminoacylation of tRNApura500. It could be possible that, with the incorporation of more FUra into tRNA, the secondary structure of tRNA might have changed leading to an altered conformation. Since the anti?tRNA antibodies recognize the conformation of tRNA also, an altered conformation of tRNA might be a reason for the failure of the antibodies to recognize the tRNA with increased substitution of FUra.
The purification of initiator tRNA from normal and FUra?treated rats was standardized by fractionating total tRNA under acidic and alkaline pH conditions on Mono Q column of FPLC, followed by fractionation on highly cross?linked polyacrylamide?7 M urea gels. FUra?substituted initiator tRNA isolated under alkaline pH conditions showed inhibition in [³?S]methionine acceptance in a dose?dependent manner over normal initiator tRNA by E. coli total AARS.
Thus, in the present study it was observed that the substitution of 5?fluorouracil into tRNA is bringing about a functional alteration in the acceptance of total as well as several of the individual amino acids. It was observed that tRNA isolated from rats injected with increasing doses of FUra showed increase in Tm indicating a probable alteration in the secondary structure of the transfer RNA. This is supported by failure of anti?tRNA antibodies to recognize the total tRNA isolated with higher doses of FUra. FUra?substituted initiator tRNA isolated from rat liver after various doses of FUra injection could not accept [³?S]methionine in a dose?dependent manner in the aminoacylation studies with E. coli total AARS. | |
