Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line.

Abstract

We studied the content and metabolism of thymidylate synthase mRNA in cultured mouse fibroblasts that were undergoing a serum-induced transition from the resting to growing state. The studies were performed with a 5-fluorodeoxyuridine-resistant 3T6 cell line (LU3-7) that overproduces the enzyme and its mRNA about 50-fold and that regulates the expression of the thymidylate synthase gene in the same manner as the parental cell line. We have previously shown that the rate of synthesis of thymidylate synthase increases at least ninefold when the serum-stimulated cells traverse the S phase. Here we show, by Northern blot analysis, that thymidylate synthase mRNA increased 20-to 40-fold as cells progressed from resting to late S phase. About 85% of poly(A)+ thymidylate synthase mRNA was associated with polysomes at all times. The increase in thymidylate synthase poly(A)+ mRNA content was the result of an eightfold increase in the rate of production of this species, as shown by pulse-labeling studies. Pulse-chase analysis revealed that the half-life of thymidylate synthase poly(A)+ mRNA was similar in resting (9 h) and growing (7 h) cells. The rate of transcription of the thymidylate synthase gene, as determined in isolated nuclei, increased only by a factor of three to four during the S phase. Since the content of the message increased to a much greater extent than the rate of transcription of the gene, posttranscriptional controls must also play a role in regulating the content of thymidylate synthase mRNA under these conditions. Our results suggest that the cell may regulate the distribution of thymidylate synthase mRNA between a relatively stable poly(A)+ RNA species and a labile poly(A)f RNA species.Thymidylate synthase (TS; EC 2.1.1.45) is the enzyme that catalyzes the reductive methylation of dUMP during the de novo synthesis of TMP. Because TS is an essential enzyme in proliferating cells, it is an important target enzyme in cancer chemotherapy (6). Many laboratories have shown that in mammalian as well as in yeast cells, the level and enzymatic activity of TS are high when cells are engaged in DNA replication but very low at other times in the cell cycle or in nondividing cells (5,13,17,22,27). We have shown that when resting (GO) mouse 3T6 fibroblasts are stimulated with serum to reenter the cell cycle, the amount of TS increases about 10-fold between 10 and 25 h following serum addition (21). Although the increase in TS activity is closely coordinated in time with DNA replication, it is not blocked by the presence of inhibitors of DNA synthesis. This shows that these two events are not directly coupled.To facilitate a more direct analysis of TS gene expression, we isolated a 5-fluorodeoxyufidine (FdUrd)-resistant 3T6 cell line (LU3-7) that overproduces TS by a factor of about 50 (23). The overproduced TS appears to be the same as the normal enzyme, as determined by several different criteria. Recently, we cloned DNA corresponding to nearly the entire length of TS mRNA into pBR322 (7). Using this plasmid as a...

miR-192/miR-215 Influence 5-Fluorouracil Resistance through Cell Cycle-Mediated Mechanisms Complementary to Its Post-transcriptional Thymidilate Synthase Regulation

“…One of the most studied mechanisms of TYMS expression variability are functional polymorphisms in the TYMS gene (15). Moreover, it is now well established that regulatory events at the level of gene amplification (16,17), transcription (18)(19)(20), posttranscription, translation, and posttranslation (21) are also involved in TYMS expression control (22). One example of TYMS posttranscriptional regulation is the rapid increase in TYMS enzyme activity described in various in vitro (23)(24)(25), in vivo, and clinical model systems following short-term exposure to fluoropyrimidines (26,27).…”

Characterization of Nuclear Factors that Bind to the Human Thymidylate Synthase Gene in HL-60 Cells Differentiated by All-trans Retinoic Acid Treatnent.

“…15) Furthermore, we identified nuclear factors (NF-TS1, NF-TS2, NF-TS3) that bind to the 5Ј-region of the human TS gene, and found that the amounts of NF-TS2 and NF-TS3 changed markedly during the differentiation of HL-60 cells into macrophage-like cells induced by VD 3 . 13,15,16) Although the functions of NF-TS2 and NF-TS3 remain to be elucidated, they were suggested to function oppositely for the regulated expression of TS gene in the differentiation of HL-60 cells induced with VD 3 ; namely, NF-TS2 enhanced the expression of the TS gene and NF-TS3 suppressed it. 13) To investigate the functions of these nuclear factors, it is necessary to examine the changes in NF-TS2 and NF-TS3 in other systems where the expression of TS changes definitely.…”

“…The gene for TS is a typical housekeeping gene and its expression is regulated depending on both the cell cycle [1][2][3] and the state of cell proliferation.4-7) The human TS gene and its cDNA were isolated [8][9][10][11] and their primary and functional structures have been extensively elucidated. …”

“…The gene for TS is a typical housekeeping gene and its expression is regulated depending on both the cell cycle [1][2][3] and the state of cell proliferation.…”

“…Human promyelocytic leukemia HL-60 cells are known to be able to differentiate into granulocyte-like and macrophagelike cells by treatment with differentiation inducers such as retinoic acid and 1,25-dihydroxyvitamin D 3 (VD 3 ). During the differentiation of the HL-60 cells, the expression of some specific genes is induced, while that of some other genes is suppressed.…”