On the mechanism of 5-bromodeoxyuridine induction of prolactin synthesis in rat pituitary tumor cells.

Abstract

GH12C1, a clonal strain of rat pituitary tumor cells in culture (GH cells), does not produce detectable amounts of prolactin. 5-Bromodeoxyuridine (BrdUrd), the thymidine analogue, at sublethal concentrations (3-5 /xg/ml) induces prolactin synthesis in these cells. BrdUrd also induces prolactin synthesis in F1BGH12C1 cells, a BrdUrd resistant (BrdUrd r) substrain isolated from GH12C1 cells. The F1BGH~2C1 strain is not drug dependent, but its resistance to BrdUrd is a stable phenotype. The significant features of the induction of prolactin synthesis in the BrdUrd r strain are the increased net synthesis of prolactin and the shortening of the lag period of prolactin induction. As BrdUrd concentration in the growth medium is increased, the rise in prolactin synthesis parallels the increased incorporation of BrdUrd into DNA. Prolactin synthesis is first detected when BrdUrd replaces 20-25% of the thymidine in DNA. BrdUrd can replace up to 75-80% of the thymidine within 2 d of treatment. Partial starvation of these cells under specified growth conditions does not affect the general growth pattern of the cells, general protein synthesis, and thymidine uptake, but does affect DNA synthesis. When cells are cultured under conditions in which DNA synthesis is preferentially inhibited, BrdUrd does not induce prolactin synthesis, suggestive of a DNA-mediated mechanism of action for the drug.KEY WORDS 5-bromodeoxyuridine prolactin gene expression 9 rat pituitary tumor ceilsThe drug 5-bromodeoxyuridine (BrdUrd), a thymidine analogue, seems to have diverse effects on the various processes of differentiation in eukaryotic cells. These may be classified into two distinct types. BrdUrd suppresses the synthesis of specific proteins in certain cells, and in other cell types the drug induces the production of certain cell-specific proteins. Rutter et al. (12) described in detail the role of BrdUrd on different eukaryotic cell systems. On the basis of the different effects of this drug, these authors discussed the possible DNA-linked and non-DNA-linked mechanisms of action of BrdUrd.Holtzer and Abbott (7) postulated that BrdUrd selectively inhibits the synthesis of luxury molecules without grossly depressing the synthesis of essential molecules of the cells. Priesler et al. (11) claimed that inhibition of differentiated function J. CELL BIOLOGY 9 The Rockefeller University Press 9

Increased level of prolactin gene sequences in bromodeoxyuridine treated GH cells

“…Number indicated by the arrow designates the size of the resolved DNA fragment as extrapolated from the sizes of the Hind III digested Lambda DNA, resolved under similar conditions. centration and duration of the drug treatment (18). Induction of PRL synthesis could also be correlated with the increased synthesis of nuclear and cytoplasmic mRNApRL sequences from undetectable to the detectable amounts (19).…”

“…Whereas the PRL-(prolactin non-producint) GH12C1 and its BrdUrdr derivative,F1BGH12C1 synthesize PRL only in the presence of the drug (17). We have reported previously that BrdUrd incorporates into cell DNA replacing thymidine,and that induction of PRL synthesis by the drug seems to be dependent on DNA synthesis and such base substitutions (18). Induction of PRL synthesis in these cells could be correlated to the increased level of nuclear and cytoplasmic RNApRL sequences (19).…”

Extent of BrdUrd-Induced Prolactin Gene Amplification in GH Cells

“…Identification of these amplification sites will be of significant interest for understanding the mechanism of gene amplification in eukaryotic cells. Since 82°7o of the thymidine in total cell DNA is substituted by BrdUrd after treatment for 48-72 hr at 300 pg/ml (Biswas et al, 1979), it may be postulated that extensive base substitution might have modified the cellular DNA to create such preferential DNA replication sites. Under such circumstances, one might expect a generalized effect on gene expression and on the copy number of other cellular genes.…”

5-Bromo-2’-deoxyuridine induces visible morphological alteration in the DNA puffs of the anterior salivary gland region of Bradysia hygida (Diptera, Sciaridae)

“…Various studies have shown that BrdUrd can induce or inhibit cellular differentiation. For example, BrdUrd was reported to induce alkaline phosphatase (1), prolactin (2), interferon β (3), and integrin (4) production in cultured cells. It induces differentiation of a cultured human small cell lung cancer cell line (5) and activates senescence-associated genes in human cells (6).…”