Thymidine Aminopterin

The cell fusion mixture is transferred to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Unflised myeloma cells are unable to grow as they lack HGPRT. Unflised normal spleen cells can grow but their proliferahon is limited and they eventually die out. The hybridoma cell can proliferate in the HAT medium as the normal spleen cell supplies the enzyme which enables the hybridoma to utilize extracellular hypoxanthine. 

Inhibition of nucleobase synthesis (2). Tetrahydrofolic acid (THF) is required for the synthesis of both purine bases and thymidine. Formation of THF from folic acid involves dihydrofolate reductase (p. 272). The folate analogues aminopterin and methotrexate (ame-thopterin) inhibit enzyme activity as false substrates. As cellular stores of THF are depleted, synthesis of DNA and RNA building blocks ceases. The effect of these antimetabolites can be reversed Ltillmann, Color Atlas of Pharmacology 2000 Thieme All rights reserved. Usage subject to terms and conditions of iicense. 

Successful fusion (2) is a rare event, but the frequency can be improved by adding polyethylene glycol (PEG). To obtain only successfully fused cells, incubation is required for an extended period in a primary culture with HAT medium (3), which contains hypoxan-thine, aminopterin, and thymidine. Amino-pterin, an analogue of dihydrofolic acid, competitively inhibits dihydrofolate reductase and thus inhibits the synthesis of dTMP (see p. 402). As dTMP is essential for DNA synthesis, myeloma cells cannot survive in the presence of aminopterin. Although spleen cells are able to circumvent the inhibitory effect of aminopterin by using hypoxanthine and thymidine, they have a limited lifespan and die. Only hybridomas survive culture in HAT medium, because they possess both the immortality of the myeloma cells and the spleen cells metabolic side pathway. 

Fusion with the cells compensates for this deficiency. When fused and unfused cells are incubated in the presence of the folic acid antagonist aminopterin, the de novo synthesis of purines and pyrimidines for DNA is blocked. Cells deficient in HGPRT die, whereas hybrid cells are able to bypass aminopterin blockage by metabolism of hypoxanthine and thymidine added to the medium. In the generation of mouse hybridomas, an number of myelomas deficient in HGPRT are available, all originating from MOPC 21, a spontaneous myeloma from the BALB/c mouse strain. 

Select HPGRT+ cells by culturmg the mixture in a medium containing HAT (hypoxanthine, aminopterin, and thymidine), which is growth inhibitory to HPGRT+ cells. Therefore, the myeloma cells will die in this medium while the hybridoma cells proliferate. The unfused lymphocytes will die due to their limited lifespan. 

Conversely, HPRT4 cells can be selected for in the presence of hypoxanthine-aminopterin-thymidine medium. It should be possible to select for HPRT cells in normal HPRT+ persons with one of the analogues, and this was one of the first systems used to detect spontaneously occurring mutants in freshly cultured cells from humans.88 388... 

HGPRT and TK enzymes are important in the synthesis of DNA from preformed nucleotides provided in the culture medium. The myeloma cells lacking these enzymes are unable to utilize the hypoxanthine or thymidine from the medium moreover, they die in the presence of aminopterin, which inhibits the de novo synthesis of DNA. Thus, in a medium containing aminopterin, hypoxanthine, and thymidine (HAT medium) only those hybridomas that receive the HGPRT or TK enzyme from the normal parents (spleen B lymphocytes) will survive. 

In practice synchronisation at the Gl/S boundary may be achieved by a number of reagents as well as thymidine, e.g. aminopterin, amethopterin, 5-fluorodeoxyuridine or hydroxyurea. 

Aminopterin and amethopterin are 4-amino analogues of folic acid (Fig. 11.5) and as such are potent inhibitors of the enzyme dihydrofolate reductase (EC 1.5.1.3) (Blakley, 1969). This enzyme catalyses the reduction of folic acid and dihydrofolic acid to tetrahy-drofolic acid which is the level of reduction of the active coenzyme involved in many different aspects of single carbon transfer. As is clear from Fig. 11.6, tetrahydrofolate is involved in the metabolism of (a) the amino acids glycine and methionine (b) the carbon atoms at positions 2 and 8 of the purine ring (c) the methyl group of thymidine and (d) indirectly in the synthesis of choline and histidine. 

Variations and other combinations of these methods are obviously possible, e.g. mitotic selection and thymidine block aminopterin block reversed with low thymidine followed by a high thymidine block reversed with deoxycytidine. 

By growing cells in the presence of increasing concentrations of aminopterin a number of resistant cells lines have been isolated (Hakala and Ishihara, 1962 Littlefield, 1969). These have been characterised as having either an altered permeability to the drug or an altered folate reductase or an increased rate of synthesis and hence increased amounts of the enzyme (Alt et al., 1976), resulting, at least in part, from a selective amplification of the dihydrofolate reductase gene (Alt et al., 1978 Schimke et al., 1988). The problem is considered in more detail in 11.8.1. The importance of the antifolates lies in their role in the HAT selection technique ( 13.5) devised by Szybalski (1962) (see also Szybalski et al., 1962 and Littlefield, 1964) for the isolation of hybrids between mutant cells defective on the one hand in thymidine kinase and on the other hand... 

Dispense lOOju.1 aliquots into the wells of a 24 well TC tray already containing 0.4 ml medium supplemented with HAT (hy-poxanthine, 100/aM aminopterin, 0.4/iM thymidine, 16jtiM see 13.2)... 

Fig. 13.3. Metabolic cooperation between BHK21/C3 cells. Growth of BHK-HPRT-and BHK-TK- cells separately and in mixed (1 1) culture, in medium containing hypoxanthine, aminopterin and thymidine (HAT medium). HPRT = hypoxanthine phosphoribosyl transferase TK = thymidine kinase. (Reproduced from Pitts, 1971, with kind permission of the author and publisher.)...

Hypoxanthine, Aminopterin, and Thymidine supplement (100X HAT, Sigma). 

Fig. 4. Multistep methods for eliciting specific antibody-producing cells (hybridomas). HAT, hypoxanthine, aminopterin, and thymidine PEG, polyethylene glycol.

HAT (HT) medium Medium containing hypoxanthine, aminopte-rin, and thymidine (HT without aminopterin)... 

---