Published in: "Proceedings of the Fourth Annual Leukocyte Culture Conference, 1969", (McIntyre RO, ed.), pp. 361-366, New York: Appleton-Century-Crofts, (1971)

Albert A. Keshgegian, Lorraine F. Meisner, and John H. Frenster,

Department of Medicine, Stanford University School of Medicine, Palo Alto, CA 94305

It has been shown that ribothymidine (5-methyl uridine) is a profound inhibitor of mitosis in proliferating lymphocytes (1). Concentrations of 10^-4M ribothymidine produce a 50% inhibition of lymphocyte mitosis, and recovery experiments suggest that such inhibition occurs at the beginning of the S phase of DNA synthesis (1).

Although thymidine has a similar effect on proliferating cells (2, 3), ribothymidine differs from thymidine in that it cannot substitute for thymidine in thymidine-requiring bacterial mutants (4), nor can it substitute for thymidine in reversing the inhibitory effects of amethopterin (5). In addition, in cell-free systems, ribothymidine triphosphate is incorporated into RNA in place of uridine triphosphate by either DNA-dependent RNA polymerase (6) or RNA-dependent RNA polymerase (7), whereas thymidine triphosphate is not utilized (8).

In support of these distinctions between ribothymidine and thymidine, we here report the ability of low concentrations of thymidine to reverse the inhibitory effects of ribothymidine in the continued presence of high concentrations of ribothymidine.

METHODS

Human lymphocytes were isolated from heparinized normal human blood by the techniques of Moorhead et al (9), and were cultured as replicates under sterile conditions in 5 ml of Medium 199 containing 20 percent autologous plasma, to which was added 300 ugm each of penicillin and streptomycin, and 0.1 ml of Phytohemagglutinin M (Difco). Replicate cultures were incubated at 37^oC in tightly-sealed 15 ml screw-cap plastic flasks. After 40 hours of such culture, blastic transformation is essentially complete, widespread DNA synthesis is underway, and the first wave of cell mitosis is apparent (10). Single nucleosides to be tested were added to the cultures between 40-72 hours of culture, the cultures were allowed to continue an additional 6-24 hours, and 1.0 ugm of colcemide was added to each flask 3-21 hours before harvesting. Cells were harvested at 100 G for 5 minutes, subjected to hypotonic lysis (9), fixed in 3:1 methanol acetic acid, spread on clean slides, and analyzed for metaphase figures and individual karyotypes. For each replicate culture flask, the number of metaphase figures observed among 5,000 cells was determined.

RESULTS

When certain of the pyrimidine nucleosides are directly compared at concentrations of 5 x 10^-3M (Table 1),

Replicate cultures of 10⁶ lymphocytes per flask were incubated for 48 hours after addition of phytohemagglutinin. Nucleosides were then added to 5 X 10^-3M concentration, the cultures were continued, colcemide was added at 69 hours of culture, and the cells were harvested at 72 hours of total culture as described in METHODS.

A further contrast between ribothymidine and thymidine is seen (Table 2),

Table 2. Reversal by Added Nucleoside of the Inhibitory Effect of Ribothymidine.

Replicate cultures as in Table 1. At 42 hours of culture, ribothymidine was added to each flask to 10^-3M concentration, at 50 hours of culture the test nucleoside was added to 0.05 X 10^-3M concentration, at 51 hours colcemide was added, and at 72 hours the cells were harvested as described in METHODS.

Since thymidine reverses most efficiently the inhibition of lymphocyte mitosis produced by ribothymidine (Table 2), it was of interest to follow the time course of the reversal process following the addition of
0.05 X 10^-3M thymidine to cultures which had been inhibited for 24 hours by 10^-3M ribothymidine (Fig. 1).

Fig. 1. Thymidine reversal of inhibition of mitosis after 24 hours of inhibition by ribothymidine. Replicate cultures as in Table 1. At 48 hours of culture, ribothymidine was added to 10^-3M concentration in each flask. At 72 hours of culture, thymidine was added to 0.05 X 10^-3M concentration in each flask. Colcemide was added 3 hours before each flask was harvested.

DISCUSSION:

In cell-free systems, ribothymidine triphosphate is incorporated into new RNA in place of uridine triphosphate by both DNA-dependent RNA polymerase (6) and by RNA-dependent RNA polymerase (7). Ribothymidine is formed by certain thymine-requiring mutants of E. coli when they are supplied with thymine or thymidine (12), but ribothymidine cannot substitute for thymidine in such mutants (4). Cell-free enzyme extracts from these mutants are able to convert thymine to ribothymidine (13).

The inhibitory effects of ribothymidine are partially reversed by the addition of each of the pyrimidine nucleosides (Table 2). This suggests that the mechanism of inhibition by ribothymidine cannot be via feedback inhibition from deoxythymidine triphosphate or other nucleoside triphosphates (Fig. 2).
Fig. 2. Biosynthesis of thymine nucleotides (see general review in reference 11). Each nucleoside can penetrate living cells and can enter the biosynthetic pathway by conversion to its respective monophosphate. Feedback pathways (------) from triphosphates result in inhibition ( X ) of specific enzymatic reactions. Ribothymidine triphosphate may compete directly with deoxythymidine triphosphate on the DNA template and, and inhibit DNA polymerase (17).

Ribothymidine inhibition of cell mitosis occurs at the beginning of the S phase of DNA synthesis, a time when the cell is beginning the transition from RNA synthesis to DNA synthesis at individual gene loci (16). Enzyme studies indicate that this is a vulnerable period in DNA template function, in that mammalian DNA polymerase appears to be especially sensitive to competitive inhibition by triphosphates of nucleosides other than deoxyribonucleosides (17). Such vulnerability suggests that ribothymidine triphosphate may compete directly with thymidine triphosphate on the DNA template for utilization by DNA polymerase (Fig. 2). If this mechanism is confirmed, it would require that all monoribonucleotides be cleared from the DNA template at the beginning of the S phase before DNA synthesis could begin. These mechanisms are currently under study in cell-free systems.

SUMMARY

Millimolar concentrations of ribothymidine result in marked inhibition of mitosis in proliferating lymphocytes, and can be overcome by lower concentrations of thymidine, in the continued presence of inhibitory concentrations of ribothymidine.

ACKNOWLEDGMENTS:

The technical assisstance of Sharon Landrum-Richardson and John Thompson was greatly appreciated.

SUPPORT

Supported in part by a NIH Public Health Service research grant CA-10174 from the National Cancer Institute. Dr. Frenster is a Research Scholar of the Leukemia Society.

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