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TCN (1 microM) totally inhibited the growth of L1210 cells in culture and caused progressive loss of cellular viability, as indicated by a decreased clonogenicity and nigrosin dye exclusion. After 24 h or more of TCN treatment, a significant fraction of the cells had shrunk in size but did not fragment into dye-impermeable vesicles as reported previously for N1S1-67 hepatoma cells (P. G. W. Plagemann, J. Natl. Cancer Inst., 57: 1283-1295, 1976). TCN-induced growth inhibition was accompanied by a block of cell cycle progression in G1 or at the G1-S boundary. At all TCN concentrations studied, progression of cells out from behind this block was evident as a depletion of the early S-phase population in comparison to controls, while increasing the concentration of TCN (0.1 to 1 microM) led to a progressive retention of cells in S phase, suggesting a slowing of progression through S phase. The fraction of S-phase cells incorporating [methyl-3H]thymidine and the amount of [methyl-3H]thymidine incorporated per labeled cell were both decreased by TCN treatment. Increasing the concentration of TCN (0.1 to 1 microM) progressively decreased DNA synthesis and increased cell lethality. Thus it appeared that inhibition of DNA synthesis might cause the retention of cells in S phase which is associated with TCN lethality.

The DNA content of murine fibrosarcoma cell lines of various metastatic potential was the subject of the current investigation. The cell lines were derived from methylcholanthrene-induced tumors as described previously (J. Varani et al., J. Natl. Cancer Inst., 71: 1281-1287, 1983). Cells were maintained in vitro and used for DNA studies no more than 48 h after passage. DNA staining was accomplished using propidium iodide and flow cytometry was used to quantitate relative amounts of DNA. Trout and chicken erythrocytes and mouse thymocytes were used as internal DNA standards for each cell line. DNA indices were calculated as the ratio of the G0-G1 peak channel number of the tumor cells to the G0-G1 peak channel number of the thymocytes. Manual chromosome counts were also obtained from each cell line using Giemsa-stained preparations. All cell lines demonstrated a single aneuploid population. The two tumor lines with the highest metastatic potential were slightly hyperdiploid whereas three low metastatic lines were near tetraploid. A sixth line of moderate metastatic potential was also found to be near tetraploid. Chromosome counts and flow cytometric analyses were in close agreement indicating that DNA content was largely due to chromosome replication. These data suggest that, in this model, metastatic potential and DNA content are inversely related once diploidy is exceeded.

A deficiency of purine nucleoside phosphorylase activity is associated with marked depletion of T-lymphocytes which is felt to be mediated by accumulation and further metabolism of the purine nucleoside phosphorylase substrate, 2'-deoxyguanosine. Human T-lymphoblasts incubated in the presence of 2'-deoxyguanosine and the purine nucleoside phosphorylase inhibitor 8-aminoguanosine accumulate deoxyguanosine 5'-triphosphate whereas B-lymphoblasts and mature T4+-cell lines accumulate GTP under identical conditions. We have compared the effects of guanine ribo- and deoxyribonucleotide accumulation on the metabolism and cell cycle of the respective cell lines. Deoxyguanosine 5'-triphosphate elevations in T-lymphoblasts are associated with inhibition of [3H]uridine incorporation into DNA and a complete block at the G1-S interface of the cell cycle. In contrast 3- to 5-fold increases in guanosine 5'-triphosphate pools in B-lymphoblasts and mature T-cell lines do not inhibit [3H]uridine incorporation into DNA or RNA but do cause a pronounced slowing in the progression of cells through S phase. B-lymphoblasts deficient in the salvage enzyme hypoxanthine guanine phosphoribosyltransferase do not accumulate guanosine 5'-triphosphate from 2'-deoxyguanosine and progress normally through the cell cycle, demonstrating a requirement for guanine salvage to inhibit cell growth. Guanine ribonucleotide accumulation was also associated with inhibition of de novo purine biosynthesis and a moderate decline in adenine nucleotide pools but not with inhibition of protein synthesis or alterations in basal levels of 3':5'-cyclic adenosine monophosphate or 3':5'-cyclic guanosine monophosphate. We conclude that the accumulation of guanine ribonucleotides by actively cycling human lymphoid cells is associated with an increase in S-phase cells and inhibition of growth. This effect is distinctly different from that produced by 2'-deoxyguanosine 5'-triphosphate and should be taken into account in pharmacological studies with 2'-deoxyguanosine and its analogues.