Differential sensitivity to 5-bromodeoxyuridine during the S phase of synchronized myogenic cells

Synchronized myogenic cell cultures have been used to demonstrate differential sensitivity to BUdR during segments of the S period. Synchronization of the cells was achieved by two methods. First, cells were initiated in medium containing FUdR, an inhibitor of DNA synthesis. Following FUdR blockade reversal with TdR after 19 hr in vitro, the synchronized cells were allowed to replicate their DNA with BUdR for periods corresponding to early and late S. Determinations of percentage labeled cells during synchronization with FUdR indicate that about 90% of the cycling population of cells accumulates at the G1/S interface of the cell-cycle and that the duration of the S period following blockade reversal with TdR is not altered. Since BUdR is pulsed to these cultures immediately after the point of synchronization, a high degree of synchrony is obtained. In the second method of synchrony, cohorts of cells which had been in G2, late S, or early S during a BUdR pulse were collected in metaphase arrest with Colcemid and selectively removed from the cultures. With the mitotic selection method the point of synchronization occurred several hours after the BUdR pulse. In both methods the cells were allowed to resume myogenesis and scored for percentage fused nuclei after approx 50 hr in vitro. With both methods of synchrony, BUdR incorporation into early replicating DNA results in a striking decline in myoblast fusion, whereas incorporation into late replicating DNA is without effect. The results cannot be attributed to a disproportionate uptake of nucleotide during early S. Further fractionation of the 4-hr S phase into 1-hr periods indicates that the BUdR sensitive target is replicated during the second hr of DNA synthesis.     

Regulation of thymidylate synthase gene expression in mouse fibroblasts synchronized by mitotic selection

Previous studies have shown that thymidylate synthase gene expression is regulated over a wide range in response to growth stimulation in cultured mouse fibroblasts. In the present study we show that the gene is also regulated during the cell cycle in continuously growing cells. Our analyses were conducted with a fluorodeoxyuridine-resistant mouse 3T6 cell line that overproduces thymidylate synthase and its mRNA by a factor of 50 due to gene amplification. Cells were synchronized by mitotic selection. RNA blot analyses showed that the amount of thymidylate synthase mRNA increased 5- to 10-fold as cells progressed from G1 through the middle of S phase. S1 nuclease protection assays showed that the pattern of 5' termini of thymidylate synthase mRNA was the same in G1 and S phase. Despite the large increase in thymidylate synthase mRNA content, the level of the enzyme increased only by a factor of 2 as cells progressed from G1 to mid S phase. This apparent discrepancy can be explained by the fact that the enzyme is highly stable.     

Stimulation of the proliferation of normal BHK21 cultured fibroblasts by plant lectins.

Concanavalin A (ConA), and the lectins from Dolichos biflorus and Robinia pseudoacacia, stimulate proliferation in cultures of BHK21 hamster fibroblasts. Both cell number and the proportion of cells incorporating acid-insoluble thymidine are increased. The proportion of labelled cells is increased over threefold (Dolichos and Robinia) and by more than 80% (ConA) at the end of the first day in culture. Optimum concentrations are 10 μg/ml, 1 μg/ml, and 0.1 μg/ml for Dolichos, Robinia, and ConA, respectively. The response of these cells to lectins is biphasic and stimulation is reduced at higher concentrations. The optimum concentrations change as the cultures begin to show density-dependent inhibition of growth and eventually, when saturation density is reached, the cultures do not respond at all. If, however, the lectin is applied while the cultures are still growing they can reach a density 40% greater than that at which saturation normally occurs. Virus-transformed BKH cells, which do not show density-dependent inhibition of growth, show none of these responses. Lectins thus alter, but do not abolish, density-dependent inhibition of growth in fibroblasts.     

Phospholipid methylation in fibroblasts of patients with trisomy 21

Comparative studies of phospholipid methylation in normal and Down syndrome (trisomy 21) fibroblasts were performed on a series of 7 pairs. Two patients showed an increased phospholipid methylation pathway as compared to the appropriate controls; one patient showed a decreased pathway; and in four patients there was no significant difference. The phospholipid methylation pathway appears to be a minor pathway, as compared to the choline pathway of phosphatidylcholine synthesis and thus does not allow the detection of an eventual disturbance of monocarbon metabolism in Down syndrome.     

Changes in ornithine decarboxylase activity and cyclic adenosine-3'-5'-monophosphate concentrations during the cell cycle of synchronized BHK cells.

BHK cells were synchronized by excess thymidine treatment, which resulted in approximately 90% synchrony. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, elevated in early S phase, decreased in G2 + M and G1 phase and then increased during late G1 approximately second round of early S phase. The concentration of cyclic adenosine-3'-5'-monophosphate (cAMP) gradually decreased during S approximately G2 + M phase and then increased during late G1 approximately second round of early S phase, preceding that of ODC activity. The data suggest that ODC activity might be regulated by cellular cAMP level.     

The distribution of spontaneous chromosome aberrations in BHK21/C13 fibroblasts.

Previous investigations of spontaneous aberrations in mammalian cells have been carried out on large heterogeneous samples of individuals, each of whom had had a different exposure to exogenous clastogens. In the present analysis using Syrian Hamster cells, a large number of metaphases were analysed from one sample of control cells. In this way all cells were exposed to the same doses of any unknown clastogens. The overall distribution of spontaneous breaks was found to be nonrandom. Breaks involved in different types of aberration had a nonrandom distribution, which was specific for each type. (e.g. terminal deletion and rearrangement).     

The detection of smooth muscle desmin-like protein in BHK21/C13 fibroblasts.

Three distinct proteins, actin (42,000 daltons), the principal form of fibroblast 10 nm filament protein (55,000 daltons), and a protein with a molecular weight of 52,000 and a pI of 5.8 were detected in nonionic detergent-insoluble cytoskeletal and 10 nm filament preparations of control BHK21/C13 and line 9 hamster fibroblasts. Cytoskeletal preparations of other hamster fibroblast cell types, such as NIL8 and primary embryo fibroblasts, contained the 55,000-dalton component but lacked the 52,000-dalton protein. A Rous sarcoma virus transformant of the BHK21/C13 line and an adenovirus transformant of line 9, resembled the NIL8 and other fibroblast types in that they contained only the 55,000- and 42,000-dalton polypeptides. The identity of the 52,000-dalton protein in BHK21/C13 cells was studied. This protein co-electrophoreses on both one- and two-dimensional polyacrylamide gels with the predominant muscle form of 10 nm filament protein. Further, one-dimensional peptide maps of the hamster smooth muscle 10 nm filament protein and the hamster fibroblast 52,000-dalton protein are identical to one another and distinct from the peptide maps of both the 42,000- and the 55,000-dalton components of the fibroblast cytoskeletal preparations. We conclude that BHK21/C13 cells contain both the fibroblast and the muscle form of 10 nm filament protein.     

Subcellular localization of three degeneration-associated phospholipids in cultured hamster fibroblasts (BHK21 cells).

Subcellular localization of bisphosphatidic acid, semilysobisphosphatidic acid and phosphatidyl-(N-acyl)ethanolamine was studied in normal and degenerating fibroblasts (BHK21 cells) by differential centrifugation. In the normal cells these lipids were highly enriched in the floating fraction consisting mainly of neutral lipid-rich lysosomes. They were also enriched in the mitochondrial fraction. In degenerating cells the high enrichment in the floating fraction was retained, but the other peak was displaced to the crude nuclear fraction. Subfractionation of the crude nuclear fraction indicated that these lipids were not enriched in the purified nuclei. Instead, their concentrations were relatively high in the other subfraction evidently enriched in the large secondary lysosomes characteristic for the degenerating cells. Neither in normal nor degenerating cells were these lipids enriched in the light mitochondrial fraction, where most of the smaller, and probably younger, lysosomes were found. On the basis of these results it is suggested that bisphosphatidic acid, semilysobisphosphatidic acid and phosphatidyl-(N-acyl)ethanolamine are lysosomal in origin. It appears possible that they are specifically associated with the organelles representing the later stages in the lysosomal lifespan.     

Isomerization of conjugated linolenic acids during methylation

Conjugated linolenic acids (CLnA) need to be converted into their methyl esters when they are analyzed by gas liquid chromatography (GLC) or high performance liquid chromatography (HPLC). We found that methylation under different conditions could cause substantial isomerization of CLnA. The present study was therefore to optimize the acid-catalyzed or base-catalyzed methylation conditions in order to minimize the artifact derived from isomerization. It demonstrated clearly that isomerization was temperature and time-dependent if methylation was conducted by acid catalysis. For the two acid-catalyzed methylation reagents, BF3/methanol caused greater isomerization than H2SO4/methanol. It was found that using H2SO4/methanol as a reagent at 40 degrees C for 10 min was most appropriate to avoid isomerization when free CLnA was methylated. In contrast, base-catalyzed methylation in NaOMe/methanol at 40 degrees C for 10 min could minimize the isomerization of CLnA in triacylglycerol form.