Physiological quiescence in plasma-derived serum: influence of platelet-derived growth factor on cell growth in culture.

A platelet-derived growth factor can be shown to be the principal stimulant of DNA synthesis in whole blood serum for those cells that require serum for maintenance and growth in culture. Cell free plasma-derived serum lacks such platelet-derived material. 3T3 cells and primate arterial smooth muscle cells can be maintained in a quiescent state in culture for as long as six weeks in plasma-derived serum. Such cells can grow logarithmically after exposure to 5% whole blood serum or as little as 100 ng/ml of partially purified platelet factor. The cell cycle of smooth muscle cells has been studied in the quiescent (5% plasma-derived serum) and growing state (5% whole blood serum or 5% plasma-derived serum plus platelet factor). The generation time of smooth muscle cells is 16 to 18 hours as shown by autoradiographic frequency of labelled mitoses. The generation time is the same for cells in the growth fraction in either 5% whole blood serum or 5% plasma-derived serum. Thus, platelet factor acts by recruiting cells into the growth fraction rather than effecting a change in the duration of the cell cycle. Flow microfluorimetry studies on cells growing logarithmically in 5% whole blood serum give the following phase durations: G1 = 5.6 hours; S = 7.6 hours; and G2 + M = 3.8 hours. Based on these studies the argument is presented that cells cultured in 5% plasma-derived serum provide a more physiological base for the study of quiescence than do cells in low concentrations of whole blood serum or confluent, density inhibited cells at high (5% or greater) concentrations of whole blood serum. Furthermore, 5% plasma-derived serum represents an appropriate state to examine the perturbation of quiescent cells.

Coordinate control of 3T3 cell proliferation by platelet-derived growth factor and plasma components.

DNA synthesis and cell division were measured in Swiss mouse 3T3 cells cultured in different concentrations of cell-free plasma-derived serum and increasing amounts of a platelet-derived growth factor. In plasma-derived serum alone, the cells were quiescent and they were arrested in the Go/G1 phase of the cell cycle. Addition of a platelet-derived growth factor to quiescent cells maintained in plasma-derived serum stimulated both DNA synthesis and cell division. When plasma components were present at high concentration (5%, vol/vol), the amount of platelet factor added to the cultures determined the number of cell doublings. Plasma-derived molecules were required for the platelet factor to stimulate DNA synthesis and cell division in the maximal number of cells. In addition, plasma components had to be present for recently divided cells to respond to the platelet factor. When 3T3 cells were cultured in excess platelet factor and limiting amounts of plasma-derived serum (0.5%, vol/vol), the cells underwent one doubling and then ceased to proliferate. Addition of fresh plasma-derived serum to these cells induced a second round of cell division. Plasma components and the platelet-derived growth factor acted in a coordinate fashion to regulate the proliferation of Swiss 3T3 cells.

Role of platelet factors in the growth of cells in culture.

The studies reported suggest that the principal mitogen(s) present in sera responsible for the proliferation of diploid cells in culture is derived from the physiologic response of platelet adherence, aggregation, and release upon their exposure to factors present in serum, such as thrombin, or in tissue, such as collagen. Since it is impossible to make whole blood serum without platelet release, all sera contain platelet mitogenic factor(s). In contrast, serum made from platelet-free plasma lacks mitogenic activity and permits maintenance of cells in culture in a quiescent state for long periods if the cells are routinely fed. The platelet factor(s) appears to be a heat-stable, basic polypeptide or protein, that upon exposure to the cells recruits them into the cell cycle, DNA synthesis, and mitosis. The factor(s) has been shown to act not only in cell culture but in vivo as well. Maintaining cells in a culture medium containing platelet-free, plasma-derived serum may be more analogous to the quiescence of adult cells in vivo, since quiescent cells in adult tissues are normally exposed to interstitial fluid that is probably more like a filtrate of plasma or lymph rather than to whole blood serum. In contrast, growth of cells in a culture medium containing whole blood serum would be more analogous to the pathologic situation that occurs during tissue injury accompanied by hemorrhage.

A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro.

Dialyzed serum from clotted monkey blood ("blood serum") promotes the proliferation of monkey arterial smooth muscle cells in culture, but dialyzed serum prepared from recalcified platelet-poor plasma ("plasma serum") is much less effective. Addition of platelets and calcium to platelet-poor plasma increases the activity of plasma serum to the same level achieved with blood serum. Furthermore, addition to plasma serum of a platelet-free supernatant prepared by exposing purified platelets to thrombin also stimulates the proliferation of smooth muscle cells. Thus, much of the growth-promoting activity of dialyzed serum is directly or indirectly derived from platelets. This finding has important implications for the response of arteries to localized injury and provides a key to further understanding of the role of factors derived from blood serum in promoting cell proliferation in vitro.

Marker effects in the genetic transduction of tryptophan mutants of E. coli.

Recombination frequencies have been determined in crosses involving 28 mutant strains for 20 of which the site of the alteration is known from studies of amino-acid substitutions in the protein products. Three of these mutants showed especially high frequencies of recombination when crossed to other single mutants or when crossed to a strain carrying two alterations at opposite ends of the trpA gene. There is no obvious molecular explanation of the high recombination of these three mutants. They include one missense mutant, one amber and one ochre. The low-frequency recombination mutants include all these same classes as well as frameshift mutants. There is nothing unique about the intragenic location of the high-recombination mutants; in each case there is at least one low-recombination mutant in the same codon.-Crosses involving mutants which were isolated in an altered wild type have shown that the behavior of a high-recombination mutant does not result from its molecular configuration alone, but from its combination with the homologous wild-type sequence from the other parent.-Several lines of evidence indicate that recombination in this system frequently involves closely-spaced double exchanges (about 40 codons apart).