R. Schindler

Studies on the division cycle of mammalian cells. Iii. Preparation of synchronously dividing cell populations by isotonic sucrose gradient centrifugation.

Abstract A method of preparing synchronous cell cultures is presented which is based on a selection of postmitotic cells by density gradient centrifugation. Gradients were prepared by appropriate mixing of normal culture medium with medium in which NaCl was replaced by sucrose at a concentration providing for isotonicity. The cells sedimenting most slowly (2–5% of the original cell number) were collected, suspended in sucrose-free medium and cultured under conditions of an optimal steady state. Cell viability, as determined by the capacity of cells to form macroscopically visible colonies in a semisolid medium, was not altered by this treatment. Rhythmic variations of mitotic activity were observed, with a first maximum at 8 h after collection of postmitotic cells. The percentage of cells in the S period was determined at regular time intervals by pulse labeling with 3H-thymidine. A high proportion of DNA-synthesizing cells was found during low mitotic activity, while a low proportion of cells in S phase was observed at the time of maximum mitotic activity.

STUDIES ON LIVER REGENERATION: I. 131IODODEOXYURIDINE AS A PRECURSOR of DNA IN NORMAL and REGENERATING RAT LIVER

Abstract. 131Iododeoxyuridine (131IDU) was injected into normal and partially hepatectomized rats, and the specificity of incorporation of this thymidine analogue into liver DNA was determined 2, 24 and 48 hr following intramuscular injection. At 2 and 24 hr after 131DU injection, a major proportion of radioactivity in the liver was in the acid‐soluble fraction, whereas 48 hr after injection the label in the acid‐soluble fraction had decreased considerably. In liver obtained 2 hr after injection of 131IDU, only 1.8–16.6% of the total radioactivity were in DNA. If, however, the tissue was subjected to formalin fixation, the acid‐soluble label was extracted selectively, and of the remaining radioactivity 64–88% was in DNA. Therefore, the radioactivity that is not extracted by formalin may be used as a measure of DNA synthesis at the time of injection of 131IDU, thus obviating time‐consuming biochemical fractionation procedures.

Participation of mitochondrial proliferation in morphological differentiation of murine mastocytoma cells

Proliferation of a cold-sensitive cell-cycle mutant isolated from an undifferentiated murine mastocytoma line is reversibly arrested at the nonpermissive temperature of 33 degrees C, and the arrested cells undergo morphological differentiation as expressed by the formation of metachromatic granules. Following transfer of these mutant cells from the permissive temperature of 39.5 to 33 degrees C, a transient increase in both cytochrome c oxidase and DNA polymerase gamma was observed, the ratio of total mitochondrial volume to cell volume nearly doubled within 6 days, and numbers of mitochondrial cross-sections per cellular cross-section as determined in electron micrographs underwent a threefold increase. Addition of chloramphenicol (100 micrograms/ml) to the mutant cell cultures 6 days prior to transfer from 39.5 to 33 degrees C prevented the increase in the ratio of total mitochondrial to cell volume. Furthermore, chloramphenicol markedly inhibited the increase in granule number per cell that normally is observed after transfer of cultures to 33 degrees C or during treatment with 1 mM butyrate, suggesting that mitochondrial proliferation may be an obligatory step in the process of morphological differentiation of these mastocytoma cells.

Studies on the division cycle of mammalian cells: II. Causal relationship between completion of DNA synthesis and onset of the G2 period☆

Abstract Cultures of a murine mast cell tumor were partially synchronized by reversible inhibition of DNA synthesis with amethopterin (in combination with hypoxanthine and glycine) during different time intervals and subsequent addition of thymidine. In these cultures, the duration of the G2 period was measured (a) by observing the time-lag between addition of thymidine and resumption of mitotic activity, (b) by evaluating the time-lag between the addition of 3 H-thymidine and the appearance of labeled mitoses. Inhibition of DNA synthesis did not significantly change the median duration of the subsequent G2 period. It is, therefore, concluded that the G2-specific processes cannot begin before DNA synthesis has been completed, and that a triggering mechanism may be responsible for the transition of a cell from the S into the G2 period.

Proliferative quiescence of normal mast cells resembles that of cold-sensitive mutant mastocytoma cells: Dominant expression of the quiescent state in heterokaryons☆

Normal murine peritoneal mast cells were fused to serum-deprived, non-proliferating cells of a cultured subline (41-SB-4) of the P-815 murine mastocytoma. Upon reincubation in medium containing 10% horse serum for 48 h, mono- and binuclear 41-SB-4 cells reentered S phase of the cell cycle, while mast cell X 41-SB-4 heterokaryons as well as mono- and binuclear mast cells remained in proliferative quiescence, indicating dominant expression of the quiescent state of mast cells. The quiescent state of normal mast cells thus resembles that of cold-sensitive (cs) mutant cells (21-F) of the undifferentiated P-815 mastocytoma: at the non-permissive temperature of 33 degrees C, the 21-F cells were found to enter a state of quiescence which is characterized by its dominant expression in heterokaryons and by morphological differentiation with the formation of metachromatically staining granules similar to those of mast cells. This suggests that the cellular control mechanisms involved in entry into proliferative quiescence and in morphological differentiation of cs 21-F cells may be analogous to those of normal mast cells and/or their precursors.

Studies on the division cycle of mammalian cells: VI. DNA polymerase activities in partially synchronous suspension cultures

Abstract Partially synchronous cultures of a murine mastocytoma were prepared by centrifugation of cells in isotonic sucrose gradients and reincubation of slowly sedimenting early interphase cells under steady state conditions. Cell multiplication in these cultures occurred in a stepwise manner with a periodicity of approx. 8 h. In aliquots that were withdrawn from synchronous cultures at 1.5 h intervals, 3 H-thymidine incorporated into DNA by intact cells and DNA polymerase activities in cell homogenates were determined. Whereas synchronous growth of the cell population was reflected by pronounced fluctuations of 3 H-thymidine incorporation into DNA of intact cells, variations with time of DNA polymerase activity, using native or denatured DNA as primer, were much smaller and parelleled those of cellular protein content. Maxima and minima of 3 H-thymidine incorporation by intact cells preceded those of DNA polymerase activity and of cellular protein content by 1.5 to 3 h. It is concluded that total DNA polymerase activity as measured in disrupted cells does not determine changes in rate of DNA synthesis in the cell culture system studied.

Comparison of 3H-cytidine and 3H-5-uridine as precursors of RNA☆

Abstract The relative incorporation into RNA and DNA of 3 H-5-uridine, 3 H-cytidine and 3 H-6-thymidine was determined in brain, liver, kidney and spleen of the mouse as well as in neoplastic cell cultures. In systems with a high proliferative activity (spleen, cell cultures), the specificity of 3 H-5-uridine as a precursor of RNA was higher than that of 3 H-cytidine, although not as high as the specificity of 3 H-thymidine as a precursor of DNA. In organs with a low proliferative activity (brain, liver and kidney), the results based on a biochemical analysis did not permit any conclusions as to the distribution of label from 3 H-5-uridine and 3 H-cytidine between RNA and DNA of the small proportion of DNA-synthesizing cells.

DNA synthesis and mitotic activity in short-term cultures of hepatocytes isolated from regenerating rat liver.

Cell suspensions were prepared from normal and regenerating liver of adult rats by perfusion with a calcium-chelating agent (EGTA), collagenase and hyaluronidase, and the cells were incubated in culture medium. In cultures prepared from regenerating liver at 20 h after partial hepatectomy, 23 ± 4% of parenchymal cells initially incorporated [3H]TdR. This incorporation was shown to reflect semiconservative DNA replication. At least some parenchymal cells were able to complete their DNA synthesis and to progress through G2 and mitosis. Numbers of hepatocytes in mitosis increased up to 12 h of culture. On the other hand, no entry of hepatocytes into the S period was detectable in cultures prepared from normal or regenerating liver.

STUDIES ON LIVER REGENERATION: II. EFFECTS of PHENOBARBITAL ON the ONSET and PATTERN of RAT LIVER REGENERATION FOLLOWING PARTIAL HEPATECTOMY

Abstract. Partially hepatectomized rats were given a single intraperitoneal injection of a phenobarbital solution or of water immediately after surgery. At various time intervals following the operation, the animals were injected with 131 iododeoxyuridine (113IDU), sacrificed 2 hr later, and radioactivity retained in formalin‐fixed liver tissue was determined as a measure of DNA synthesis at the time of administration of the labeled precursor. In control animals without phenobarbital treatment, 131IDU incorporation into liver began to increase between 14 and 16 hr after partial hepatectomy. Phenobarbital treatment (0.1 mg per g of body weight) resulted in a delay of the increase in 131IDU incorporation by several hours. This delay was observed in animals subjected to partial hepatectomy in the morning as well as in those operated on in the evening. After phenobarbital treatment, the increase of mitotic activity was either delayed or occurred more slowly. the results are compared with the reported effects of partial hepatectomy on the time course of microsomal enzyme induction by phenobarbital.

Proliferation and specific functions of neoplastic mast cells in culture: Effects of amino acid deprivation

Abstract Cultures in vitro of a murine mastocytoma were incubated in a series of semisynthetic media. In order to produce specific nutritional deficiencies media were used in which one amino acid ( l -leucine, l -histidine or l -tryptophan, respectively) was present in optimal and various suboptimal concentrations. Appropriate reduction of the concentration of l -leucine or l -histidine resulted in a decrease of cell multiplication and in an increase of the relative number of dead cells, whereas omission of l -tryptophan had no appreciable effect on cell proliferation during the observation period of 96 h. After incubation of cells in these media during 4 days, the mean cellular histamine and 5-hydroxytryptamine contents as well as the incorporation of 35 SO 4 into the protein-bound heparin fraction were determined. The mean cellular content of histamine or 5-hydroxytryptamine was reduced in media containing suboptimal concentrations of the corresponding precursor amino acid ( l -histidine or l -tryptophan, respectively). In contrast, appropriate reduction of the l -leucine level in the culture medium resulted in an increase of the mean cellular 5-hydroxytryptamine content and an increased 35 SO 4 incorporation.