Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. II. Ribosome complement in resting and early G1 cells

Using a new, sensitive and quantitative technique for determining the ribosomal-RNA content of a measured number of cells, the cellular ribosome complement was compared for cultured hamster embryo cells in the stationary growth phase and in the early G₁ phase of the cell cycle. Cells from stationary phase cultures were found to contain less than 70% of the ribosome complement of the early G₁ phase cells, though the volumes of the two cell types were similar. This would imply that the stationary phase cell is physiologically different from a cell merely arrested at some point in the cell cycle.     

Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. 3. Electrophoretic patters of newly synthesized proteins in synchronized proliferating cells and resting cells

Electrophoretic patterns of newly synthesized proteins have been compared for hamster embryo fibroblasts in asynchronous cultures, mitotically synchronized cultures, and stationary phase cultures. Only proteins with molecular weight between 30,000 and 150,000, comprising 60–70% of the total cell proteins and excluding histones and collagen were included in the comparison. Although no significant differences could be detected between such patterns for cells at different stages of the cell cycle, significant differences were detected between patterns for cells in stationary phase and for proliferating asynchronous or synchronous cells in any stage of the cell cycle. These differences amounted to at least 5% of the newly synthesized cellular proteins. Much larger differences were detected between patterns from a nuclear fraction of proliferating and resting cells. These results indicate that normal cells in stationary phase are arrested in a state distinct from any phase of the normal cell cycle and may provide a biochemical marker for resting cells.     

Effect of serotonin on proliferating and resting cells in culture

The influence of exogenous serotonin on cell division of L929 and L-41 cell strains has been investigated under various conditions of cell growth in culture (the incubation either in 10% serum medium without changing the medium, or in the medium with 0.5% serum). The data obtained show that serotonin in physiological concentration (10(-7) M) stimulates proliferation of resting cells. In proliferating cells, compared to resting ones, the sensitivity to exogenous amine appeared statistically non-significant. Exogenous serotonin is suggested to be a proliferating stimulus for resting cells.     

Glycerolization of Chinese hamster ovary cells in monolayer culture

CHO monolayer cultures were used as a model system to examine the kinetics of cell glycerolization and deglycerolization. Both influx and efflux of [³H]glycerol appear to be first-order processes, the rate of flux being proportional to glycerol concentration. When net flux ceases, the intracellular and extracellular concentrations of glycerol appear to be identical. Influx can be described by a single first-order curve, whereas efflux requires at least two such functions. Flux rates are sensitive to changes in osmolality, the fast efflux component accelerating and the slow component decelerating with increased application of osmotic stress. The rate of influx slows as the temperature drops and as the viscosity of the medium increases. Cells can tolerate a much steeper deglycerolizing gradient at 37 °C than at 0 °C.     

Amitrole-induced cell transformation and gene mutations in Syrian hamster embryo cells in culture

Amitrole, a widely used herbicide, is an animal carcinogen and an inducer of cell transformation. However, it is inactive as a mutagen in bacterial test systems. Thus, it has been suggested that amitrole is a non-mutagenic carcinogen. Over the dose range that induces morphological transformation of Syrian hamster embryo cells in culture, amitrole induced gene mutations at the Na+/K+ ATPase and hypoxanthine phosphoribosyl transferase loci measured concomitantly in the same cells. These results indicate that amitrole may act via a mutational mechanism.     

Metabolism of proliferating and resting cells]

This review concerns the modern trends and experimental approaches to the study of cell cycle (cell population kinetics, cell structure and functions at various steps of the cycle,, etc.) and their input into the current views of cell proliferation controls. The resting state of the cell is considered and metabolic features of proliferating and resting cells are compared. Evidence is presented that resting cells are metabolically active and less resistant to the damaging factors that it has been previously supposed. The importance of this finding for biology and medicine, especially for cancer chemotherapy, is discussed.     

Characterization of an unscheduled DNA synthesis assay with Syrian hamster embryo cells

The Syrian hamster embryo cell transformation assay is widely used for studies of carcinogenesis. The characterization of an unscheduled DNA synthesis (UDS) assay for these cells is reported. Benzo[a]pyrene, aflatoxin B1 and UV light induced UDS in the cells in a dose-dependent manner without exogenous metabolic activity. Nitrosopiperidine induced UDS as well as gene mutations and cell transformation only in the presence of an exogenous metabolic activation system. The utility of this UDS assay with these cells is discussed.     

Pepsinogen synthesis in monolayer culture of human and rabbit gastric mucosal cells

We have established monolayer cultures of human and rabbit gastric mucosal cells and of isolated rabbit gastric chief cells. These cultures were capable of de novo pepsinogen synthesis and secretion, demonstrated by electrophoresis and subsequent autoradiography of cell lysates and growth medium after culture in the presence of 14C-labelled amino acids. Cultures could be maintained for 1 week without overgrowth by fibroblasts.     

Regulation of macromolecular synthesis in reovirus-infected L-929 cells I. Effect of L-histidinol.

The histidine analogue L-histidinol, reported by Vaughan and Hansen (1973) to establish a potent, readily reversible inhibition of eukaryotic protein synthesis in vivo, was used to investigate the regulation of macromolecular synthesis in reovirus-infected L-929 cells. The addition of L-histidinol to normal L cells led to a total inhibition of protein synthesis. The inhibition appeared to be a consequence neither of isotope dilution resulting from elevated endogenous amino acids nor of an inability of treated cells to accumulate exogenous amino acids. Addition of L-histidine to histidinol-arrested cells resulted in a complete recovery of protein synthesis. Similarly, protein synthesis in reovirus-infected L cells examined 17 h postinfection (31 C) was totally inhibited by histidinol treatment and was readily reversed by the addition of histidine. Reovirus-infected cells treated with histidinol had an essentially unaltered capacity to synthesize reovirus single-stranded RNA relative to unperturbed cultures but a diminishing ability to maintain genome RNA synthesis. Addition of L-histidine to arrested cultures led to a complete recovery of genome RNA synthesis. The L-histidinol-mediated arrest of protein synthesis was both very effective and easily reversed, suggesting the general applicability of this novel inhibitor to investigations of regulation of macromolecular synthesis in both normal and virus-infected eukaryotic cells.     

The effect on macromolecular synthesis of amino acid deprivation of hamster kidney cells

Since asparagine has been found to inhibit growth of some tumors and to inhibit or delay mitotic activity in other cells, we have studied the effect of asparaginase and of deprivation of some essential amino acids (Arg, Asn, Leu, Ile, Trp) on nucleic acid and protein synthesis in an asparagine-requiring strain of BHK/21 cells. We find that: (1) there is no essential difference in the pattern of synthesis following deprivation of any of the amino acids we tested; (2) that the effect of asparaginase is similar to that of amino acid deprivation; (3) that RNA synthesis is inhibited more rapidly than DNA or protein synthesis; (4) that after 10 hr of amino acid starvation, DNA synthesis is almost totally (reversibly) inhibited while RAN synthesis continues at about 30-50% and protein at about 100% of the initial value.     

Characterization of differentiated syrian golden hamster pancreatic duct cells maintained in extended monolayer culture

Summary Epithelial cells isolated from fragments of hamster pancreas interlobular ducts were freed of fibroblast contamination by plating them on air-dried collagen, maintaining them in serum-free Dulbecco's modified Eagle's (DME):F12 medium suppleneted with growth factors, and selecting fibroblast-free aggregates of duct cells with cloning cylinders. Duct epithelial cells plated on rat type I collagen gel and maintained in DME:F12 supplemented with Nu Serum IV, bovine pituitary extract, epidermal growth factor, 3,3′, 5-triodothyronine, dexamethasone, and insulin, transferrin, selenium, and linoleic acid conjugated to bovine serum albumin (ITS⁺), showed optimal growth as monolayers with a doubling time of about 20 h and were propagated for as long as 26 wk. Early passage cells consisted of cuboidal cells with microvilli on their apical surface, complex basolateral membranes, numerous elongated mitochondria, and both free and membrane-bound ribosomes. Cell grown as monolayers for 3 mo. were more flattened and contained fewer apical microvilli, mitochondria, and profiles of rough surfaced endoplasmic reticulum; in addition, there were numerous autophagic vacuoles. Functional characteristics of differentiated pancreatic duct cells which were maintained during extended monolayer culture included intracellular levels of carbonic anhydrase and their capacity to generate cyclic AMP (cAMP) after stimulation by 1×10⁻⁶ M secretin. From 5 to 7 wk in culture, levels of carbonic anhydrase remained stable but after 25 to 26 wk decreased by 1.9-fold. At 5 to 7 wk of culture, cyclic AMP increased 8.7-fold over basal levels after secretin stimulation. Although pancreatic duct cells cultured for 25 to 26 wk showed lower basal levels of cAMP, they were still capable of generating significant levels of cAMP after exposure to serretin with a 7.0-fold increase, indicating that secretin receptors and the adenyl cyclase system were both present and functional. These experiments document that pancreatic duct monolayer cultures can be maintained in a differentiated state for up to 6 mo. and suggest that this culture system may be useful for in vitro physiologic and pathologic studies. This research was supported by grant CA34051 from the National Cancer Institute, Bethesda, MD.     

Effects of bradykinin on DNA synthesis in resting NIL8 hamster cells and human fibroblasts

Bradykinin stimulates [3H]thymidine incorporation and DNA synthesis in resting, serum-deprived NIL8 hamster cells. The ED50 for this stimulation is 4.52 +/- 2.91 nM. Other kinin peptides including lys-bradykinin (kallidin) and met-lys-bradykinin also stimulate [3H]thymidine incorporation in the NIL8 cells, whereas desarg9-bradykinin is without effect, suggesting action of the kinin peptides through type B2 receptors. Bradykinin also stimulates DNA synthesis in IMR-90 human fibroblasts; however, this effect is observed only in the presence of indomethacin, which blocks prostaglandin synthesis. These results suggest that prostaglandins act as negative modulators of the growth-stimulatory effects of bradykinin in the fibroblasts. This conclusion is supported by the observation that exogenously added PGE1, PGE2, PGA1, PGA2, PGB1, and PGB2 strongly inhibit [3H]thymidine incorporation in the human fibroblasts. The direct effect of bradykinin observed in the NIL8 cells may be attributable to the relative resistance of these cells to growth inhibition by prostaglandins.     

Interrelationships between Sertoli cells and germ cells in the Syrian hamster

The interrelationships of the Sertoli cells and germ cells in the Syrian hamster were examined using the electron microscope. Demosome-like junctions were observed attaching Sertoli cells to spermatogonia and spermatocytes. In the region of the junctions dense plaques lay on the cytoplasmic surfaces of the plasmalemma of the opposing cells. Sertoli cell cytoplasmic filaments converged in the area of the junctions and inserted into the subsurface densities. Filaments were not observed associated with the subsurface densities of the germ cells. In the region of the junctions a 15...20 nm gap, filled with an attenuate amorphous substance, separated the plasmalemmata. Another attachment device termed "junctional specialization" occurred between Sertoli cells, and preleptotene spermatocytes and all successive developmental steps in the germ cell line in the hamster. The junctional specializations consisted of a mantel of Sertoli cell cytoplasmic filament lying subjacent to the Sertoli cell plasmalemma and an opposed cisterna of the endoplasmic reticulum. In stages VII-VIII preleptotene supermatocytes were observed in transit from the basal compartment to the adluminal compartment. While Sertoli-Sertoli junctions adluminal to the spermatocytes remained intact, typical Sertoli-Sertoli junctions formed between opposed Sertoli cell processes basal to the spermatocytes. It is proposed that, during the passage of spermatocytes in to the adluminal compartment, junctional specializations associated with preleptotene spermatocytes in the basal compartment migrate basal to the spermatocytes and contribute to formation of Sertoli-Sertoli junctions. Treatment of seminiferous tubules with hypertonic media was used to demonstrate that the junctional specializations function in cell-to-cell adhesion. Data indicated that these junctions function to retain the developing spermatids within the seminiferous epithelijm until the time of spermiation. At spermination the junctional specializations disappear and the spermatids drift off into the tubule lumen.     

Identification of adenovirus 2 early genes required for induction of cellular DNA synthesis in resting hamster cells.

tsAF8 cells are temperature-sensitive (ts) mutants of BHK-21 cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. When made quiescent by serum restriction, they can be stimulated to enter the S phase by 10% serum at 34 degrees C, but not at 40.6 degrees C. Infection by adenovirus type 2 or type 5 stimulates cellular DNA synthesis in tsAF8 cells at both 34 and 40.6 degrees C. Infection of these cells with deletion Ad5dl312, Ad5dl313, Ad2 delta p305, and Ad2+D1) and temperature-sensitive (H5ts125, H5ts36) mutants of adenovirus indicates that the expression of both early regions 1A and 2 is needed to induce quiescent tsAF8 cells to enter the S phase at the permissive temperature. This finding has been confirmed by microinjection of selected adenovirus DNA fragments into the nucleus of tsAF8 cells. In addition, we have shown that additional viral functions encoded by early regions 1B and 5 are required for the induction of cellular DNA synthesis at the nonpermissive temperature.     

Effect of low-intensity periodic-impulse laser UV radiation on the nucleic acid synthesis rate in proliferating and resting cells

A study was made of the effect of the low intensity (10(-6)--10(3)/cm3) high repetition rate (pulse duration 18 nseconds, repetition rate 10 KHz) UV radiation (2nd harmonic of Cu vapour laser, lambda = 271.2 nm) on proliferating and resting cells (HeLa and fibroblast-like cells of Chinese hamster 431). The treatment within the dose range from 0.05 to 5 J/m3 stimulates DNA synthesis in the resting cells rather than in the proliferating ones. It was shown autoradiographically that, within the same dose range, the number of synthesizing cells increased in 2.5 hour.