摘要:

AbstractChinese hamster embryo cells transformed with the tsA 58 mutant of Simian virus 40 express the transformed phenotype at the permissive temperature (33°C or 37°C) and a “normal” phenotype at the nonpermissive temperature (40.5°C). Immunofluorescence and immunoprecipitation of T antigens demonstrated that the “T” antigen (100 K) has an increase rate of synthesis and degradation at 40.5°C. However, the cells continue to replicate at the nonpermissive temperature when assayed by flow cytometry and autoradiography. This DNA synthesis was cellular, not viral, and not owing to an increase in DNA repair. When the cell cycle distributions of G1, S, and G2+ M were assayed by the fraction labeled mitoses method, no differences were evident at the permissive and nonpermissive temperature; however, the doubling time was lengthened at 40.5°C, the tsA transformed cells are cycling and dying. However, if the transformed cells are seeded onto monolayers of normal Chinese hamster cells at 40.5°C, the cells are growth arrested when measured by growth assays, flow cytometry, autoradiography, and immunofluorescence for T antigen. Therefore, growth arrest can be obtained in tsA 58 transformed Chinese hamster cells when cocultured with normal Chinese

ISSN:0021-9541    

DOI:10.1002/jcp.1041110302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe following evidence suggests that inhibition of hepatoma cell (HTC) growth by cyclic nucleotides is an adenosine‐like effect that is greatly modified by the type and treatment of serum used in the culture medium and is probably not mediated by cyclic AMP‐dependent protein kinase: (1) Heating serum reduces its phospho‐diesterase content, thereby slowing metabolism of cyclic AMP and reducing the inhibition of HTC cell growth by cyclic AMP; (2) Using medium that contains phosphodiesterase but lacks adenosine deaminase causes adenosine to accumulate from cyclic AMP and increases the toxicity of cyclic AMP; 3) Uridine or cytidine reverse the growth inhibition caused by adenosine, 5'‐AMP or cyclic AMP; 4) adenosine, 5'‐AMP and N6‐(δ2‐isopentenyl) adenosine are more toxic for HTC cells than is cyclic AMP, and N6, O2‐dibutyryl cyclic AMP is not toxic; and 5) N6, O2'‐dibutyryl cyclic AMP inhibits growth of Reuber H35 cells, but uridine prevents this inhibition of growth. We conclude that most, if not all, of the inhibitory effects of cyclic AMP and N6, O2'‐dibutyryl cyclic AMP on HTC and Reuber H35 hepatoma cell growth are due to the generation

ISSN:0021-9541    

DOI:10.1002/jcp.1041110303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractWe characterized murine hemopoietic colonies consisting of granulocytes, macrophages, megakaryocytes, and blast cells and yet lacking erythroid elements. Mouse marrow or spleen cells were cultured in methylcellulose media in the presence of 10% (v/v) pokeweek mitogen‐stimulated spleen cell‐conditioned medium (PWM‐SCM) and 2 units/ml erythropoietin for 8 days. Granulocyte‐macrophage‐megakaryocyte (GEMM) colonies could be distinguished from granulocyte‐erythrocyte‐macrophage‐megakaryocyte (GEMM) colonies because the former lacked the typical appearance of bursts with red color. Analysis of Y‐chromosomes in mixing experiments with male and female marrow cells confirmed the clonal nature of the GMM colonies. Differential counts of GMM colonies revealed varying, but significant, numbers of blast cells in all of the day‐8 and day‐12 colonies and in seven out of ten day‐14 GMM colonies. In general, the percentages of blast cells were inversely related to the length of incubation in culture. Replating experiments confirmed the absence of late erythroid precursors such as CFU‐E and normoblasts in all of the 50 day‐8 GMM colonies. However, six out of the 50 GMM colonies contained early progenitors capable of erythroid expression, such as BFU‐E, CFU‐EM, CFU‐GEM, and CFU‐GEMM. In contrast, the three day‐14 GMM colonies which did not reveal blast cells failed to produce secondary colonies. Thus, while the progenitors for the latter colonies are restricted to only granulocyte‐macrophage‐megakaryocyte differentiation, some of the apparent GMM colonies containing blast cells may have originated in early progenitors close to pluripotent stem cells. Detailed cytological analyses and replating experiments are necessary for characterization of true differentiatio

ISSN:0021-9541    

DOI:10.1002/jcp.1041110304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractFibroblast growth and synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin‐P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose‐dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80°C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (α‐LT). It was not cytotoxic, as shown by the51chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G0or G1stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA Synthesis but cannot divide, and they accumulate in G2. The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory pr

ISSN:0021-9541    

DOI:10.1002/jcp.1041110305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractAutoradiography of colony replicas immobilized on filter paper was used to isolate a Chinese hamster ovary cell line deficient in incorporation of radiolabeled fucose into a trichloroacetic acid‐insoluble fraction. This cell line, called 62.1, has the same growth rate at 37°C as wild‐type cells, but incorporates five times less fucose into acid‐insoluble radioactivity. Chemical analysis of fucose bound to macromolecules also showed a fivefold reduction in the mutant. The fucoproteins of the mutant cell line differ qualitatively from those of wild‐type cells as visualized by SDS gel electrophoresis fluorography; no differences were detected between total proteins as visualized by coomassie blue staining. The macromolecular sialic acid content of the mutant was somewhat higher than the wild type (20%).Studies of the synthesis of the glycoprotein of vesicular stomatitis virus in mutant and wild‐type cells showed that the mutant is unable to synthesize complex‐type N‐linked oligosaccharides. Enzyme assays show that this defect in the mutant is due to reduction in UDP‐N‐acetylglucosamine‐glycoprotein N‐acetyl‐glucos‐aminyltransferase, a key enzyme in the assembly of complex glycopeptides.Hybridization studies have shown that mutant 62.1 has common mutations belonging to the same complementation group as mutant PHaR1‐1. This latter mutant was previously isolated using lectin resistance by Stanley et al. (1975) and was also deficient in the above N‐

ISSN:0021-9541    

DOI:10.1002/jcp.1041110306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractUsing fibroblastic CHO cells, we have examined (1) the internalization and redistribution of surface binding sites for the lectins Concanavalin A and wheat germ agglutinin and (2) the sensitivity of these processes to putative inhibitors of cytoskeletal activity. Following initial exposure to fluorescein conjugated Con A (CAF) or WGA (WGAF) at 4° C, kinetic analysis of internalization and intracellular aggregation of lectin at 37°C indicated more rapid aggregate formation in the case of WGA than in the case of Con A. Treatment with tertiary amine local anesthetics (tetracaine, dibucaine, and xylocaine) or with a lysosomatrophic amine, m‐dansyl cadaverine, blocked internalization of Con A but not of WGA. Similar differential effects on redistribution of Con A and WGA were not however observed with the antimicrotubule agents colchicine and nocodazole. Simultaneous treatment of cells with WGAF and with rhodamine labeled Con A (CAR) resulted in the accumulation of both labels in a central perinuclear aggregate; a similar experiment in the presence of local anesthetic resulted in a diffuse peripheral distribution of CAR and a central aggregate of WGAF. These results suggest (1) CHO cells possess at least two distinct pathways for lectin internalization and redistribution, which can be discriminated in terms of drug sensitivity; (2) CHO cells can sort out and independently internalize different populations and lectin binding sites; and (3) different pathways may be a manifestation of biochemically distinct linkages between cytoskeletal elements and various groups of surface glycoproteins. Present findings concur with our previous results concerning the mutual independence of the surface binding sites for Con A and WGA (Emerson and Juliano, 19

ISSN:0021-9541    

DOI:10.1002/jcp.1041110307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractGranulocyte‐macrophage colony formation by C57BL bone marrow cells was initiated in agar cultures either by the granulocyte‐macrophage stimulus, GM‐CSF, or by the predominantly macrophage stimulus, M‐CSF. After 24 hours, paired daughter cells of granulocyte‐macrophage colony‐forming cells (GM‐CFC) were separated by micromanipulation and one cultured in GM‐CSF, the other in M‐CSF. From the differentiation pattern of the resulting colonies, irreversible commitment of some cells occurred during the first 24 hours and completion of the first cell division. A similar result was obtained using granddaughter cells present after 24 hours of incubation. However, when intact developing day 2 and days 3 clones were cross‐transferred to GM‐CSF or M‐CSF recipient cultures, irreversible commitment was more obvious. Most M‐CSF‐initiated clones exhibited irreversible commitment to macrophage formation in GM‐CSF cultures and a high proportion of GM‐CSF‐initiated clones continued to produce granulocyte progeny after transfer to M‐CSF.The results indicated that GM‐CSF and M‐CSF can irreversibly commit the progeny of GM‐CFC respectively to granulocyte or macrophage production. While for some GM‐CFC this occurs within 24 hours and one cell division, for many cells, the process is slower and requires an incubation period of up to 48 hours and/or several cell divisions.Calculations from the data indicated that two‐thirds of GM‐CFC in adult C57BL marrow are biresponsive and

ISSN:0021-9541    

DOI:10.1002/jcp.1041110308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe regulation of cell growth can be achieved at many levels but ultimately the regulatory factors must alter protein synthesis since growing cells always exhibit an increased rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis is directly dependent on mRNA content. Other studies have shown that growing and resting cells have similar amounts of mRNA and that protein synthesis is regulated by the proportion of mRNA in polysomes. We have analyzed mRNA content in growing and resting epithelial cells ofXenopus laevis. Quantitation of poly(A)+mRNA by uniform labeling with3H‐uridine and by3H‐poly(U)hybridization demonstrated a direct relationship between mRNA content and the relative rate of protein synthesis in growing and resting cells. Likewise, after serum stimulation of resting cells the increase in mRNA content closely paralleled the increase in protein synthesis. Our results suggest that control of protein synthesis in growing and nongrowing cells is exerted before the translational le

ISSN:0021-9541    

DOI:10.1002/jcp.1041110309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractA novel synergistic effect of several purine derivatives such as adenine, adenosine, hypoxanthine, and guanine on the toxicity of nucleoside analogs pyrazofurin and 6‐azauridine towards cultured Chinese hamster ovary (CHO) cells has been observed. The presence of the above purine derivatives enhanced the toxicity of pyrazofurin and 6‐azauridine, in a dose dependent manner. The growth inhibitory effects of these nucleoside analogs either alone or in combination with the purine derivatives were reversed by uridine and cytidine, providing evidence that the synergistic effect of the purine derivatives was exerted at the level of pyrimidine nucleotide biosynthesis. Studies with mutant cells lacking various purine phosphorylating enzymes show that phosphorylation of purine derivatives through reactions utilizing phosphoribosylpyrophosate (PRPP) is essential for observing the synergistic response. It is suggested that the above purine derivatives (including adenosine, via conversion to hypoxanthine) exert their synergistic effects by depleting the cellular pool of PRPP by two separate mechanisms (direct utilization and feedback inhibition of its synthesis), which as a result becomes rate limiting in the synthesis of orotidine monophosphate (OMP). The reduced levels of OMP, which is a competing substrate with pyrazofurin‐ and 6‐azauridine‐5'‐monophosphates for binding to the target enzyme OMP decarboxylase, could then account for the inhibition of the enzyme at lower concentrations of th

ISSN:0021-9541    

DOI:10.1002/jcp.1041110310

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractRous sarcoma virus (RSV)‐infected chicken embryo cells were used to study the effect of viral transformation on the hormone‐stimulated synthesis of cyclic AMP. Transformation by RSV in response to the β‐adrenergic agonist isoproterenol as compared to untransformed cells. This enhancement was observed in both intact cells and in membranes prepared from these cells. The inclusion of guanosine 5'‐0‐(3‐thiotriphosphate), a nonhydrolyzable analogue of GTP, in assays of adenylate cyclase activity did not abolish the quantitative differences between the transformed and normal cell membranes. Infection of cells by Rous‐associated virus, which lacks the oncogenesrc, did not induce this hyperresponsiveness thus indicating the probable involvement of thesrcgene product in this phenomenon. The duration of the isoproterenol‐induced cyclic AMP elevation was longer in the transformed than in the untransformed cells; transformed cells, unlike untransformed cells, required at least 120 min before full desensitization became established. Membranes prepared from transformed cells specifically bound more than 5 times the quantity of the β;‐adrenergic radiolabeled antagonist (‐)3H‐dihydroalprenolol and125I‐iodocyanopindolol compared to the untransformed cell membranes. Thus, it appears that major differences between the transformed and normal phenotypes reside in the concentration of membrane β‐adrenergic receptors and the inability of RSV‐transformed cells to self‐limit their respo

ISSN:0021-9541    

DOI:10.1002/jcp.1041110311

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY