B cells in the appendix and other lymphoid organs of the rabbit: Stimulation of DNA synthesis by anti-immunoglobulin

Lymphocytes from rabbit lymphoid organs were cultured in the presence of class specific anti-immunoglobulin sera and of anti-allotype sera. Stimulation of tritiated thymidine uptake into DNA was taken to indicate the presence of the corresponding immunoglobulins on the cell surfaces. Thymus and bone marrow lymphocytes were unresponsive to all reagents tested. Popliteal lymph node contained cells responsive to anti-μ, anti-γ, and anti-α and therefore presumably bearing IgM, IgG, and IgA. Spleen had only IgM- and IgG-bearing-cells, and the appendix contained cells with IgM and IgA receptors only. The lymph node, spleen, and appendix cells of rabbits depleted of B lymphocytes by irradiation (900 R) and injection of thymocytes were unresponsive to anti-immunoglobulin but were stimulated at almost normal levels by PHA and Con A. T cell-depleted animals (thymectomy, irradiation with three divided doses of 450 R and bone marrow shielding) had immunoglobulin-bearing lymphocytes but were unresponsive to the mitogens. However a moderate level of mitogen-responsiveness reappeared by 3–4 wk after irradiation. Cells of morphologically distinct regions of the appendix, separated manually, showed different responses corresponding to the inferred origins of these anatomic areas. The “dome” and “corona” contained functional IgM- and IgA-bearing cells. The “TDA” reacted well to PHA, Con A, and PWM, but was depleted of immunoglobulin-bearing cells. The “follicle” cells, which are almost all in active DNA synthesis or mitosis, were relatively unresponsive to either T or B cell stimuli. Anti-allotype serum stimulated the same populations which responded to class-specific heteroantisera but at a slightly lower level. It was inferred that gut-associated lymphoid tissues like the appendix may play a special role as an amplification site for B-cells destined to produce IgM and IgA elsewhere in the organism.     

Significant non-S-phase DNA synthesis visualized by flow cytometry in activated and in malignant human lymphoid cells

The development of a monoclonal antibody to the deoxynucleoside bromodeoxyuridine (BrdU), combined with two parameter flow cytometry, has allowed us to examine large numbers of cells for non-S-phase DNA synthesis. Three human lymphoid cell populations were studied to determine the level of deoxynucleoside (dN) incorporation as a function of DNA content. In each population, non-S-phase DNA synthesis was observed. In a rapidly growing human T-lymphoblastoid cell line (CCRF-CEM), 53% of dN incorporation occurred in G0/G1 plus G2 + M. In chronic lymphocytic leukemia (CLL) cells stimulated with tetradecanoylphorbol acetate (TPA), 45% of the observed burst in thymidine incorporation was found to be localized to G0/G1 cells. Non-S-phase incorporation was not, however, limited to neoplastic cells. Normal human peripheral blood B cells treated with the Cowan strain of Staphylococcus aureus (CSA) undergo a transient burst in thymidine incorporation, but do not go on to divide in the absence of other stimuli. Flow-cytometric analysis showed that 80% of this CSA-stimulated dN incorporation was into G0/G1 cells. These data are consistent with a more dynamic state of DNA synthesis than usually envisioned. Furthermore, the data show that although thymidine incorporation levels are related to incorporation of dN into DNA, they can be unrelated to cell proliferation.     

Increased clastogenicity and decreased inhibition of DNA synthesis by neocarzinostatin and tallysomycin in ataxia telangiectasia lymphoid cells

Cytogenetic damage in cells cultured from normal individuals and patients with ataxia telangiectasia (A-T) and xeroderma pigmentosum (XP) was induced by the chemotherapeutic antibiotics neocarzinostatin (NCS), tallysomycin (TLM) and bleomycin (BLM). Chromosomal breakage was specifically elevated in A-T cells when compared to the other genotypes tested. Similar results were not observed with the clastogens mitomycin C (MMC) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) as all cells responded similarly. All 5 chemical agents caused a marked suppression of de novo DNA synthesis in normal and XP long-term lymphoid cell lines while the A-T cells seemed resistant to this effect of NCS, TLM and BLM.     

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors from spontaneously proliferating and mitogen-activated lymphoid cells

Cytoplasmic extracts, prepared from continuously proliferating lymphoblastoid cells, as well as mitogen-activated normal lymphocytes, contain an extractable factor capable of inducing DNA synthesis in isolated quiescent nuclei. This factor is not detectable in resting cells. It is nondialyzable, precipitable by 30-50% saturated ammonium sulfate, and inactivated by trypsin. It is heat sensitive, but stable to cold and lyophilization. The molecular weight of the factor is greater than 100,000. This cytoplasmic activator of nuclear DNA replication is not released from the cell, and has no effect on intact cells. This suggests that it serves as an intracellular mitogenic signal in replicating cells.     

Macrophage regulation of DNA synthesis in lymphoid cells: effects of a soluble factor from macrophages

Addition of rat peritoneal macrophages to nonadherent rat spleen cells in culture results in enhancement or suppression of DNA synthesis depending on the ratio of macrophages to lymphocytes. At high ratios of macrophages to lymphocytes (1:5), suppression can be observed as early as four hours. Macrophages suppress incorporation of thymidine (TdR) by nonadherent spleen, thymus and bone marrow cells, in most instances, to less than 5% of that observed in culture to which macrophages were not added. In the presence of macrophages, incorporation of [³H]uridine and [¹⁴C] amino acids by spleen cells was also moderately suppressed. Based on ⁵¹Chromium release and dye exclusion assays, it appears that suppression is not due to cytotoxicity. Furthermore, suppression of [³H]TdR incorporation by nonadherent spleen cells is reversible, in the presence of an antigenic stimulus, following removal of the macrophages from the cultures. The suppressive effects are not elicited by extracts of macrophages, freeze-thawed or heated macrophages, but appear to be due to a low molecular weight, heat stable factor released into the macrophage culture fluid.     

Alterations of G-protein coupling function in phosphoinositide signaling pathways of cells transformed by ras and other membrane-associated and cytoplasmic oncogenes.

We showed previously that transformation by cytoplasmic and membrane-associated oncogenes including ras results in uncoupling between surface stimulation by platelet-derived growth factor, bombesin, and serum and activation of intracellular phospholipase C (PLC); this uncoupling does not involve alterations at the receptor or effector enzyme levels (T. Alonso, R. O. Morgan, J. C. Marvizon, H. Zarbl, and E. Santos, Proc. Natl. Acad. Sci. USA 85:4271-4275, 1988). In this study, we stimulated normal and oncogene-transformed NIH 3T3 cells with fluoroaluminate (AIF4-), thus directly activating PLC-associated G protein(s) and bypassing the receptor step. A1F4(-)-elicited PLC responses were significantly impaired in transformed cells when compared with those in their normal counterparts, suggesting that the uncoupling of PLC is the result, at least in part, of functional impairment at the G-protein level. Transformation by ras oncogenes has also been reported to result in enhanced PLC response to bradykinin resulting from increased receptor numbers (G. Parries, R. Hoebel, and E. Racker, Proc. Natl. Acad. Sci. USA 84:2648-2652, 1987; J. Downward, J. de Gunzburg, R. Riehl, and R. Weinberg, Proc. Natl. Acad. Sci. USA 85:5774-5778, 1988). We demonstrate here that transformation by other membrane-associated and cytoplasmic oncogenes also results in increased responsiveness to bradykinin ("supercoupling") and enhanced receptor numbers. However, there is no direct correlation between the number of receptors and the enhancement in responsiveness, suggesting that other factors besides receptor number are also involved in the enhanced responses. We propose that a common effect of transformation by cytoplasmic and membrane-associated oncogenes is functional alteration of coupling G proteins and that a similar modification of different kinds of G proteins may account for the pleiotropic alterations of signal transduction (uncoupling and supercoupling) observed.     

Alterations in the phosphorylation and activity of DNA polymerase alpha correlate with the change in replicative DNA synthesis as quiescent cells re-enter the cell cycle

The regulation of DNA polymerase alpha was examined in quiescent, human fibroblast cells stimulated to re-enter the cell cycle by subculturing in fresh serum-containing medium. The level of DNA polymerase alpha activity was measured in cell lysates and after specific immunoprecipitation. DNA polymerase alpha activity increased approximately 10-fold during the period of measurement. The activity increase was coincident with an approximately 60-fold increase in thymidine incorporation in the whole cells representing the first S phase. The large increase in polymerase alpha activity was not predominantly the result of synthesis of new polymerase, since the abundance of the enzyme changed less than 2-fold over the measured period. The quantity of [32P]phosphate incorporated into two subunits (180 and 68 kilodaltons) of DNA polymerase alpha increased approximately 10-fold in parallel with the increase in polymerase activity. The specific activity of the cellular ATP pool remained nearly constant over the period of measurement, indicating that the increase in labeling reflects a true increase in incorporation of phosphate. Results from other laboratories indicate that phosphorylation of DNA polymerase alpha increases its catalytic activity. Our results then suggest that the activity increase observed in DNA polymerase alpha when quiescent, human fibroblasts are stimulated to proliferate is largely caused by a phosphorylation-dependent regulatory process.     

Alterations to controls of cellular DNA synthesis by adenovirus infection.

Human adenovirus type 5 and temperature-sensitive mutants ts36, ts37, and ts125 induced cellular DNA synthesis in quiescent rodent cells at both permissive and nonpermissive temperatures. Cellular DNA synthesis induced by adenovirus type 5 or by serum required protein synthesis for both initiation and continuation, whereas viral DNA synthesis was not dependent upon continued protein synthesis once it was initiated. Both cellular and viral DNA replication was induced in adenovirus type 5-infected cells in the presence of dibutyryl cyclic AMP at concentrations which inhibited induction by serum which suggested that some of the controls of DNA synthesis in serum-treated and virus-infected cells are different. After adenovirus infection of quiescent cells, there was a decrease in the number of cells with G1 DNA content and an increase in cells with G2 diploid and greater DNA contents. Thus, adenovirus type 5 induces a complete round of cellular DNA replication, but in some cells, it induces a second round without completion of a normal mitosis. These results suggest that adenovirus type 5 is able to alter cell growth cycle controls in a way which may be related to its ability to transform cells.     

Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.     

Pituitary hormones regulate c-myc and DNA synthesis in lymphoid tissue

Hypophysectomy of Fischer 344 rats of both sexes led to a rapid involution of the thymus and spleen which was associated with a profound decrease in spontaneous DNA synthesis in these organs. The proportion of B lymphocytes in the spleen, of T cells and their subsets (CD4+/CD8+) in spleen and thymus, and the histological structure of the involuted organs remained normal. Treatment of hypophysectomized animals with growth hormone (GH) or prolactin (PRL) stimulated the expression of the c-myc proto-oncogene and DNA synthesis and reversed the involution in these organs. Replacement doses of adrenocorticotrophic hormone, follicle-stimulating hormone, luteinizing hormone, or thyroid-stimulating hormone had no influence on thymus or spleen size and DNA synthesis. A rapid expression of c-myc was also observed in thymuses and spleens of intact rats after the injection of GH or PRL. In vitro physiological concentrations (2.5 ng/ml) of either ovine or rat PRL or GH stimulated the incorporation of [3H]thymidine by thymus and spleen cells. These results indicate that GH and PRL regulate lymphocyte growth. This regulatory role is likely to serve as the principal mechanism of immunoregulation by these hormones.     

Rate of DNA synthesis as a function of temperature in cultured hamster fibroblasts (V-79) and HeLa-S3 cells

The rates of intracellular DNA synthesis at various temperatures between 39 ° and 31 °C were determined in hamster fibroblasts and HeLa cells by measuring average amounts of ³H-thymidine incorporated per cell in S phase per unit of time. The energy of activation and Q₁₀ for intracellular DNA synthesis were calculated from the slopes of the relative rates of DNA synthesis in HeLa cells and hamster fibroblasts vs. time, plotted on Arrhenius coordinates. In both cell types the incorporation of thymidine into DNA is characterized by an energy of activation of 21 000 calories/mole and a Q₁₀ of 2.94. The absolute rates of DNA synthesis were determined in hamster cells at various temperatures, with values ranging from 1.44 to 0.60 × 10⁻¹⁴ g DNA/ min/cell at 39 ° to 31 °C, respectively. The length of the S phase of the hamster cell was calculated over a 39 ° to 31 °C range, and found to be 5.0 to 11.9 h, respectively. It is concluded that the S phase length is partly determined by the rate of temperature-dependent DNA synthesis.     

Alterations of protein synthesis in arbovirus-infected L cells

Lust, George (Fort Detrick, Frederick, Md.). Alterations of protein synthesis in arbovirus-infected L cells. J. Bacteriol. 91:1612-1617. 1966.-Cellular protein synthesis and ribonucleic acid (RNA) synthesis in mouse L cells were markedly depressed 1 hr after infection with Venezuelan equine encephalomyelitis virus. Host RNA and protein synthesis were inhibited more rapidly by the virus infection than by actinomycin D. In cells infected 4 hr, a cytoplasmic RNA polymerase was demonstrated which was absent in uninfected cells. At this time, deoxyribonucleic acid-directed RNA synthesis catalyzed by the nuclear RNA polymerase was inhibited in vitro in enzyme preparations from nuclei of virus-infected cells. For optimal activity, the cytoplasmic RNA polymerase required the four nucleoside triphosphates, Mg(++), and RNA. The enzyme was insensitive to actinomycin D and deoxyribonuclease, indicating that it catalyzed RNA-directed RNA synthesis. Attempts to purify the induced polymerase further were unsuccessful. Fresh preparations had to be used because the enzymatic activity was unstable.     

Alterations in liver mitochondrial function as a result of fasting and exhaustive exercise

Two-dimensional displays of the restriction fragments from the DNA of Mus musculus revealed a complex species-specific pattern produced from nonsatellite repetitive sequences. The patterns have been used as a guide in the direct purification of a group of broadly interspersed repeated DNA sequences (characterized by a 1350-bp Eco-Bam fragment) that have been studied by molecular cloning, restriction mapping and genomic Southern blotting. These studies show that the cloned representatives originate from an abundant group of sequences that share homology with about 2% of the mouse genome. The sequences do not appear to share homology with mouse-interspersed-family-1 (MIF-1) nor with the major AT-rich satellite sequences of mouse. They appear to be part of a group of larger repetitive elements that is both broadly interspersed and heavily methylated in normal mouse tissue.