"Numatrin," a nuclear matrix protein associated with induction of proliferation in B lymphocytes

To detect nuclear proteins that might be involved in induction of cellular mitogenesis, we examined the effect of various mitogens on early changes in synthesis of nuclear proteins in murine B lymphocytes. Using two-dimensional gel electrophoresis, we found that activation of B cells by mitogens (anti-immunoglobulin antibody, lipopolysaccharide, phorbol 12-myristate 13-acetate (PMA)/A23187) was associated with a rapid and prominent (5-20-fold) increase in the synthesis of a 40-kDa/pI 5.0 nuclear protein, here termed numatrin. Numatrin was found to be absent from the cytosol (soluble fraction) of resting as well as activated B cells and was markedly resistant to DNase/RNase digestion and 2 N NaCl extraction, indicating that this protein is tightly bound to the nuclear matrix. Kinetic studies showed that the increase in synthesis of numatrin was detected 60-120 min following mitogen activation, reached a peak at 16 h, and declined to almost control level by 48 h, correlating with the peak of cellular DNA synthesis. The increase in synthesis of numatrin in normal B cells was found to be associated exclusively with cellular commitment for mitogenesis because activation of B cells by stimuli such as B cell stimulating factor 1, PMA alone, and calcium ionophore A23187, which do not stimulate an increase in DNA synthesis, also failed to induce an increase in the synthesis of numatrin. Inhibition of anti-Ig-induced proliferation (by PMA pretreatment) was associated with a 63% inhibition in the synthesis of numatrin. Addition of 8-mercaptoguanosine to these PMA-treated cells was associated with restoration of the increase in synthesis of numatrin, concomitant with induction of proliferation. Elevated synthesis of numatrin was also detected in the malignant B lymphoma cells: Raji, BAL-17, and WEHI-231. Taken collectively, these results suggest that numatrin, a tightly bound nuclear matrix protein, is a growth-regulated protein which might have an important role in regulation of cellular mitogenesis in normal and malignant B lymphocytes.     

The nuclear matrix protein, numatrin (B23), is associated with growth factor-induced mitogenesis in Swiss 3T3 fibroblasts and with T lymphocyte proliferation stimulated by lectins and anti-T cell antigen receptor antibody

Numatrin is a tightly bound nuclear matrix protein (40 kD/pI-5) whose synthesis is markedly and promptly increased in association with cellular commitment for mitogenesis in B lymphocytes. (Feuerstein, N., and J.J. Mond. 1987. J. Biol. Chem. 262:11389-11397). To study whether this event is exclusively associated with proliferation of B lymphocytes, we examined the synthesis of numatrin in T lymphocytes (murine and human) activated by lectins or by anti-T cell antigen receptor monoclonal antibody and in Swiss 3T3 fibroblasts stimulated by growth factors. We showed a close correlation between induction of DNA synthesis and induction of numatrin synthesis in T lymphocytes stimulated by concanavalin A, anti-T cell antigen receptor monoclonal antibody, and IL-2 in murine T cells. Similar results were observed in Swiss 3T3 fibroblasts, thus only combinations of growth factors (insulin/EGF or insulin/B subunit of cholera toxin) or serum, which induced a significant increase in DNA synthesis, were also associated with a significant increase in synthesis of numatrin. Similar to B cells, the increase in numatrin synthesis in fibroblasts was found to occur at early G1 phase. The calcium ionophores, A23187 and ionomycin, previously shown to induce an increase in c-myc and c-fos mRNA levels in fibroblasts, induced a marked increase in the synthesis of a nuclear protein at 80 kD/pI-5 but failed to induce an increase in the synthesis of numatrin indicating that an increase in intracellular Ca++ level is not sufficient for induction of the synthesis of numatrin. This further indicates that the increase in synthesis of numatrin may be more closely correlated with cellular commitment for mitogenesis as compared with other biochemical parameters. Using a polyclonal numatrin antibody we demonstrated that mitogen stimulation is also associated with a marked increase in numatrin abundance, which reached a peak at the onset of S phase and declined at the end of S phase. Evidence is presented to show that numatrin synthesis and abundance is elevated in various lymphoma cell lines. Using indirect immunofluorescence assays we showed that numatrin is abundant in other malignant cells: KB, epidermoid carcinoma, and Hep2 human hepatoma. Immunofluorescence studies further showed that mitogen stimulation of B lymphocytes induced a marked accumulation of numatrin in the nucleoli. This observation is in accord with the recent finding of identity of numatrin with the nucleolar protein B23 (Feuerstein et al. 1988. J. Biol. Chem. 263:10608-10612).(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in phospholipid metabolism during B lymphocyte activation

We have examined phospholipid metabolism in murine B lymphocytes stimulated with anti-Ig bound to Sepharose. T cell-depleted splenic B lymphocytes cultured with Sepharose-coupled, affinity-purified goat anti-mouse Ig (GAMIg) increased the incorporation of 32PO4 into phosphatidic acid and phosphatidylinositol within 3 hr and increased [3H]-thymidine uptake at 48 hr. No increase in labeling was observed in phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. Based on both negative and positive selection procedures, it was demonstrated that these responses occurred in B lymphocytes. In contrast to the thymidine uptake response of the GAMIg-stimulated B lymphocytes, the phospholipid response did not require the presence of accessory cells or exogenous cytokines. The same selective changes in phospholipid metabolism were observed in neoplastic B lymphocytes (BCL1) after treatment with Sepharose anti-mu, but not with Sepharose anti-Ia or Sepharose normal Ig. The dose-response relationships of 32PO4 incorporation into phosphatidic acid and phosphatidylinositol and [3H] thymidine uptake were nearly identical in BCL1 cells. The results of these experiments indicate that interaction of B lymphocytes with insolubilized anti-Ig results in prompt and selective changes in phospholipid metabolism that appear to be correlated with B lymphocyte proliferation.     

Activation of human B cells. Comparison of the signal transduced by IL-4 to four different competence signals

The effects of the cytokine IL-4 on resting and activated human B cells were compared with the effects of known "competence" signals able to drive resting B cells into the cell cycle, including anti-Ig, PMA, anti-CD20, and a recently described competence signal, anti-Bgp95. In proliferation assays, IL-4 was costimulatory with anti-Ig and anti-Bgp95 but not with anti-CD20 or PMA. IL-4 alone triggered increases in expression of class II DR/DQ and CD40, but it did not trigger increases in intracellular free calcium [Ca2+]i in resting B cells or induce resting B cells to leave G0 and enter the G1 phase of the cell cycle. Although IL-4 has some characteristics of competence signals, it was most effective if added to B cells up to 12 h after anti-Ig or anti-Bgp95 rather than before, and thus, in this respect, works more like a progression signal. Like IL-4, all four competence signals for B cells triggered increases in class II and CD40, but only IL-4 consistently induced increases in CD23 surface levels. IL-4 was costimulatory only with anti-Ig and anti-Bgp95, each of which can trigger increases in [Ca2+]i and new protein synthesis of the proto-oncogene c-myc, and can increase attachment of protein kinase C to the plasma membrane. IL-4 was not costimulatory with signals that 1) did not affect [Ca2+]i yet induced c-myc protein synthesis (anti-CD20), 2) only stimulated the translocation of protein kinase C (PMA), or 3) only stimulated increases in [Ca2+]i (calcium ionophore). These results suggest that resting human B cells require at least two intracytoplasmic signals before IL-4 can effectively promote B cell proliferation.     

Early activation and cell cycle entry of resting B cells after Fab-anti-Ig treatment: role of receptor crosslinking

Comparison of the effect of goat anti-rabbit Ig (GARIg) and its monovalent fragment (Fab-GARIg) demonstrates that surface Ig (sIg) crosslinking is not necessary to effect G0 to G1 transition in rabbit peripheral blood B cells but is required for induction of DNA synthesis. Five micrograms per milliliter or more of GARIg is sufficient to induce DNA synthesis but up to 50 micrograms/ml of Fab-GARIg is not. However, the monovalent reagent induces microscopically observable cytoplasmic and nuclear changes (blast transformation) in a dose-dependent manner. These differ qualitatively and quantitatively from the morphological changes seen with comparable doses of GARIg; Fab anti-Ig produces "small blasts" whereas complete GARIg induces large blasts. The monovalent reagent, in a wide range of concentrations, is as effective as the complete antibody in modulating sIg from rabbit B cells. Fab-GARIg treatment modulates sIg in a biphasic manner. It clears the high-density sIg within 5 min, whereas the remaining low-density receptors disappear after 4 hr. Cytosolic protein kinase C levels decline equally after treatment with either Fab-GARIg or whole anti-Ig. RNA synthesis, as measured by [3H]uridine incorporation, increases for the first 12 hr in cells activated with either reagent. It declines to basal levels in Fab-GARIg stimulated cells, but a continuous increase occurs in cells stimulated with 5 and 50 micrograms/ml of complete antibody. Simultaneous addition of 50 micrograms/ml Fab-GARIg with 5 microgram/ml of GARIg causes greater RNA synthesis for 12 hr after stimulation than is caused by GARIg alone. After 12 hr the monovalent reagent has an inhibitory effect on RNA synthesis. Fluorescence-activated cell sorter analysis of acridine orange-stained cells shows that Fab anti-Ig-stimulated cells have higher RNA content than resting cells, but lower than GARIg-activated cells. These findings suggest that rabbit B cells can be activated from the G0 stage of cell cycle to G1 by monovalent anti-Ig reagents but further cell cycle progression requires maintenance signals provided by receptor crosslinking. The implications of these results for B cell activation signalling are discussed in the context of the floating receptor model.     

Identification of a prominent 85-kDa cAMP-dependent phosphoprotein associated with late G1 phase in mitogen-stimulated B lymphocytes

In order to elucidate late regulatory events which may be involved in the onset of S phase in B lymphocytes, we studied the effect of anti-Ig on phosphorylation of soluble proteins at late G1 phase. Stimulation of murine splenic B lymphocytes with anti-Ig and other mitogens for 18 h was found to be associated with a major increase in phosphorylation of an 85 kDa/pI approximately 5.3 cytosolic protein, conversely, stimulation of the cells with non-mitogenic stimuli did not induce the phosphorylation of pp85. The increase in phosphorylation of pp85 could not be detected after 30 min, was barely detectable after 6 h, but was very prominent after 18 h of stimulation with anti-Ig. Thus, the increase in phosphorylation of pp85 is not an early signal but is rather correlated with the late G1 phase. pp85 could not be detected in the nuclei of either control or stimulated cells. Stimulation of B cells for 30 min with forskolin induced the phosphorylation of pp85, while phorbol ester did not have any effect. The phosphorylation of pp85 was induced by the catalytic subunit of cAMP protein kinase. Comparison of the phosphopeptide map of pp85 phosphorylated by anti-Ig in intact cells to the phosphopeptide map phosphorylated by forskolin or by the catalytic subunit of cAMP protein kinase, showed a striking similarity indicating that cAMP protein kinase may be involved in phosphorylation of pp85 in mitogen-stimulated cells. An increase in intracellular cAMP levels at late G1 phase was found in B cells stimulated by mitogens. These results implicate an important role for cAMP-dependent phosphorylation events, specifically the phosphorylation of pp85/pI 5.3, at late G1 phase during the cell cycle.     

Anergy in immature B lymphocytes. Differential responses to receptor-mediated stimulation and T helper cells

We have compared the responses of purified neonatal and adult B lymphocytes to stimulation by anti-Ig antibodies, which are functional analogues of Ag, and by Th cells. Neonatal B cells are markedly deficient in proliferative responses to anti-Ig antibodies + IL-4 or to anti-Ig conjugated to dextran, both of which induce strong proliferation of adult B cells in the absence of T lymphocytes. Anti-Ig antibodies actually inhibit the functional responses of neonatal B cells, even to polyclonal stimuli such as LPS. However, Th cells induce both proliferation and Ig secretion by neonatal B cells in the presence of Ag that bind to B cell Ig and are subsequently presented by the B cells. Thus, in neonatal B lymphocytes, cross-linking of membrane Ig in the absence of Th cells has a net inhibitory effect, and this inhibition is overcome by T cell help. These results also suggest that unresponsiveness or tolerance to thymus-independent Ag is induced in the B cells themselves, but tolerance to thymus-dependent proteins resides primarily in the T cell compartment.     

A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N∶2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53^−/− cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.     

Anti-Ig-stimulated B lymphoblasts can be restimulated via their surface Ig

Engaging AgR (surface Ig) on B lymphocytes leads to rapid inositol phosphate turnover and elevation of intracellular [Ca2+]. Continuous receptor occupancy (greater than 18 h) by anti-Ig leads to transit of most B lymphocytes from G0 to G1 stage of the cell cycle (blast transformation); a fraction of cells continue into S phase but do not proliferate continuously in the absence of growth factors. Prolonged exposure to ligand can induce receptor desensitization of some receptors. We therefore investigated whether such desensitization occurs in B cells activated by insolubilized anti-Ig. Resting B cells and anti-Ig-activated blasts were examined for their potential to elevate [Ca2+]i, maintain viability, and synthesize DNA in response to reexposure to anti-Ig. B cells and anti-Ig blasts had similar basal [Ca2+]i levels. Anti-Ig blasts retained the capacity to increase [Ca2+]i in response to anti-Ig; the magnitude of the increase was equal to or greater than that observed with resting B cells and occurred in more than 90% of cells. Isolated anti-Ig blasts subcultured in the presence of T cell-derived growth factors for 3 to 5 days responded to restimulation by anti-Ig with an increase in [Ca2+]i similar to that observed in freshly isolated blasts. The B cell and B lymphoblast ion channels were found to be permeable to Ca2+ but impermeable to Mn2+. Finally, blasts restimulated by anti-Ig retained viability and incorporated low levels of [3H]thymidine for 24 h. These results suggest that AgR on activated B lymphocytes can remain functionally coupled to intracellular signaling pathways and can participate in immune responses subsequent to initial activation.     

Characterization of immunosuppressive substances in the basic protein fraction of uterine secretions from pregnant ewes

The basic protein fraction of ovine uterine secretions collected late in pregnancy (Days 125-140) contains a substance capable of inhibiting in vitro blastogenic responses of lymphocytes to phytohemagglutinin (PHA) or mixed lymphocyte reactions. In this study, the immunosuppressive substance in the basic protein fraction of uterine secretions was further defined by gel filtration. The immunosuppressive activity resided in a group of high molecular weight proteins eluting at the void volume of Sephacryl S-200 and Sepharose CL-6B columns. For example, incorporation of thymidine by PHA-stimulated lymphocytes incubated with 20, 40, 80, and 120 micrograms/ml of protein from the void volume of Sepharose CL-6B was 65, 28, 2, and 0 percent of control lymphocytes, respectively. Based on polyacrylamide-gel electrophoresis in the presence of sodium dodeylsulfate (SDS), the immunosuppressive fraction from Sepharose CL-6B chromatography contained aggregates of uterine milk proteins (UTM-proteins) and a pair of proteins running at the top of a 5% (w/v) polyacrylamide gel. Other protein peaks resolved by Sephacryl S-200 and Sepharose CL-6B contained aggregates of UTM-proteins but were not immunosuppressive. The substance inhibiting in vitro lymphocyte function was not of conceptus origin, because it was found in fluid from the ligated uterine horn of unilaterally pregnant ewes and from the uterus of an ovariectomized ewe treated for 60 days with progesterone and estrone.     

Identification of numatrin, the nuclear matrix protein associated with induction of mitogenesis, as the nucleolar protein B23. Implication for the role of the nucleolus in early transduction of mitogenic signals

We have previously described and characterized a nuclear protein at 40 kDa/pI 5 termed "numatrin" which is tightly bound to the nuclear matrix. We demonstrated that a rapid increase in the synthesis of numatrin at early G1 phase is closely correlated with receptor-mediated induction of cellular proliferation by various mitogens and that elevated amounts of numatrin are found in tumor cells, suggesting that numatrin may have an important role in regulation of cellular growth in normal and malignant cells. Further experiments were undertaken to compare the biochemical characteristics of numatrin to those of other known proteins that are associated with cellular mitogenesis. Comparison of the electrophoretic mobility of numatrin with the proliferation cell nuclear antigen/cyclin showed that these proteins are not identical. However, numatrin had an identical electrophoretic migration on two-dimensional gel electrophoresis to that of a previously described nucleolar protein B23. The tryptic digest peptide map of 125I-labeled B23 was identical to that of numatrin on two-dimensional thin layer electrophoresis/chromatography. Labeling of cells with 32P further showed that numatrin is a major phosphoprotein as previously reported for protein B23. Using the protocol for purification of B23, we purified numatrin from nucleoli of HL-60 cells and produced two polyclonal antibodies (303 and 339) to this protein. We further show that numatrin is recognized by anti-B23 monoclonal antibody as well as by polyclonal antibodies 303 and 339 in enzyme-linked immunosorbent assay. Conversely, these anti-numatrin polyclonal antibodies cross-react with protein B23 as shown in immunoblot analysis. These results, taken collectively, prove that numatrin is identical to the nucleolar protein B23 and thus suggest that protein B23 and events which occur at the nucleolus might have an important role in early transduction of mitogenic signals at the G1 phase of the cell cycle.     

Reorganization of the protein composition of the nuclear matrix of hepatoma cells after inhibition of DNA synthesis with novobiocin

The nuclear matrix of Zajdela hepatoma cells, in which DNA synthesis was blocked by novobiocin, contained 2.5-3.0 times more DNA and protein not dissociating in 2 M NaCl than the nuclear matrix of control cells. Chromatography of nuclear matrix preparations on Sepharose 2B-CL resulted in isolation of tightly bound DNA-protein complexes which did not dissociate in 8 M urea or 0.1% SDS. Subsequent elution of DNA-protein complexes on a hydroxylapatite column with a buffer containing 4 M guanidine hydrochloride and 5 M urea caused partial dissociation of the complexes. Electrophoretic analysis revealed essential changes in the composition of proteins DNA-protein complexes of hepatoma cells nuclear matrix during inhibition of DNA synthesis.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

Proteomic analysis of p16ink4a-binding proteins

The p16(ink4a) tumor suppressor protein plays a critical role in cell cycle control, tumorogenesis and senescence. The best known activity for p16(ink4a) is the inhibition of the activity of CDK4 and CDK6 kinases, both playing a key role in cell cycle progression. With the aim to study new p16(ink4a) functions we used affinity chromatography and MS techniques to identify new p16(ink4a)-interacting proteins. We generated p16(ink4a) columns by coupling the protein to activated Sepharose 4B. The proteins from MOLT-4 cell line that bind to p16(ink4a) affinity columns were resolved by SDS-PAGE and identified by MS using a MALDI-TOF. Thirty-one p16(ink4a) -interacting proteins were identified and grouped in functional clusters. The identification of two of them, proliferating cell nuclear antigen (PCNA) and minichromosome maintenance protein 6 (MCM6), was confirmed by Western blotting and their in vivo interactions with p16(ink4a) were demonstrated by immunoprecipitation and immunofluorescence studies. Results also revealed that p16(ink4a) interacts directly with the DNA polymerase delta accessory protein PCNA and thereby inhibits the polymerase activity.     

Lymphoma models for B cell activation and tolerance. VI. Reversal of anti-Ig-mediated negative signaling by T cell-derived lymphokines

We have recently described three "immature" B cell lymphomas which are exquisitely sensitive to growth inhibition by anti-Ig reagents and may serve as models for tolerance induction in normal B cells. These cells are inhibited from cell cycle progression into S after receiving a negative signal in early G1. In this paper, we demonstrate that the growth inhibition by anti-Ig can be prevented and reversed by the addition of supernatants from T cell lines. One such line, called Tova, produces factors which restore normal levels of DNA synthesis in the presence of concentrations of anti-Fab or anti-kappa immunoglobulins which cause up to a 90% inhibition of thymidine incorporation in a 2- to 3-day culture period. This factor is at least partially effective when added up to 24 hr after anti-Ig to unsynchronized lymphoma cells and it does not alter the growth of control cultures. Studies using synchronized lymphoma cells indicated that the T cell factor permitted cycle progression into S when added during the early G1 exposure to anti-kappa and was less effective when added late in G1. Preliminary characterization suggests that both B cell growth factor II (interleukin 5) and B cell stimulatory factor 1 (interleukin 4) have additive activity in this system, although another unidentified lymphokine may also be involved. The relevance of T cell reversal of Ig receptor-mediated negative signaling to neonatal B cell tolerance is emphasized.     

Cross-linking of surface Ig receptors on murine B lymphocytes stimulates the expression of nuclear tetradecanoyl phorbol acetate-response element-binding proteins

Cross-linking of sIg on primary B lymphocytes leads to increased nuclear DNA-binding activity specific for the tetradecanoyl phorbol acetate-response element (TRE), as judged by gel mobility shift assays. Stimulation of B cells to enter S phase of the cell cycle by treatment with the combination of phorbol ester plus calcium ionophore also stimulated nuclear TRE-binding activity within 2 h, with maximal expression at 4 h; however, phorbol ester and calcium ionophore were not as effective in stimulating binding activity when examined separately. Stimulated nuclear expression of TRE-binding activity appears to require protein synthesis. Fos- and Jun/AP-1-related proteins participate directly in the identified nucleoprotein complex, as shown by the ability of c-fos- and c-jun-specific antisera to either alter or completely abolish electrophoretic migration of the complex in native gels. Further, UV photo-cross-linking studies identified two major TRE-binding protein species, whose sizes correspond to TRE-binding proteins derived from HeLa cell nuclear extracts. The results suggest that in primary B cells nuclear TRE-binding activity represents a "downstream" signaling event that occurs subsequent to changes in protein kinase C activity and intracellular Ca2+ but that can be triggered "physiologically" through sIg.