Quasi-real-time range monitoring by in-beam PET: a case for 15O.

A fast and reliable range monitoring method is required to take full advantage of the high linear energy transfer provided by therapeutic ion beams like carbon and oxygen while minimizing damage to healthy tissue due to range uncertainties. Quasi-real-time range monitoring using in-beam positron emission tomography (PET) with therapeutic beams of positron-emitters of carbon and oxygen is a promising approach. The number of implanted ions and the time required for an unambiguous range verification are decisive factors for choosing a candidate isotope. An experimental study was performed at the FRS fragment-separator of GSI Helmholtzzentrum für Schwerionenforschung GmbH, Germany, to investigate the evolution of positron annihilation activity profiles during the implantation of [Formula: see text]O and [Formula: see text]O ion beams in a PMMA phantom. The positron activity profile was imaged by a dual-panel version of a Siemens Biograph mCT PET scanner. Results from a similar experiment using ion beams of carbon positron-emitters [Formula: see text]C and [Formula: see text]C performed at the same experimental setup were used for comparison. Owing to their shorter half-lives, the number of implanted ions required for a precise positron annihilation activity peak determination is lower for [Formula: see text]C compared to [Formula: see text]C and likewise for [Formula: see text]O compared to [Formula: see text]O, but their lower production cross-sections make it difficult to produce them at therapeutically relevant intensities. With a similar production cross-section and a 10 times shorter half-life than [Formula: see text]C, [Formula: see text]O provides a faster conclusive positron annihilation activity peak position determination for a lower number of implanted ions compared to [Formula: see text]C. A figure of merit formulation was developed for the quantitative comparison of therapy-relevant positron-emitting beams in the context of quasi-real-time beam monitoring. In conclusion, this study demonstrates that among the positron emitters of carbon and oxygen, [Formula: see text]O is the most feasible candidate for quasi-real-time range monitoring by in-beam PET that can be produced at therapeutically relevant intensities. Additionally, this study demonstrated that the in-flight production and separation method can produce beams of therapeutic quality, in terms of purity, energy, and energy spread.

Precision of the PET activity range during irradiation with10C,11C, and12C beams.

Objective. Beams of stable ions have been a well-established tool for radiotherapy for many decades. In the case of ion beam therapy with stable12C ions, the positron emitters10,11C are produced via projectile and target fragmentation, and their decays enable visualization of the beam via positron emission tomography (PET). However, the PET activity peak matches the Bragg peak only roughly and PET counting statistics is low. These issues can be mitigated by using a short-lived positron emitter as a therapeutic beam.Approach.An experiment studying the precision of the measurement of ranges of positron-emitting carbon isotopes by means of PET has been performed at the FRS fragment-separator facility of GSI Helmholtzzentrum für Schwerionenforschung GmbH, Germany. The PET scanner used in the experiment is a dual-panel version of a Siemens Biograph mCT PET scanner.Main results.High-quality in-beam PET images and activity distributions have been measured from the in-flight produced positron emitting isotopes11C and10C implanted into homogeneous PMMA phantoms. Taking advantage of the high statistics obtained in this experiment, we investigated the time evolution of the uncertainty of the range determined by means of PET during the course of irradiation, and show that the uncertainty improves with the inverse square root of the number of PET counts. The uncertainty is thus fully determined by the PET counting statistics. During the delivery of 1.6 × 107ions in 4 spills for a total duration of 19.2 s, the PET activity range uncertainty for10C,11C and12C is 0.04 mm, 0.7 mm and 1.3 mm, respectively. The gain in precision related to the PET counting statistics is thus much larger when going from11C to10C than when going from12C to11C. The much better precision for10C is due to its much shorter half-life, which, contrary to the case of11C, also enables to include the in-spill data in the image formation.Significance. Our results can be used to estimate the contribution from PET counting statistics to the precision of range determination in a particular carbon therapy situation, taking into account the irradiation scenario, the required dose and the PET scanner characteristics.

Induction of autoimmunity in a transgenic model of B cell receptor peripheral tolerance: changes in coreceptors and B cell receptor-induced tyrosine-phosphoproteins.

Abrogation of peripheral tolerance in transgenic mice that express a uniform B-cell receptor may create a powerful tool to examine the molecular mechanisms that underlie the autoimmune response in B cells. Here we report that processes that induce a systemic lupus erythematosus-like syndrome in normal mice, namely chronic graft vs host reaction, trigger systemic autoimmunity in a well-established transgenic mice model of B cell receptor peripheral tolerance. The induction of graft vs host reaction in mice that carry both a rearranged B cell Ag receptors specific for hen egg lysozyme and expressing chronically circulating hen egg lysozyme Ag resulted in induction of high and sustained levels of circulating anti-hen egg lysozyme autoantibodies and glomerulonephritis with proteinuria. This was associated with marked changes in expression of cell-surface proteins, such as CD23 and complement receptor 2. B cells from the graft vs host-induced mice could proliferate in vitro in response to self-Ag, and upon stimulation with anti-IgD demonstrated rapid phosphotyrosine phosphorylation of specific proteins, which could not be induced in the anergic double transgenic B cells. Conversely, loss of tolerance was not associated with a higher induction in the level of Syk kinase phosphorylation following stimulation with anti-IgD. Taken collectively, these data establish that 1) processes that induce a systemic lupus erythematosus-like syndrome in normal mice can abrogate peripheral tolerance in transgenic mice expressing self-tolerized B cells, and that 2) loss of tolerance in this model is associated with marked changes in surface expression of B cell coreceptors as well as with selective changes in IgD-induced signaling by discrete tyrosine-phosphoproteins, but not Syk kinase.

Association of activating transcription factor 2 (ATF2) with the ubiquitin-conjugating enzyme hUBC9. Implication of the ubiquitin/proteasome pathway in regulation of ATF2 in T cells.

Activating transcription factor 2 (ATF2) is regulated by phosphorylation via the Jun N-terminal kinase, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V. (1996) J. Immunol. 156, 4582-4593). To identify proteins that interact specifically with ATF2 in lymphocytes, the yeast two-hybrid interaction system was employed using ATF2 cDNA as a "bait." In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the ubiquitin-conjugating enzyme family. An identical sequence was recently reported as the human homolog of the yeast UBC9, hUBC9. Northern blot analysis revealed a 1.3-kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes. An antibody that was generated against the bacterially expressed glutathione S-transferase-hUBC9 detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells. Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of hUBC9 with ATF2 and lack of interaction with lamin or control vectors. Two other cyclic AMP-responsive element-binding transcription factors, CREB and ATF1, also showed significant levels of interaction with hUBC9. However, this interaction was severalfold lower as compared with ATF2. Far Western blot analysis confirmed the specific binding of ATF2 and hUBC9 also in vitro. Evidence is presented that indicates a physiological significance for the interaction of hUBC9 with ATF2. (a) We show that ATF2 is ubiquitinated in vivo and in vitro, and (b) ATF2 ubiquitination in vitro is facilitated by addition of purified hUBC9. (c) ATF2 is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of ATF2 phosphorylation. (d) A proteasome inhibitor delays the down-regulation of ATF2 phophorylation after T cell activation. Taken collectively, these results implicate a role for hUBC9 and the ubiquitin/proteasome pathway in regulation of ATF2 in T cells.

Late induction of CREB/ATF binding and a concomitant increase in cAMP levels in T and B lymphocytes stimulated via the antigen receptor.

Transcription factors of the cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family were implicated in the expression of T cell-specific genes and in the expression of oncogenic retroviruses associated with leukemia in T and B lymphocytes. To study the regulation of CREB/ATF transcription factors during lymphocyte activation, studies were pursued in primary cultures of resting murine splenic T and B lymphocytes stimulated via the Ag receptor. Using consensus/CRE and proliferating cell nuclear Ag (PCNA)/CRE as probes in the DNA binding assay, we showed that a marked induction of CRE binding is associated with activation of splenic T lymphocytes with anti-CD3 Ab. CRE binding was markedly induced after 48 h; it gradually declined at 72 h, but remained elevated above control levels after 120 h. Most significant, activation by anti-CD3 was associated with a marked induction of cAMP levels that preceded the onset of DNA synthesis and the induction of IL-2 secretion and reached a peak after 48 h (9.5- to 11-fold), concomitant with the peak in CRE binding. Rapamycin, a potent immunosuppressant, inhibited the induction of cAMP levels by anti-CD3 concomitant with inhibition of CRE binding activity and arrest of DNA synthesis. A marked induction in CRE binding after 48 h was also found in splenic B lymphocytes stimulated by LPS and anti-Ig and was correlated with a 3- to 4-fold increase in the intracellular levels of cAMP. Two inducible CRE complexes were found to bind to consensus/CRE and PCNA/CRE; the major complex contained primarily CREB homodimers and was constitutively expressed in resting lymphocytes. Conversely, stimulation of lymphocytes was associated with formation of a new, slow migrating CRE complex that demonstrated high inducibility in both consensus/CRE and PCNA/CRE. We show that this de novo inducible CRE complex contains CREB and ATF2, but not ATF1. Taken collectively, these results suggest that recruitment of CREB and ATF2 to the promoter of genes is tightly regulated during activation of T and B lymphocytes and implicate a cross-talk of cAMP and non-cAMP pathways in the regulation of transcriptional processes at late stages of activation in T and B lymphocytes stimulated via the Ag receptor.

Rapamycin selectively blocks interleukin-2-induced proliferating cell nuclear antigen gene expression in T lymphocyte. Evidence for inhibition of CREB/ATF binding activities.

The macrolide rapamycin arrests T lymphocytes stimulated by interleukin-2 (IL-2) at G1/S. We have recently found that IL-2 induced an increase in the binding of discrete transcription factors of the ATF/cAMP-responsive element binding factor (CREB) family at G1/S, and that this effect was inhibited by rapamycin (Feuerstein, N., Huang, D., Hinrichs, S. H., Orten, D. J., Aiyar, N., and Prystowsky, M. B. (1995) J. Immunol. 154, 68-79). We now show, by using high resolution two-dimensional gel electrophoresis, that rapamycin inhibited selectively the synthesis of three discrete IL-2-induced soluble proteins (35 kDa/pI approximately 5, 68 kDa/pI approximately 4, 110 kDa/pI approximately 4.3). Analysis of nuclear proteins demonstrated that rapamycin selectively blocked the expression of proliferating cell nuclear antigen (PCNA), an obligate cofactor of DNA polymerase-delta, an important component for DNA replication. Rapamycin inhibited the IL-2-induced PCNA mRNA, and the murine PCNA promoter activity in IL-2-stimulated cells. Inducible CRE-binding proteins were shown previously to be required for PCNA promoter activity in IL-2-stimulated T lymphocytes. Using DNA binding gel mobility shift assay we demonstrated that rapamycin potently inhibited the binding of CREB/ATF transcription factors to CRE elements in the murine proximal PCNA promoter. These results suggest that PCNA is a preferred target in a rapamycin-sensitive transduction pathway, and that the mechanism by which rampamycin inhibits PCNA gene expression may involve the inhibition of the interaction of CREB/ATF transcription factors with CRE elements in the proximal PCNA promoter.

PKC activity and protein phosphorylation in regulation of sIg mediated B cell activation.

The inhibitory and stimulatory elements of cellular signalling associated with activation of protein kinase C (PKC) in murine B lymphocytes were investigated by employing two PKC activators with opposing effects on cell proliferation. Being an inhibitor of anti-Ig mediated proliferation, the phorbol ester PDBU induced a more substantial translocation of cytosolic PKC activity than the alkaloid PKC activator indolactam, which enhances anti-Ig mediated B cell proliferation. PDBU and indolactam were equally effective kinase activators, as determined by 32P incorporation of the substrate proteins. Concentrations of indolactam which induced an inhibition of anti-Ig mediated B cell proliferation also induced a precipitous decline in detergent soluble cellular PKC activity, which was comparable with 1 microM PDBU. The induced phosphoprotein patterns were similar, with an exception of the nuclear envelope protein lamin B, which was prominently phosphorylated by PDBU but not by stimulatory concentrations of indolactam. The enhanced phosphorylation of lamin B was associated with cellular growth arrest: inhibitory concentrations of indolactam induced the phosphorylation of lamin B equal to PDBU, whereas an increased phosphorylation of lamin B was never observed upon stimulation with anti-Ig. Together, inhibition of anti-Ig mediated B cell proliferation was related to down-regulation of cytoplasmic PKC and induction of nuclear PKC-dependent phosphorylation.

Regulation of cAMP-responsive enhancer binding proteins during cell cycle progression in T lymphocytes stimulated by IL-2.

IL-2 stimulates the proliferative response of various lymphoid cells. Previous studies showed an increase in intracellular levels of cAMP concomitant with an increase in phosphorylation of discrete proteins by protein kinase A at late G1 phase in mitogen-stimulated lymphocytes. Thus, experiments were undertaken to study nuclear proteins that bind to the cAMP-responsive enhancer (CRE) in cloned T lymphocytes stimulated with IL-2. With the use of a 32P-labeled CRE consensus sequence in a DNA binding gel mobility shift assay, we showed that IL-2 stimulation resulted in the induction of two major DNA-protein complexes at late G1/S during the cell cycle. This binding was competed in a dose-dependent manner by a nonlabeled CRE oligonucleotide but was not competed by a nonlabeled AP-1 oligonucleotide. Rapamycin, a potent immunosuppressant, which arrests IL-2-stimulated T lymphocytes at G1/S, inhibited the IL-2-induced CRE binding activities concomitantly with inhibition of DNA synthesis. By using specific Abs in a gel mobility shift assay, we identified two known CREB/ATF transcription factors in the IL-2-induced CRE complexes: the CRE binding factor (CREB), and ATF1. The induction of CREB binding by IL-2 was not associated with an increase in its abundance but was associated with a major increase in CREB phosphorylation that was particularly prominent at late G1/S. However, we found that G1/S progression induced by IL-2 was not associated with an increase in the intracellular levels of cAMP. These results suggest that 1) the transcription factors CREB and ATF1 and possibly other CRE binding proteins may have an important role in the modulation of specific gene expression at G1/S during cell cycle progression induced by IL-2. 2) The involvement of these CRE binding transcription factors in IL-2-stimulated cells is regulated via a mechanism that is not cAMP dependent.

Interleukin-2-induced lung injury. The role of complement.

Pulmonary edema and sepsis-like syndrome are grave complications of interleukin-2 (IL-2) therapy. Recent animal studies have suggested IL-2-induced microvascular injury as the underlying mechanism. Since complement factors have been shown to mediate increased vascular permeability in diverse conditions that lead to pulmonary injury and recombinant human IL-2 is known to activate the complement system in patients undergoing IL-2 therapy, we hypothesized that complement factors play a pivotal role in the development of increased vascular permeability after IL-2 treatment. To test this hypothesis, we evaluated the capacity of recombinant soluble human complement receptor type 1 (sCR1, BRL 55730), a new highly specific complement inhibitor, to attenuate IL-2-induced lung injury in the rat. Recombinant human IL-2 (intravenously for 60 minutes) at 10(6) U per rat (n = 4) elevated lung water content (37 +/- 6%, P < .05), myeloperoxidase activity (162 +/- 49%, P < .05), and serum thromboxane B2 (30 +/- 1 pg/100 microL, P < .01) and had no effect on serum tumor necrosis factor-alpha sCR-1 at 30 mg/kg (n = 5), but not at 10 mg/kg (n = 6), attenuated the elevation of lung water content (18 +/- 2%, P < .05) and myeloperoxidase activity (42 +/- 9%, P < .05) but failed to alter serum thromboxane B2 response to IL-2. These data suggest the involvement of complement in the pathogenesis of IL-2-induced pulmonary microvascular injury and point to the potential therapeutic capacity of complement inhibitors in combating this toxic effect of IL-2 therapy.

Phosphorylation of numatrin and other nuclear proteins by cdc2 containing CTD kinase cdc2/p58.

Numatrin is a nuclear matrix phosphoprotein whose synthesis and abundance were shown to be regulated during the cell cycle in mitogen-stimulated lymphocytes (Feuerstein, N., and Mond, J. (1987) J. Biol. Chem. 262, 11389-11397). We examined the effect of (a) CTD-kinase, which contains the cdc2 catalytic component (p34) in a complex with a p58 subunit (cdc2/p58) and (b) the M phase-specific histone H1 kinase, which contains the cdc2 kinase in association with a p62 subunit (cdc2/p62), on phosphorylation of numatrin. We show that both cdc2 kinase complexes can phosphorylate numatrin. However, cdc2/p58 at conditions that caused a similar effect to cdc2/p62 on phosphorylation of histone H1 (dpm/micrograms of substrate/micrograms of enzyme) was found to have a 5-25-fold higher catalytic activity in the phosphorylation of numatrin. Analysis of the tryptic phosphopeptide map of numatrin phosphorylated by these cdc2 kinase complexes showed that both kinase complexes phosphorylated two major identical peptides, but minor additional peptides were differentially phosphorylated by each of these kinases. This indicates that under certain experimental conditions cdc2/p58 and cdc2/p62 may express some differences in their catalytic activity. In vitro phosphorylation by CTD kinase of a whole nuclear protein extract from murine fibroblasts showed that numatrin is the most prominent substrate for CTD kinase in this nuclear extract. CTD kinase cdc2/p58 was found to induce significantly the phosphorylation of five other discrete nuclear substrates. Particularly, two nuclear proteins at 75 kDa/pI approximately 6.5 and 85 kDa/pI approximately 5.3, which were not Coomassie Blue stainable, were found to be markedly phosphorylated by CTD kinase. The results of this study call for further study of the role of CTD kinase cdc2/p58 in the phosphorylation of numatrin under physiological conditions and to further characterization of the other nuclear substrates for CTD kinase.

In vivo and in vitro phosphorylation studies of numatrin, a cell cycle regulated nuclear protein, in insulin-stimulated NIH 3T3 HIR cells.

Numatrin, a nuclear matrix protein has been implicated to be involved in mitogenesis of normal and malignant cells (Feuerstein and Mond, J. Biol. Chem. 262, 11389, 1987) and was later found to be identical to the nuclear phosphoprotein, B23. To study whether phosphorylation of numatrin is regulated by mitogenic stimulation, we examined the effect of phosphorylation of numatrin in the insulin-responsive cells, NIH 3T3 HIR. We found that an increase in phosphorylation of numatrin was associated with stimulation of the cells with insulin for 4 h and that the level of phosphorylation remained elevated after 8 h. By this time there was no increase in numatrin abundance as shown by Coom-massie blue stain and Western blot analysis. The induction in phosphorylation of numatrin could not be detected after 30 min stimulation with insulin, thus, indicating that the increase in phosphorylation of numatrin is not a rapid event. Analysis of the phosphopeptides by thin layer chromatography indicated four peptides that were phosphorylated in numatrin (one major and three minor). Stimulation with insulin was associated primarily with an increase in phosphorylation of the minor phosphopeptides. The phosphopeptide map of numatrin was identical after 4, 8, 17, 24, and 32 h stimulation with insulin, indicating that identical sites are phosphorylated at different phases of the cell cycle. In a search for the protein kinase which is involved in phosphorylation of numatrin we found that numatrin is a most prominent substrate for the cell cycle regulated cdc2 (p 34) kinase. However, the major phosphopeptides which were phosphorylated by this kinase did not comigrate with either of the phosphopeptides phosphorylated in insulin-stimulated intact cells. This may indicate that it is unlikely that cdc2 kinase may account for the mechanism(s) associated with phosphorylation of numatrin by insulin under physiological conditions.

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.

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.

Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein.

Stimulation of various cell types with growth factors is associated with a rapid induction in the synthesis of a nuclear matrix protein, termed 'numatrin' which was shown to be identical to the nucleolar protein B23. The abundance of numatrin was shown to be correlated with entry and progression through the S-phase. Thus, experiments were undertaken to examine whether numatrin also has DNA binding activity. Using whole nuclear extract, we showed that numatrin binds to both double-stranded (DS) DNA and to single-stranded (SS) DNA cellulose columns. Purified numatrin, which was extracted either under native conditions (in oligomeric form) or under urea conditions (in monomeric form), demonstrated significant binding to either [3H]DS-DNA or [3H]DS-DNA as shown by nitrocellulose filter binding assay. However, numatrin binding to DS-DNA was qualitatively and quantitatively different from its binding to SS-DNA. Thus, the binding of numatrin was several fold higher to DS-DNA as compared to SS-DNA. The binding to DS-DNA was reduced by 77% in the presence of 0.5 M NaCl, while the binding to SS-DNA was not affected under this condition. Furthermore, treatment of the native numatrin under conditions which caused monomerization of the protein resulted in a significant enhancement of numatrin binding to SS-DNA but did not affect the binding to DS-DNA. Following heparin-Sepharose chromatography purification (under native conditions), numatrin at picomole amounts showed significant binding to both DS-DNA and SS-DNA. Finally, numatrin was found to copurify with the complex of DNA polymerase alpha primase together with other proteins required for SV-40 in vitro replication activity. These results demonstrate that numatrin has DNA binding activity, and imply a possible role for numatrin/B23 in DNA-associated processes.

Protein kinase C activation in B cells by indolactam inhibits anti-Ig-mediated phosphatidylinositol bisphosphate hydrolysis but not B cell proliferation.

In order to examine the role of phosphatidylinositol bisphosphate (PIP2) hydrolysis in B cell activation, we studied the effect of various classes of protein kinase C (PKC) activators on anti-Ig-mediated B cell stimulation. Anti-Ig-stimulated PIP2 hydrolysis, elevations in [Ca2+]i, and induction of DNA synthesis were inhibited by PMA (a phorbol ester) as previously reported. In contrast, indolactam (an alkaloid PKC activator) inhibited PIP2 hydrolysis and elevations in [Ca2+]i, but stimulated rather than inhibited cellular proliferation. In order to examine whether the binding avidity of the PKC activators to PKC played a role in determining their activity to stimulate or inhibit B cell activation, we studied two other PKC activators, bryostatin and mezerein. Again, both inhibited anti-Ig mediated PIP2 hydrolysis and elevations in [Ca2+]i, whereas only the former inhibited induction of DNA synthesis. These data suggest that a) high levels of PIP2 hydrolysis and elevations in [Ca2+]i are not essential for anti-Ig-mediated induction of B cell DNA synthesis and b) activation of PKC may induce both stimulatory and inhibitory pathways of B cell activation, and whether stimulation or inhibition of cell activation is observed may depend on the combined intensity of these two signals.

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)

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.

Anti-Ig-mediated proliferation of human B cells in the absence of protein kinase C.

Cross-linking of surface Ig has been shown to stimulate phosphatidylinositol hydrolysis in murine B cells, leading to increases in [Ca2+]i and activation of protein kinase C (PKC). Preliminary evidence suggests that a similar activation mechanism occurs in human B cells. We wished to examine whether anti-Ig antibody-stimulated human B cell proliferation is as dependent upon the presence of PKC as is anti-Ig-mediated murine B cell proliferation. Using highly purified, small, dense peripheral-blood B lymphocytes from healthy adult donors, we confirmed that PMA, a direct activator of PKC, is a potent mitogen for human B cells that synergizes with anti-mu antibody. Furthermore, we demonstrated that PMA treatment abolishes detectable cellular stores of immunoreactive PKC. However, after such depletion of cellular PKC, anti-mu antibody is still capable of delivering a proliferative signal to human B cells. It is unlikely that this signal occurs solely on the basis of increases in [Ca2+]i, because the calcium ionophore A23187 does not induce a proliferative response in PMA-treated B cells similar in magnitude to that seen with anti-mu. Additionally, the finding that pretreatment of B cells with PMA ablates the ability of anti-Ig antibody to mobilize intracellular and extracellular calcium also suggests that the ability of PMA to enhance anti-Ig mediated stimulation does not depend on elevations of [Ca2+]i induced by anti-Ig. Together, these observations suggest that anti-Ig signaling of human B cells may occur via other pathways in addition to the phosphatidylinositol system of calcium influx and PKC activation.

B-lymphocyte activation mediated by anti-immunoglobulin antibody in the absence of protein kinase C.

B-cell activation induced by crosslinking of surface immunoglobulin is known to stimulate hydrolysis of phosphatidylinositol to diacylglycerol and inositol trisphosphate. We now provide evidence that alternative pathways of activation may also be recruited during such activation. We utilized depletion of protein kinase C activity to determine whether this enzyme is required under all conditions for anti-immunoglobulin-stimulated B-cell activation. Although anti-immunoglobulin does not induce B-cell proliferation in protein kinase C-depleted cells, it stimulates an earlier event in B-cell activation as reflected by its ability to enhance the expression of major histocompatibility complex-encoded class II molecules. Furthermore, the ribonucleoside 8-mercaptoguanosine restores the ability of anti-immunoglobulin to induce B-cell proliferation in protein kinase C-depleted cells. This restoration is also demonstrated by an enhancement of synthesis of a nuclear protein that we find is increased during B-cell mitogenesis. These results indicate that B-cell activation stimulated by anti-immunoglobulin may recruit pathways in addition to the one dependent on protein kinase C.

Identification of a prominent nuclear protein associated with proliferation of normal and malignant B cells.

Experiments were undertaken to identify nuclear proteins that might be involved in regulation of the mitogenic process in B lymphocytes. Murine splenic B lymphocytes were purified and cultured with anti-Ig insolubilized onto Sepharose (anti-Ig/Sepharose) for 16 hr and labeled with [35S]methionine. Nuclei were isolated and the nuclear proteins were analyzed by two-dimensional gel electrophoresis. Anti-Ig/Sepharose induced a prominent increase in the synthesis and abundance of a 40 kDa/pI 5 nuclear protein (p40/pI-5). Inhibition of anti-Ig/Sepharose-induced mitogenesis by pretreatment of the cells with phorbol-12-myristate-13-acetate was associated with a specific inhibition (63%) of p40/pI-5. Subcellular fractionation experiments showed that p40/pI-5 is not detected in the soluble fraction of resting or activated B cells, indicating that this protein is located exclusively in the nucleus. Analysis of the expression of p40/pI-5 relative the cell cycle showed that the synthesis of this protein was increased during G1 phase and gradually reduced during S phase of the cell cycle. Abundant amounts of p40/pI-5 were also found in the rapidly proliferating B lymphoma cells, WEHI-231, and growth arrest of these cells by anti-mu was found to be associated with a marked inhibition (68%) of this protein. Taken collectively these results suggest that the nuclear protein p40/pI-5 may have an important role in regulation of the proliferation of normal and malignant B lymphocytes.