Degradation of cyclin D3 independent of Thr-283 phosphorylation.

Cyclin D3 has been shown to play a major role in the regulation of cell cycle progression in lymphocytes. It is therefore important to understand the mechanisms involved in the regulation of this protein. We have previously shown that both basal and cAMP-induced degradation of cyclin D3 in Reh cells is dependent on Thr-283 phosphorylation by glycogen synthase kinase-3beta (GSK-3beta). We now provide evidence of an alternative mechanism being involved in the regulation of cyclin D3 degradation. Treatment of lymphoid cells with okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), induces rapid phosphorylation and proteasomal degradation of cyclin D3. This degradation is not inhibited by the GSK-3beta inhibitors lithium or Kenpaullone, or by substitution of Thr-283 with Ala on cyclin D3, indicating that cyclin D3 can be degraded independently of Thr-283 phosphorylation and GSK-3beta activity. Interestingly, in vitro experiments revealed that PP1, but not PP2A, was able to dephosphorylate cyclin D3 efficiently, and PP1 was found to associate with His-tagged cyclin D3. These results support the hypothesis that PP1 constitutively keeps cyclin D3 in a stable, dephosphorylated state, and that treatment of cells with OA leads to phosphorylation and degradation of cyclin D3 through inhibition of PP1.

Vitamin A regulates proliferation and apoptosis of human T- and B-cells.

Vitamin A is known to protect against infections, but it is not established how vitamin A metabolites stimulate the immune system. We have investigated the effects of physiological levels of retinoic acid on the function of normal human T- and B-cells. Surprisingly, we found that the proliferation of B-cells was inhibited by retinoids, and that this was due to rapid inhibition of the cell cycle machinery regulating G(1)-to-S transition. In contrast, the proliferation of T-cells was enhanced by physiological levels of retinoic acid, and the effect was due to induction of IL-2 (interleukin 2). The 'non-death-receptor'-mediated apoptosis of normal T-cells induced by prolonged (but single) stimulation of the cells was also prevented by retinoid acid, and also this effect was mediated via enhanced production of IL-2. The induction of IL-2 was at the transcriptional level, and all the effects of vitamin A on both B-and T-cells were mediated via the nuclear retinoic acid receptors (RARs), and not retinoid X receptors (RXRs).

[Evaluation of the new doctoral degree program at the Faculty of Medicine in Oslo 1993-98]. / Evaluering av doktorgradsprogrammet ved Det medisinske fakultet i Oslo 1993-98.

BACKGROUND: In 1993, the doctoral degree programme in the Faculty of Medicine of the University of Oslo was substantially revised to include coursework and supervision of thesis work. PhD students were expected to complete their work towards the doctorate in three years, and funding was only provided for this period. MATERIAL AND METHODS: In spring 1999, all doctoral candidates, their supervisors and members of the adjudicating committees were invited to reply to a questionnaire with the purpose of evaluating the results of the new programme over the 1993-99 period. RESULTS: Only a few doctoral students had been able to obtain their degrees in three years, the defined length of the programme. The mean age for new PhDs was 38; however, physicians obtained their PhD at a later age than the other life scientists enrolled in the programme, and the percentage of PhDs with a medical background declined from 71% in 1993-95 to 51% in 1996-98. INTERPRETATION: The doctoral programme should be extended from three to four years. More physicians should go into research soon after graduating from medical school and more openings for postdocs should be created. More time for research in the university clinics is also needed.

Expression of a novel factor, com1, is regulated by 1,25-dihydroxyvitamin D3 in breast cancer cells.

Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumor growth and metastasis. We recently identified a nuclear factor, termed com1, that is up-regulated in human breast carcinoma cells on formation of experimental metastatic tumors and is assumed to act as a growth-promoting factor in breast cancer. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is a potent inhibitor of growth in breast cancer both in vitro and in vivo. We compared the growth-regulatory mechanisms of nontumorigenic and estrogen-dependent MCF-7 cells with those of the tumorigenic and tamoxifen-resistant subline MCF7/ LCC2 in the presence of 1,25(OH)2D3. Proliferation of MCF7/LCC2 cells, which revealed constitutive com1 expression, was inhibited by 1,25(OH)2D3 (10(-7) M). This was strongly associated with cell cycle arrest in G1 phase, consistent with accumulation of the hypophosphorylated form of the retinoblastoma protein as well as the induction of the cyclin-dependent kinase inhibitor p21. These cell cycle events were preceded by a transient up-regulation (5-8-fold) of com1 mRNA. Furthermore, clonal growth of the MCF7/LCC2 cells was also inhibited by 1,25(OH)2D3 (10(-7) M), and when the com1-negative MCF-7 cells were stably transfected with com1, the resulting MCF7/com1 cells showed a significant decrease in colony formation. These results seem to indicate that rather than promoting growth, com1 may participate in the regulatory pathway involved in cellular growth inhibition when recruited by inhibitory signals.

Differential expression of bcl-2 homologs in human CD34(+) hematopoietic progenitor cells induced to differentiate into erythroid or granulocytic cells.

The Bcl-2 family of proteins has been shown to play a central role in the regulation of apoptosis. We have examined the expression of several Bcl-2 homologs upon stimulation of CD34(+) human hematopoietic progenitor cells. CD34(+) cells were induced to differentiate into predominantly erythroid cells in the presence of erythropoietin (Epo) and stem cell factor (SCF), while the addition of G-CSF and SCF led to differentiation predominantly into granulocytic cells, as demonstrated by immunophenotyping and morphological examination of cultured cells. In Epo- and SCF-stimulated cells, we found a marked increase in the level of Bcl-x(L) protein expression and downregulation of Bax expression, apparent from day 4 and more pronounced on days 8 and 21. In contrast, Bcl-x(L) protein expression was downregulated in G-CSF- and SCF-stimulated cells compared with cells cultured in medium alone, whereas there was no sign of change in the level of Bax. Mcl-1 expression showed a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl-1 levels initially decreased in granulocytic progenitor cells and increased in erythroid progenitor cells. Finally, Bcl-2 expression was significantly downregulated in both Epo and SCF and G-CSF- and SCF-stimulated cells. The role of the distinct upregulation of Bcl-x(L) in early erythroid differentiation was further examined by use of specific ribozymes against Bcl-x(L). Addition of Bcl-x(L) ribozymes promoted a clear increase in cell death of Epo- and SCF-stimulated cells, while erythroid differentiation was not affected. In conclusion, we found a distinct regulation of several Bcl-2 family members in CD34(+) cells dependent on the cytokine stimulation given. The use of Bcl-x(L)-specific ribozymes suggested that Bcl-x(L) is important for survival but not for differentiation of erythroid progenitor cells.

cAMP-mediated growth inhibition of lymphoid cells in G1: rapid down-regulation of cyclin D3 at the level of translation.

cAMP is an important physiological mediator of lymphoid growth inhibition. The purpose of the present study was to establish the link between cAMP and the cell cycle machinery leading to inhibition of G1/S transition in human peripheral blood lymphocytes (PBL). To unravel immediate effects of cAMP on this part of the cell cycle machinery, lymphocytes were synchronized in mid to late G1 after stimulation with phytohemaglutenin (PHA) for 32 h. We report that addition of forskolin or cAMP analogues to the cells resulted in dephosphorylation of retinoblastoma protein commencing as early as 30 min. A rapid effect of forskolin was noted on the activity of cyclin-dependent kinase (cdk) 4, which decreased significantly within 30 min of treatment. The decrease in cdk4 activity was concurrent with reduced levels of cyclin D3 protein and a decrease in the fraction of cdk4 associated with cyclin D3. The down-regulation of cyclin D3 was at the level of translation, and this event was preceded by a pronounced inhibition of Akt/protein kinase B phosphorylation at Ser 473. Taken together, our data imply that cyclin D3 is a major effector of cAMP-mediated inhibition of cell cycle progression in PBL, and that cAMP exerts its effect on cyclin D3 expression at the level of translation.

Co-induction of Mad1 and c-Myc in activated normal B lymphocytes.

The purpose of the present study was to examine the expression of the Myc network proteins c-Myc, Mad1 and Max in normal cells under different growth and differentiation conditions. A dominant view has been that Mad1 as a c-Myc antagonist plays a role in growth inhibition linked to differentiation. Of particular interest to us was therefore to study the regulation of Mad1 in cells undergoing differentiation in the absence of growth cessation. To do so we utilized normal B lymphocytes isolated from peripheral blood. The cells were induced to concomitant proliferation and differentiation by stimulation with a combination of anti-IgM antibodies (anti-mu) and the phorbol ester TPA. Thus, by 72 h of stimulation the percentage of plasmablasts increased from 3 to 17%, and the percentage of lymphocytes decreased from 89 to 27%. The most intriguing observation we made using this cell system was a pronounced coinduction of Mad1 and c-Myc. The levels of c-Myc and Mad1 mRNAs and proteins increased within 3 h of anti-mu stimulation, and the levels were further enhanced by TPA. Furthermore, the expressions of both c-Myc and Mad1 were reduced by forskolin, which also inhibited the anti-mu + TPA driven growth and differentiation of the B lymphocytes. The level of Max remained virtually unchanged. Taken together, our results indicate that a high level of Mad1 in normal human B cells is linked to differentiation and not to growth inhibition. Furthermore, the results demonstrate that Mad1 and c-Myc are not necessarily expressed in a reciprocal manner, which underlines an independent role of Mad1 unrelated to its function as a c-Myc antagonist.

Lovastatin inhibits G1/S transition of normal human B-lymphocytes independent of apoptosis.

Lovastatin is a potent inhibitor of protein prenylation, and it has been reported to have pleiotropic cellular effects. In the present study we have elucidated the effects of lovastatin on cell cycle progression and apoptosis of normal human B-lymphocytes. When added to B-lymphocytes stimulated with anti-immunoglobulin (anti-mu) and SAC, lovastatin (20 microM) inhibited the cells in the late G1 phase of the cell cycle. Thus, no early activation parameters such as Ca(2+) flux or MYC induction were affected by lovastatin, whereas progression of cells into the second cell cycle as well as DNA synthesis was markedly reduced. We therefore examined the effects of lovastatin on components of the cell cycle machinery responsible for regulating the G1/S transition. We demonstrated that pRB phosphorylation, cdk2 activity needed for this phosphorylation, and the levels of cyclin A, D, and E were inhibited after 24 h of lovastatin treatment, while the levels of p27(Kip1) were elevated. There was no effect on p21(Cip1), cyclin D2, cdk4, and cdk6. These data are consistent with the cells being inhibited by lovastatin between 24 and 32 h into G1. Lovastatin added to stimulated B-cells in late G1 still inhibited the DNA synthesis by 60%, but at this point only minor effects were noted on the cell cycle machinery. We therefore looked for induced apoptosis as an explanation for reduced S-phase entry of the cells. However, despite the ability to enhance the apoptosis of unstimulated B-cells from 48 to 61% as judged by the TUNEL method, lovastatin only marginally affected apoptosis when administered to stimulated B-cells. Thus, it appears that accelerated apoptosis cannot account for the effect of lovastatin on cell cycle progression.

Fas ligand promotes cell survival of immature human bone marrow CD34+CD38- hematopoietic progenitor cells by suppressing apoptosis.

Fas (CD95, APO-1) is a member of the TNF receptor family, and engagement of Fas by its ligand, Fas ligand (FasL), can induce apoptotic death of Fas expressing cells. Signaling through Fas has previously been shown to induce apoptosis of CD34+ human hematopoietic progenitor cells after exposure to IFN-gamma or TFN-alpha. In contrast, we found that FasL promoted a significantly increased viability of primitive CD34+CD38- cells. Thus, incubation with FasL for 48 hours reduced cell death from 46 to 29% compared to cells cultured in medium alone as measured by propidium iodide (PI) incorporation (n = 8, p < 0.02). Inhibition of apoptosis was confirmed by morphological analysis and by the Nicoletti technique. Furthermore, by using a delayed addition assay at the single cell level we found that sFasL treatment had a direct viability-promoting effect on CD34(+)CD38(-) cells. The effect of sFasL was completely blocked by NOK-1, a neutralizing mAb against FasL. In agreement with previous reports, FasL alone slightly increased cell death of more mature CD34(-)CD38+ cells, indicating an interesting shift in the responsiveness to FasL during early hematopoiesis.

Retinoic acid prevents phosphorylation of pRB in normal human B lymphocytes: regulation of cyclin E, cyclin A, and p21(Cip1).

The mechanisms underlying the growth-inhibitory effect of retinoids on normal human B lymphocytes are not well understood. We addressed this issue by examining the effect of retinoic acid on the cell cycle machinery involved in G1/S transition. When retinoic acid was administered to B cells stimulated into mid to late G1 by anti-IgM antibodies (anti-mu) and Staphylococcus aureus crude cell suspension (SAC), the phosphorylation of pRB required for S-phase entry was prevented in a time- and dose-dependent manner. Thus, 2-hour treatment with retinoic acid at the optimal concentration of 1 micromol/L prevented phosphorylation of pRB, and effects were noted at concentrations as low as 10 nmol/L. Based on our results, we suggest that the rapid effect of retinoic acid on pRB phosphorylation is due primarily to the reduced expression of cyclin E and cyclin A in late G1. This could lead to the diminished cyclin E- and cyclin A-associated kinase activities noted as early as 2 hours after addition of retinoic acid. Furthermore, our results imply that the transient induction of p21(Cip1) could also be involved. Thus, retinoic acid induced a rapid, but transient increased binding of p21(Cip1) to CDK2. The retinoic acid receptor (RAR) agonist TTNPB mimicked the key events affected by retinoic acid, such as pRB phosphorylation, cyclin E expression, and expression of p21(Cip1), whereas the RAR-selective antagonist Ro 41-5253 counteracted the effects of retinoic acid. This implies that retinoic acid mediates its growth-inhibitory effect on B lymphocytes via the nuclear receptors.

Activation of the cAMP signaling pathway increases apoptosis in human B-precursor cells and is associated with downregulation of Mcl-1 expression.

During B- and T-cell ontogeny, extensive apoptosis occurs at distinct stages of development. Agents that increase intracellular levels of cAMP induce apoptosis in thymocytes and mature B cells, prompting us to investigate the role of cAMP signaling in human CD10+ B-precursor cells. We show for the first time that forskolin (which increases intracellular levels of cAMP) increases apoptosis in the CD10- cells in a dose-dependent manner (19%-94% with 0-1,000 microM forskolin after 48 hours incubation, IC50 = 150 microM). High levels of apoptosis were also obtained by exposing the cells to the cAMP analogue 8-chlorophenylthio-cAMP (8-CPT-cAMP). Specific involvement of cAMP-dependent protein kinase (PKA) was demonstrated by the ability of a cAMP antagonist, Rp-isomer of 8-bromo-adenosine- 3', 5'- monophosphorothioate (Rp-8-Br-cAMPS), to reverse the apoptosis increasing effect of the complementary cAMP agonist, Sp-8-Br-cAMPS. Furthermore, we investigated the expression of Bcl-2 family proteins. We found that treatment of the cells with forskolin or 8-CPT-cAMP for 48 hours resulted in a fourfold decline in the expression of Mcl-1 (n = 6, P = 0.002) compared to control cells. The expression of Bcl-2, Bcl-xL, or Bax was largely unaffected. Mature peripheral blood B cells showed a smaller increase in the percentage of apoptotic cells in response to 8-CPT-cAMP (1.3-fold, n = 6, P = 0.045) compared to B-precursor cells, and a smaller decrease in Mcl-1 levels (1.5-fold, n = 4, P = 0.014). Taken together, these findings show that cAMP is important in the regulation of apoptosis in B-progenitor and mature B cells and suggest that cAMP-increased apoptosis could be mediated, at least in part, by a decrease in Mcl-1 levels.

Retinoic acid induces apoptosis of human CD34+ hematopoietic progenitor cells: involvement of retinoic acid receptors and retinoid X receptors depends on lineage commitment of the hematopoietic progenitor cells.

Retinoids are bifunctional regulators of growth and differentiation of hematopoietic cells. In this study we explored the effects of retinoic acid (RA) on apoptosis of human CD34+ hematopoietic progenitor cells isolated from normal bone marrow. RA (100 nM) induced an increase in the percentage of dead cells from 24% to 44% at day 6 (p < 0.05, n = 6) as compared to control cells cultured in medium alone. The effect was dose dependent and appeared relatively late. Significant differences were observed from day 4 onward. Apoptosis, or programmed cell death, was demonstrated as the mode of cell death by using the TUNEL assay, which detects single strand nicks in DNA, or by the Nicoletti technique demonstrating a subdiploid population by DNA staining. RA previously was found to inhibit granulocyte colony-stimulating factor--and not granulocyte-macrophage colony-stimulating factor--stimulated proliferation of CD34+ cells. However, we found that RA opposed anti-apoptotic effects of G-CSF and GM-CSF on CD34+ cells (G-CSF: 8% dead cells at day 6; G-CSF + RA: 20%; GM-CSF: 12%; GM-CSF + RA: 27%). Moreover, RA induced apoptosis of CD34+ cells and CD34+CD71+ cells stimulated with erythropoietin. To explore the receptor signaling pathways involved in RA-induced apoptosis, we used selective ligands for retinoic acid receptors (RARs; RO13-7410) and retinoid X receptors (RXRs; RO 25-6603). We found that RARs were involved in RA-mediated apoptosis of myeloid progenitor cells, whereas RARs as well as RXRs were involved in RA-mediated apoptosis of erythroid progenitor cells.

Mad1 expression in the absence of differentiation: effect of cAMP on the B-lymphoid cell line Reh.

The protein Mad1 heterodimerizes with Max to form an E-box binding complex able to interfere with the transcriptional and transforming activities of c-Myc. Downregulation of c-Myc accompanied by induction of Mad1 upon differentiation has fueled the notion that Mad1 may play a role in the cessation of proliferation associated with the differentiation process. Since studies on Mad1 expression have so far been limited to cells undergoing differentiation, it was of interest to examine Mad1 expression in a cell system unable to differentiate. To do so, we utilized the leukemia-derived B-precursor cell line, Reh, and studied the expressions of Mad1, c-Myc, Mxil, and Max during cAMP-mediated growth inhibition of these cells. Thus, the adenylate cyclase activator forskolin induced growth inhibition of the cells in the G1 phase of the cell cycle. This growth inhibition was associated with transient increased expression of Mad1 concomitant with transient downregulation of c-Myc. The Mad1 protein levels essentially paralleled those of mRNA, with peak levels at 4 h of forskolin treatment. By coimmunoprecipitation we detected increased binding of Mad1 to Max in forskolin-treated cells, indicating that the changes in Mad1 protein levels had functional implications. By continually treating Reh cells with forskolin for 72 h, we observed a sustained elevated expression of Mad1 concomitant with downregulated c-Myc expression, still without changing the differentiation profile of the Reh cells. Interestingly, we showed that other known cell cycle regulatory proteins also were transiently regulated by forskolin. To this extent, following forskolin treatment of Reh cells, cyclin E-cdk2 activity was transiently reduced concomitant with dephosphorylation of pRB. We suggest that the early changes in Mad1 and the cell cycle regulatory proteins initiate a chain of events resulting in permanent growth arrest. Thus, the increased expression of Mad1 in the absence of differentiation indicates that Mad1 expression in Reh cells is linked to growth arrest per se.

Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines.

Activation of the cAMP signaling pathway in lymphoid cells is known to inhibit cell proliferation of T and B cells as well as cytotoxicity of natural killer (NK) cells. In order to find suitable model systems to study cAMP-mediated processes, we have examined the expression of cAMP-dependent protein kinase (PKA), endogenous levels of cAMP, and cell proliferation in eight cell lines of B lineage origin, four cell lines of T lineage origin, and normal human B and T cells. We demonstrated that the expression of mRNA and protein for one of the regulatory (R) subunits of PKA (RIalpha) was present in all the cells investigated, in contrast to the other R subunits (RIbeta, RIIalpha, and RIIbeta). Furthermore, three T cell lines and one B cell line expressed only RIalpha and C, implying these cells to contain solely PKA type I. Moreover, for the RI subunit, we observed an apparent reciprocal relationship between levels of mRNA and protein. Generally, RIalpha protein was low in cell lines where mRNA was elevated and vice versa. This was not the case for the RII subunits, where high levels of mRNA were associated with elevated levels of protein. Interestingly, we demonstrated an inverse correlation between levels of endogenous cAMP and cell growth as determined by [3H]-thymidine incorporation and cell-doubling rate (P < 0.05). Taken together, our results demonstrate great differences in PKA isozyme composition, which should be taken into consideration when using lymphoid cell lines as model system for cAMP/PKA effects in normal lymphocytes.

Multiplication and death-type of leukemia cell lines exposed to very long-chain polyunsaturated fatty acids.

Polyunsaturated fatty acids (PUFA) may reduce cell multiplication in cultures of normal, as well as transformed, white blood cells. We assessed the sensitivity of 14 different leukemia cell lines to PUFA by measuring cell number after 3 days of incubation. Ten of the examined cell lines were sensitive to 30, 60 and/or 120 microM of arachidonic, eicosapentaenoic and docosahexaenoic acid, whereas four cell lines were resistant. The sensitivity to PUFA was not associated with any particular cell lineage, clinical origin or specific mRNA pattern of bcl-2 and c-myc. Effects on cell viability were assessed by studying cell membrane integrity, DNA fragmentation and cell morphology. The sensitive cell lines Raji and Ramos died by necrosis and apoptosis, respectively, during incubation with eicosapentaenoic acid, whereas the viability of the resistant U-698 cell line was unaffected. The effects of EPA on Raji cells, was counteracted by vitamin E, indicating that lipid peroxidation was involved. However, apoptosis induced by eicosapentaenoic acid in Ramos cells, was unaffected by vitamin E, as well as eicosanoid synthesis inhibitors. In conclusion, our results indicate that a majority of leukemia cell lines are sensitive to PUFA. This sensitivity may be caused by induction of apoptosis or necrosis by very long-chain polyunsaturated fatty acids.

[Tumor suppressors--genes and proteins]. / Tumorsuppressorer--gener og proteiner.

Cancer is generally understood to be a genetic disease in the sense that somatic mutations are the cause of tumour initiation and development. Our knowledge of cancer-associated genes and gene products has evolved mainly over the past 20 years. The identification and characterization of tumour suppressor genes (TSGs) as normal growth-inhibiting or apoptosis-inducing genes have helped us to understand how mutations are tumorigenic. Various TSG encoding membrane-, cytosol-, or nuclear proteins have been identified. Tumor suppressor genes are often functionally inactive in cancer cells because of mutations of both parental gene copies. Many TSGs are associated with hereditary cancer diseases or syndromes caused by the existence of one mutant allele in the germ-line. Individuals who carry only one functional gene copy, are therefore at great risk of developing cancer. Several TSGs, such as TP53, RB1 and CDKN2A, encode proteins that are significant to the cell cycle. TP53 is the most frequently mutated gene in human cancer, showing changes in more than 50% of all solid tumours. Both DNA repair and apoptosis are stimulated by p53-induced transcription of genes involved in the two processes. The characterization of TSGs and their gene products has led to the identification of a number of new diagnostic and prognostic molecular genetic parameters in oncology. Furthermore, some TSGs are potentially among the most promising and important targets for gene therapy in cancer and other hyperproliferative diseases.

RAR-, not RXR, ligands inhibit cell activation and prevent apoptosis in B-lymphocytes.

We have previously shown that retinoids inhibit activation of human peripheral blood B-lymphocytes. In the present paper, we wished to explore the involvement of nuclear retinoid-specific receptors in this process by using ligands specific for the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We found that the RAR-specific ligand TTAB reduced anti-IgM-induced B-cell activation in a dose-dependent manner. Thus, at 100 nM of TTAB, DNA synthesis was reduced by approximately 60%. In contrast, the RXR-selective ligand SR11217 had no effect on DNA synthesis. Similar findings were obtained when the expression of the activation antigen CD71 (appears late in G1) was examined. The role of retinoids in apoptosis of resting peripheral blood B-lymphocytes was examined using the same receptor-selective ligands. Again, we found that the RAR-selective ligands were more potent effectors than were the RXR-selective ligands. In spite of the inhibitory effects of retinoids on B-cell proliferation, the same retinoids significantly promoted the survival of the cells. Thus, 10 nM TTAB significantly reduced spontaneous apoptosis of in vitro cultured B-cells at day 3 from 45% to 30%, as determined by vital dye staining and DNA end-labeling. Again, the RXR-specific ligand SR11217 had no effect. Interestingly, we found that CD40 ligand was able to potentiate the retinoid-mediated inhibition of apoptosis. By reverse transcriptase polymerase chain reaction (PCR), we found that peripheral blood B-lymphocytes expressed RARalpha, RARgamma, and RXRalpha, but not RARbeta, RXRbeta, or RXRgamma. Hence, the lack of effect of the RXR-specific ligand SR11217 on growth and apoptosis was not due to absence of RXRs. In conclusion, the ability of retinoids to inhibit growth and prevent apoptosis of normal human B-lymphocytes indicates a dual role of retinoids in this cell compartment, and it appears that both effects of retinoids are mediated via RARs and not RXRs.

Interleukin-13 in combination with CD40 ligand potently inhibits apoptosis in human B lymphocytes: upregulation of Bcl-xL and Mcl-1.

Interleukin-13 (IL-13) is a novel T-cell-derived cytokine with IL-4-like effects on many cell types. In human B lymphocytes, IL-13 induces activation, stimulates proliferation in combination with anti-IgM or anti-CD40 antibodies, and directs Ig isotype switching towards IgE and IgG4 isotypes. We show here that IL-13 also regulates human B-cell apoptosis. IL-13 reduced spontaneous apoptosis of peripheral blood B cells in vitro, as shown by measurement of DNA fragmentation using the TUNEL and Nicoletti assays. The inhibition of cell death by IL-13 alone was significant but modest, but was potently enhanced in combination with CD40 ligand (CD40L), a survival stimulus for B cells by itself. Interestingly, IL-13 increased the expression of CD40 on peripheral blood B cells, providing a possible mechanism for the observed synergy. IL-13 alone was a less potent inhibitor of apoptosis than IL-4. Moreover, there was no additive effect of combining IL-4 and IL-13 at supraoptimal concentrations, which is consistent with the notion that the IL-4 and IL-13 binding sites share a common signaling subunit. The combination of IL-13 with CD40L augmented the expression of the Bcl-2 homologues Bcl-xL and Mcl-1, suggesting this as a possible intracellular mechanism of induced survival. By contrast, levels of Bcl-2, and two other Bcl-2 family members, Bax and Bak, remained unaltered. Given the importance of the CD40-CD40L interaction in B-cell responses, these results suggest a significant role of IL-13 in the regulation of B-cell apoptosis.

Uncoupling of the order of the S and M phases: effects of staurosporine on human cell cycle kinases.

The protein kinase inhibitor staurosporine (SSP) was employed to study the involvement of kinases in human cell cycle progression. Thirty to 100 ng/ml SSP blocks entry into S phase and M phase. Lack of entry into S phase is due to impaired activity of the retinoblastoma protein kinase. The requirement for any of the SSP-sensitive kinases for cell cycle progression can be abrogated in tumour cells. Therefore, these kinases act in a checkpoint network negatively controlling the initiation of S phase, M phase and cytokinesis, rather than being inherent parts of a substrate-product chain required for the initiation of the cell cycle phases. As a consequence of the lack of certain checkpoint effectors, tumour cells may endoreduplicate or binucleate in the presence of SSP. The latter processes, as well as meiosis, are naturally occurring in specialized cell types, leading to the idea that this checkpoint network controls the order of the cell cycle phases in normal cells. A model is presented where the cell cycle is envisioned as two independently running cycles, the S and the M cycle, which are controlled by intra and intercycledependent checkpoints in human somatic cells. The model accounts for the dependency of S and M phase initiation on the successful completion of the previous M and S phase, respectively, as well as entry into a resting state.

CDw78--a determinant on a major histocompatibility complex class II subpopulation that can be induced to associate with the cytoskeleton.

In the present study we demonstrate that CDw78 monoclonal antibody (mAb) recognizes a distinct subpopulation of major histocompatibility complex (MHC) class II molecules. We show that the CDw78 epitope is present on less than 10% of the total number of MHC class II molecules expressed on different cells, is not linked to a single isotype, and exhibits a characteristic expression pattern in tonsils. While mAb against MHC class II (DR, DP and DQ) stained the majority of cells both in the mantle zone and in germinal centers, the CDw78 staining was more heterogeneous with the strongest reactivity and the highest number of positive cells in the mantle zone and in the light centrocyte-rich part of the germinal centers. Antibodies to this MHC class II subpopulation (e.g. FN1) induced association with the cytoskeleton and a subsequent capping in more than 90% of peripheral blood B cells. In contrast, mAb against MHC class II (DR, DP and DQ) did not induce association with the cytoskeleton and only 10-20% of B cells were induced to cap, suggesting that CDw78 defines a population of MHC class II molecules functionally different from the majority of these antigens. Scatchard plot analysis indicates that FN1 mAb is of relatively low affinity (Ka = 1.5 x 10(8) M(-1)) and monovalent Fab fragments fail to bind to the cell surface with measurable affinity. Our data seen in the context of the ability of FN1 to co-stimulate B cells with a suboptimal dose of anti-mu suggest that CDw78 mAb might recognize a functional important subpopulation of MHC class II molecules so far not described. It seems likely that this subpopulation represents dimerized or aggregated MHC class II molecules that can selectively bind this low-affinity mAb.