Dan H. Schulze

Cell cycle regulation of mouse H3 histone mRNA metabolism.

The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3H]uridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.

Isopentenyl Pyrophosphate–Activated CD56+ γδ T Lymphocytes Display Potent Antitumor Activity toward Human Squamous Cell Carcinoma

Purpose: The expression of CD56, a natural killer cell–associated molecule, on αβ T lymphocytes correlates with their increased antitumor effector function. CD56 is also expressed on a subset of γδ T cells. However, antitumor effector functions of CD56+ γδ T cells are poorly characterized. Experimental Design: To investigate the potential effector role of CD56+ γδ T cells in tumor killing, we used isopentenyl pyrophosphate and interleukin-2–expanded γδ T cells from peripheral blood mononuclear cells of healthy donors. Results: Thirty to 70% of expanded γδ T cells express CD56 on their surface. Interestingly, although both CD56+ and CD56− γδ T cells express comparable levels of receptors involved in the regulation of γδ T-cell cytotoxicity (e.g., NKG2D and CD94), only CD56+ γδ T lymphocytes are capable of killing squamous cell carcinoma and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules because CD56+ γδ T lymphocytes expressed higher levels of CD107a compared with CD56− controls following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanamycin A and a combination of anti-γδ T-cell receptor + anti-NKG2D monoclonal antibody, suggesting that the lytic activity of CD56+ γδ T cells involves the perforin-granzyme pathway and is mainly γδ T-cell receptor/NKG2D dependent. Importantly, CD56-expressing γδ T lymphocytes are resistant to Fas ligand and chemically induced apoptosis. Conclusions: Our data indicate that CD56+ γδ T cells are potent antitumor effectors capable of killing squamous cell carcinoma and may play an important therapeutic role in patients with head and neck cancer and other malignancies.

Protein Kinase A Hyperphosphorylation Increases Basal Current but Decreases β-Adrenergic Responsiveness of the Sarcolemmal Na+-Ca2+ Exchanger in Failing Pig Myocytes

Abstract— The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by &bgr;-adrenergic receptor (&bgr;-AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni2+-sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF (P <0.05 versus control). Despite this modest increase in protein, the basal peak outward NCX current was increased almost 5-fold in HF (P <0.05 versus control). Stimulation with isoproterenol, however, increased the control currents to a significantly greater extent than HF (500% increase in control versus 100% increase in HF, P <0.01); peak stimulated current was not different in HF and control. This reduction in responsiveness to &bgr;-AR stimulation was refractory to forskolin, 8-Br-cAMP, or okadaic acid stimulation. In vitro protein kinase A back-phosphorylation revealed higher phosphorylation capacity of NCX protein in control versus HF, consistent with increased phosphorylation in vivo (hyperphosphorylation) in HF. Protein phosphatase type 1 exposure resulted in a significant reduction (73%) in peak basal current in HF (compared with no significant difference in controls), confirming that the increased basal NCX current in HF is predominately attributable to hyperphosphorylation. NCX expression and activity are thus increased in HF, although &bgr;-AR responsiveness is decreased because of NCX hyperphosphorylation.

Novel subunit composition of a renal epithelial KATP channel.

Unique ATP-inhibitable K+channels (KATP) in the kidney determine the rate of urinary K+ excretion and play an essential role in extracellular K+ balance. Here, we demonstrate that functionally similar low sulfonylurea affinity KATP channels are formed by two heterologous molecules, products of Kir1.1a and cystic fibrosis transmembrane conductance regulator (CFTR) genes. Co-injection of CFTR and Kir1.1a cRNA into Xenopus oocytes lead to the expression of K+ selective channels that retained the high open probability behavior of Kir1.1a but acquired sulfonylurea sensitivity and ATP-dependent gating properties. Similar to the KATP channels in the kidney but different from KATP channels in excitable tissues, the Kir1.1a/CFTR channel was inhibited by glibenclamide with micromolar affinity. Since the expression of Kir1.1a and CFTR overlap at sites in the kidney where the low sulfonylurea affinity KATP are expressed, our study offers evidence that these native KATP channels are comprised of Kir1.1a and CFTR. The implication that Kir subunits can interact with ABC proteins beyond the subfamily of sulfonylurea receptors provides an intriguing explanation for functional diversity in KATP channels.

Fc dependent expression of CD137 on human NK cells: insights into "agonistic" effects of anti-CD137 monoclonal antibodies

CD137 (4-1BB) is a costimulatory molecule that can be manipulated for the treatment of cancer and autoimmune disease. Although it is known that agonistic antibodies (mAbs) against CD137 enhance the rejection of murine tumors in a natural killer (NK) cell- and T cell-dependent fashion, the mechanism for NK dependence is poorly understood. In this study, we evaluated the ability of 2 different glycoforms of a chimerized antihuman CD137 mAb, an aglycosylated (GA) and a low fucose form (GG), to react with human NK cells. Both mAbs bound similarly to CD137 and partially blocked the interaction between CD137 and CD137 ligand. However, unlike GA mAb, immobilized GG mAb activated NK cells and enhanced CD137 expression. These effects were seemingly dependent on Fc interaction with putative Fc receptors on the NK-cell surface, as only the immobilized Fc-fragment of GG was required for CD137 expression. Furthermore, CD137 expression could be enhanced with antibodies directed against non-CD137 epitopes, and the expression levels directly correlated with patterns of Fc-glycosylation recognized to improve Fc interaction with Fc gamma receptors. Our data suggest that CD137 can be enhanced on NK cells in an Fc-dependent fashion and that expression correlates with phenotypic and functional parameters of activation.

Functional differences between cardiac and renal isoforms of the rat Na+‐Ca2+ exchanger NCX1 expressed in Xenopus oocytes

1 The transcript of the Na+‐Ca2+ exchanger gene NCX1 undergoes alternative splicing to produce tissue‐specific isoforms. The cloned NCX1 isoforms were expressed in Xenopus oocytes and studied using a two‐electrode voltage clamp method to measure Na+‐Ca2+ exchanger activity. 2 The cardiac isoform (NCX1.1) expressed in oocytes was less sensitive to depolarizing voltages and to activation by [Ca2+]i than the renal isoform (NCX1.3). 3 The cardiac isoform of NCX1 is more sensitive to activation by protein kinase A (PKA) than the renal isoform which may be explained by preferential phosphorylation. The cardiac isoform of NCX1 is phosphorylated to a greater extent than the renal isoform. 4 The action of PKA phosphorylation which increases the activity of the cardiac isoform of the Na+‐Ca2+ exchanger in oocytes was confirmed in adult rat ventricular cardiomyocytes by measuring Na+‐dependent Ca2+ flux. 5 We conclude that alternative splicing of NCX1 confers distinct functional characteristics to tissue‐specific isoforms of the Na+‐Ca2+ exchanger.

Tumor-Induced Senescent T Cells with Suppressor Function: A Potential Form of Tumor Immune Evasion

Senescent and suppressor T cells are reported to be increased in select patients with cancer and are poor prognostic indicators. Based on the association of these T cells and poor outcomes, we hypothesized that tumors induce senescence in T cells, which negatively effects antitumor immunity. In this report, we show that human T cells from healthy donors incubated with tumor for only 6 h at a low tumor to T-cell ratio undergo a senescence-like phenotype, characterized by the loss of CD27 and CD28 expression and telomere shortening. Tumor-induced senescence of T cells is induced by soluble factors and triggers increases in expression of senescence-associated molecules such as p53, p21, and p16. Importantly, these T cells are not only phenotypically altered, but also functionally altered as they can suppress the proliferation of responder T cells. This suppression requires cell-to-cell contact and is mediated by senescent CD4(+) and CD8(+) subpopulations, which are distinct from classically described natural T regulatory cells. Our observations support the novel concept that tumor can induce senescent T cells with suppressor function and may effect both the diagnosis and treatment of cancer.

CD137 Promotes Proliferation and Survival of Human B Cells

CD137 (4-1BB)-mediated costimulation plays an important role in directing the fate of Ag-stimulated T cells and NK cells, yet the role of CD137 in mediating B cell function is unknown. We found that CD137 is expressed in vitro on anti-Ig–stimulated peripheral blood B cells and in vivo on tonsillar B cells with an activated phenotype. In vitro CD137 expression is enhanced by CD40 stimulation and IFN-γ and is inhibited by IL-4, -10, and -21. The expression of CD137 on activated human B cells is functionally relevant because engagement with its ligand at the time of activation stimulates B cell proliferation, enhances B cell survival, and induces secretion of TNF-α and -β. Our study suggests that CD137 costimulation may play a role in defining the fate of Ag-stimulated human B cells.

Sodium/calcium exchanger expression in the mouse and rat olfactory systems

Sodium/calcium (Na+/Ca2+) exchangers are membrane transport systems that regulate Ca2+‐homeostasis in many eukaryotic cells. In olfactory and vomeronasal sensory neurons ligand‐induced olfactory signal transduction is associated with influx and elevation of intracellular Ca2+, [Ca2+]i. While much effort has been devoted to the characterization of Ca2+‐related excitation and adaptation events of olfactory chemosensory neurons (OSNs), much less is known about mechanisms that return [Ca2+]i to the resting state. To identify proteins participating in the poststimulus Ca2+‐clearance of mouse OSNs, we analyzed the expression of three potassium (K+)‐independent (NCX1, 2, 3) and three K+‐dependent (NCKX1, 2, 3) Na+/Ca2+ exchangers. In situ hybridization showed that mRNAs of all six Na+/Ca2+ exchangers coexist in neurons of the olfactory and vomeronasal systems, and that some are already detectable in the embryo. Of these, NCX1 and NCKX1 represent the most and least abundant mRNAs, respectively. Moreover, immunohistochemistry revealed that the NCX1, 2, and 3 proteins are expressed in nearly all neurons of the olfactory epithelium, the vomeronasal organ, the septal organ of Masera, and the Grueneberg ganglion. These three exchanger proteins display different expression profiles in dendrites, knobs, and plasma membranes of OSNs and in sustentacular cells. Furthermore, we show that NCX1 mRNA in rat olfactory mucosa is expressed as 8 alternative splice variants. This is the first comprehensive analysis of Na+/Ca2+ exchanger expression in the mammalian olfactory system. Our results suggest that Ca2+‐extrusion by OSNs utilizes multiple different Na+/Ca2+ exchangers and that different subtypes are targeted to different subcellular compartments. J. Comp. Neurol. 501:944–958, 2007.

Functional Regulation of Alternatively Spliced Na+/Ca2+ Exchanger (NCX1) Isoforms

Abstract: Alternative splicing of RNA transcripts is a general characteristic for NCX genes in mammals, mollusks, and arthropods. Among the family of three NCX genes in mammals, the NCX1 gene contains six exons, namely, A, B, C, D, E, and F, that make up the alternatively spliced region. Studies of the NCX1 gene transcripts suggested that 16 distinct gene products can be produced from the NCX1 gene. The exons A and B are mutually exclusive when expressed. Generally, exon A‐containing transcripts are predominantly found in excitable cells like cardiomyoctes and neurons, whereas exon B‐containing transcripts are mostly found in nonexcitable cells like astrocytes and kidney cells. Other alternatively spliced exons (C‐F) appear to be cassette‐type exons and are found in various combinations. Interestingly, exon D is present in all characterized transcripts. The alternatively spliced isoforms of NCX1 show tissue‐specific expression patterns, suggesting functional adaptation to tissues. To investigate functional differences among alternatively spliced isoforms of NCX1, we expressed an exon A‐containing transcript present in cardiac tissue (NCX1.1) and an exon B‐containing transcript found in the kidney (NCX1.3) in Xenopus oocytes. We demonstrated that the Na+/Ca2+ exchangers expressed by exon A‐ and exon B‐containing transcripts display differences in activation by PKA and by [Ca2+]i. We also observed that these two isoforms show differences in voltage dependence. Suprisingly, the alternatively spliced isoforms of NCX1 display greater functional differences among themselves than the products of different gene loci, NCX1, NCX2, and NCX3.