Adaptor FYB (Fyn-binding protein) regulates integrin-mediated adhesion and mediator release: differential involvement of the FYB SH3 domain.

Aggregation of the high-affinity IgE receptor (FcepsilonRI) on mast cells activates a tyrosine phosphorylation cascade that is required for adhesion and degranulation events leading to the release of histamine and other inflammatory mediators. The full range of intracellular mediators that regulate this process is unknown. Recent studies have identified a group of immune cell-specific adaptor proteins that include linker for activation of T-cell (LAT), SH2-domain-containing leukocyte protein (SLP-76), and Fyn-T-binding protein (FYB)/SLP-76-associated protein (SLAP). In this study, we demonstrate that FYB can up-regulate integrin-mediated adhesion to fibronectin and mediator release in RBL-2H3 mast cells. The regulation of these two events could be distinguished from each other by the requirement of the FYB SH3 domain in beta-hexosaminidase release, but not adhesion, and the up-regulation of mediator release by FYB in nonadherent cells. FcepsilonRI aggregation increased FYB tyrosine phosphorylation, whereas confocal immunofluorescence microscopy showed that FYB colocalizes with F-actin in membrane ruffles and plaques. Our findings identify FYB as a regulator of integrin-mediated adhesion and degranulation events, which, in the case of mast cells, has potential applications to inflammatory and allergic responses.

Stability of biodegradable radioactive rhenium (Re-186 and Re-188) microspheres after neutron-activation.

Our objective was to determine if microspheres made from the biodegradable polymer poly(lactic acid) that contained rhenium could withstand the conditions of direct neutron activation necessary to produce therapeutic amounts of radioactive rhenium. The radiation damage of the polymer produced by gamma-doses of up to 1.05 MGy from Re-186 and Re-188 was examined by scanning electron microscopy and size exclusion chromatography. At a thermal neutron flux of 1.5 x 10(13)n/cm2/s the microspheres melted after 3 h in the nuclear reactor, but suffered little damage after 1 h of radiation and released less than 5% of the radioactivity during incubation in buffer at 37 degrees C. The radioactive microspheres produced in this manner have a specific activity too low for radioembolization for treatment of liver tumors, but could be injected directly into tumors or applied topically to the wound bed of partially resected tumors.

Phosphorylation of a conserved integrin alpha 3 QPSXXE motif regulates signaling, motility, and cytoskeletal engagement.

Integrin alpha 3A cytoplasmic tail phosphorylation was mapped to amino acid S1042, as determined by mass spectrometry, and confirmed by mutagenesis. This residue occurs within a "QPSXXE" motif conserved in multiple alpha chains (alpha 3A, alpha 6A, alpha 7A), from multiple species. Phosphorylation of alpha 3A and alpha 6A did not appear to be directly mediated by protein kinase C (PKC) alpha, beta, gamma, delta, epsilon, zeta, or mu, or by any of several other known serine kinases, although PKC has an indirect role in promoting phosphorylation. A S1042A mutation did not affect alpha 3-Chinese hamster ovary (CHO) cell adhesion to laminin-5, but did alter 1) alpha 3-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin (in the presence or absence of phorbol 12-myristate 13 acetate stimulation), and p130(CAS) (in the absence of phorbol 12-myristate 13 acetate stimulation), 2) the shape of cells spread on laminin-5, and 3) alpha 3-dependent random CHO cell migration on laminin-5. In addition, S1042A mutation altered the PKC-dependent, ligand-dependent subcellular distribution of alpha 3 and F-actin in CHO cells. Together, the results demonstrate clearly that alpha 3A phosphorylation is functionally relevant. In addition, the results strongly suggest that alpha 3 phosphorylation may regulate alpha 3 integrin interaction with the cytoskeleton.

Expression of cyclooxygenase 2 (COX-2) in human glioma and in vitro inhibition by a specific COX-2 inhibitor, NS-398.

The up-regulation of cyclooxygenase 2 (COX-2) expression is a frequent occurrence in a variety of different tumors. In this study, COX-2 protein expression was investigated in 50 glioma and 3 normal brain specimens by immunohistochemistry. Expression of COX-2 protein was observed in all normal brain and glioma specimens by immunohistochemistry, regardless of histological grade. The immunoreactive score was significantly higher in high-grade glioma than low-grade glioma and normal brain specimens. For a subset of these tumors (nine gliomas and three normal brain), Western blot analysis was also performed. COX-2 protein was detected in all specimens by Western blot analysis. The effect of the specific COX-2 inhibitor NS-398 on monolayer cell cultures and three-dimensional glioma spheroids was investigated using U-87MG and U-251MG human glioblastoma cell lines. The proliferation rate was assessed in monolayer cultures. In addition, a growth assay, a migration assay, an apoptosis assay, and a tumor invasion assay were performed in a three-dimensional spheroid culture system. NS-398 was able to reduce the proliferation of monolayer cell cultures, as well as the growth of spheroids and tumor cell migration, in a dose-dependent manner. There was also a moderate increase in the number of apoptotic cells in the treated spheroids. NS-398 did not have an inhibitory effect on tumor invasion in the coculture spheroid system. Our study provides evidence that COX-2 is up-regulated in the majority of high-grade gliomas and that a potential role of COX-2 inhibitors as an adjuvant therapy for brain tumors may exist.

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

Detection and analysis of cancer cells in blood and bone marrow using a rare event imaging system.

An automated rare event detection system (Rare Event Imaging System) is described for the recognition of cancer cells that appear at low frequencies (1 in 1 million) in peripheral blood (PB) or bone marrow (BM). The instrumentation includes an automated fluorescence microscope (Nikon Microphot-FXA) with a cooled charge coupled device camera and a 60-MHz Pentium personal computer. Main features of the system are rapid analysis of large microscopic fields, including a total cell count, detection of fluorescently labeled cells, and a display of digitally stored images of the detected cells. Furthermore, the X,Y coordinates of each identified object are stored and can be recalled for morphological analysis of the cell using higher magnification or different fluorescent filter sets. The preparation of the blood or BM samples for automated analysis consists of lysis of the RBCs, attachment of sample cells onto adhesion slides, fixation, and fluorescent labeling with anticytokeratin antibodies. Cytokeratin-positive cells, however, were detected in 17% of the samples from healthy blood donors using this procedure (mean number, approximately 7/10(6) mononuclear cells in positive samples). To improve the specificity of the rare event detection, a double-labeling protocol combining intracellular cytokeratin with epithelial cell adhesion molecule (Ep-CAM) (breast, ovarian, colon, and lung carcinoma antigen) or disialo-ganglioside (GD2) antigen (small cell lung carcinoma, neuroblastoma, melanoma antigen) was developed. Examples of doubly labeled cultured cells and cancer cells from breast and small cell lung cancer patients are shown. Using the double-labeling protocol, no "positive" cells were seen in samples of healthy blood donors. Automated rare event detection (cytokeratin single-staining) was applied to 355 PB, BM, and stem cell (SC) samples from breast cancer patients before autologous BM transplantation. Cytokeratin-positive cells were found in 52% of BM, 35% of PB, and 27% of SC samples at frequencies of 1-1020 positive cells/10(6) mononuclear cells, thereby establishing the efficacy of the technique in the detection of rare cancer cells in hematopoietic tissue samples of cancer patients.

The BCR/ABL oncogene alters the chemotactic response to stromal-derived factor-1alpha.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is a chemoattractant for CD34(+) progenitor cells, in vitro and in vivo. The receptor for SDF-1alpha, CXCR-4, is a 7 transmembrane domain receptor, which is also a coreceptor for human immunodeficiency virus (HIV). Here we show that transformation of hematopoietic cell lines by BCR/ABL significantly impairs their response to SDF-1alpha. Three different hematopoietic cell lines, Ba/F3, 32Dcl3, and Mo7e, were found to express CXCR-4 and to respond to SDF-1alpha with increased migration in a transwell assay. In contrast, after transformation by the BCR/ABL oncogene, the chemotactic response to SDF-1alpha was reduced in all 3 lines. This effect was directly due to BCR/ABL, because Ba/F3 cells, in which the expression of BCR/ABL could be regulated by a tetracycline-inducible promoter, also had reduced chemotaxis to SDF-1alpha when BCR/ABL was induced. The reduced response to SDF-1alpha was not due to an inability of BCR/ABL-transformed cell lines to migrate in general, as spontaneous motility of BCR/ABL-transformed cells was increased. In mice, injection of SDF-1alpha into the spleen resulted in a transient accumulation of untransformed Ba/F3 cells, but not Ba/F3. p210(BCR/ABL) cells administered simultaneously. The mechanism may involve inhibition of CXCR-4 receptor function, because in BCR/ABL-transformed cells, CXCR-4 receptors were expressed on the cell surface, but SDF-1alpha calcium flux was inhibited. Because SDF-1alpha and CXCR-4 are felt to be involved in progenitor cell homing to marrow, the abnormality decribed here could contribute to the homing and retention defects typical of immature myeloid cells in chronic myelogenous leukemia.

HRad17 colocalizes with NHP2L1 in the nucleolus and redistributes after UV irradiation.

The rad17 gene of Schizosaccharomyces pombe plays an important role as a checkpoint protein following DNA damage and during DNA replication. The human homologue of S. pombe rad17, Hrad17, was recently identified, but its function has not yet been established. Using the yeast two-hybrid system, we determined that HRad17 can interact with a nucleolar protein, NHP2L1. This interaction was also demonstrated biochemically, in human cells. Immunofluorescence studies revealed that HRad17 and NHP2L1 colocalize to the nucleolus, and immunogold labeling further resolved the location of NHP2L1 to the dense fibrillar component of the nucleolus. Interestingly, the localization of HRad17 in the nucleolus was altered in response to UV irradiation. These results provide some insight into the DNA damage and replication checkpoint mechanisms of HRad17.

Molecular cloning and characterization of human trabeculin-alpha, a giant protein defining a new family of actin-binding proteins.

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.

Novel isoform of lymphoid adaptor FYN-T-binding protein (FYB-130) interacts with SLP-76 and up-regulates interleukin 2 production.

T-cell activation involves the participation of protein-tyrosine kinases p56(lck) and ZAP-70/SYK as well as lymphoid proteins such as SLP-76 and FYB/SLAP. FYB/SLAP has the hallmarks of an adaptor protein that binds to the SH2 domains of the Src kinase FYN-T and SLP-76. Whereas two forms of FYB at 120 and 130 kDa have been identified biochemically, a cDNA encoding only the lower molecular weight isoform has been cloned (termed FYB-120 or SLAP-130). In this study, we report the isolation of an alternative isoform of FYB with a molecular mass of 130 kDa (FYB-130) that has the same structure as FYB-120 except for an insertion of 46 amino acids toward the carboxyl-terminal region of the protein. FYB-120 and FYB-130 share an ability to bind to the SH2 domains of FYN-T and SLP-76, to act as substrates for p59(FYN-T), and to be expressed in the cytoplasm and nucleus of T-cells. Differences were noted between the isoforms in the efficiency of binding to SLP-76 and in the preferential expression of FYB-130 in mature T-cells. When co-expressed together with FYN-T and SLP-76, FYB-130 caused a significant increase in anti-CD3-driven NF-AT transcription. Finally, fluorescence in situ hybridization analysis localized the FYB gene to human chromosome 5 at position p13.1. FYB-130 therefore represents a novel variant of FYB protein that can up-regulate T-cell receptor-driven interleukin 2 production in mature T-cells.

HRad17, a human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, is overexpressed in colon carcinoma.

Using the palindromic PCR-cDNA display method, we have cloned a novel gene overexpressed by human colon carcinoma relative to normal colon. Among normal tissues examined, only testis expresses it at a high level. Sequence analysis revealed its extensive homology with checkpoint genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces cerevisiae. This novel gene designated as hRad17 is localized to chromosome 5q12,13.1, a region known to be deleted in a variety of human cancers. Promoter region and one pseudogene of hRad17 have been identified. Whereas the increased expression of hRad17 by human colon carcinomas may be related to the known resistance of these cells to DNA-damaging agents during therapy, the deletion of hRad17 in a variety of cancers may predispose them to increased rate of mutation and heightened sensitivity to DNA-damaging agents, including radiation and anticancer drugs.

Clioquinol-zinc chelate: a candidate causative agent of subacute myelo-optic neuropathy.

BACKGROUND: 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol) was used clinically three decades ago as an oral antiparasitic agent and to increase intestinal absorption of zinc in patients with acrodermatitis enteropathica, a genetic disorder of zinc absorption. Use of clioquinol was epidemiologically linked to subacute myelo-optic neuropathy (SMON), characterized by peripheral neuropathy and blindness, which affected 10,000 patients in Japan. Discontinuation of oral clioquinol use led to elimination of SMON, however, the mechanism of how clioquinol induces neurotoxicity is unclear. MATERIALS AND METHODS: We tested the effect of clioquinol-metal chelates on neural crest-derived melanoma cells. The effect of clioquinol chelates on cells was further studied by electron microscopy and by a mitochondrial potential-sensitive fluorescent dye. RESULTS: Of the ions tested, only clioquinol-zinc chelate demonstrated cytotoxicity. The cytotoxicity of clioquinol-zinc chelate was extremely rapid, suggesting that its primary effect was on the mitochondria. Electron microscopic analysis demonstrated that clioquinol-zinc chelate caused mitochondrial damage. This finding was further confirmed by the observation that clioquinol-zinc chelate caused a decrease in mitochondrial membrane potential. CONCLUSIONS: We demonstrate that clioquinol, in the presence of zinc, is converted to a potent mitochondrial toxin. The phenomenon of clioquinol mediated toxicity appears to be specific to zinc and is not seen with other metals tested. Since clioquinol has been shown to cause increased systemic absorption of zinc in humans, it is likely that clioquinol-zinc chelate was present in appreciable levels in patients with SMON and may be the ultimate causative toxin of SMON.

BAX expression is associated with enhanced intracellular accumulation of paclitaxel: a novel role for BAX during chemotherapy-induced cell death.

SW626 cells that overexpress BAX are sensitized to the cytotoxic effects of paclitaxel and vincristine. It has been assumed that BAX mediates these effects through its ability to alter mitochondrial function, specifically by promoting the release of cytochrome c and facilitating the mitochondrial permeability transition. However, we have found that several early paclitaxel-mediated events are enhanced in SW626 transfectants that overexpress BAX, including G2-M-phase arrest, tubulin polymerization, and BCL-2 phosphorylation. We now demonstrate that these seemingly disparate effects are explained by an enhanced accumulation of paclitaxel in BAX-overexpressing cells, an effect due to diminished drug efflux. In contrast, drug efflux is increased in cells that do not overexpress BAX, resulting in low intracellular paclitaxel levels and relative resistance to the effects of this drug. Drug efflux in SW626 cells is mediated by a verapamil-inhibitable, non-MDR-1, non-MRP-1 transporter whose function or expression may be inhibited by BAX. These data suggest that stable transfectants that overexpress BAX may be sensitized to apoptotic cell death through a novel mechanism involving the enhancement of intracellular levels of naturally occurring toxins such as alkaloid derivatives.

Inactivation of DNA-dependent protein kinase by protein kinase Cdelta: implications for apoptosis.

Protein kinase Cdelta (PKCdelta) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCdelta in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCdelta (PKCdelta CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCdelta cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCdelta associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCdelta CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCdelta CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCdelta CF. These findings support the hypothesis that functional interactions between PKCdelta and DNA-PK contribute to DNA damage-induced apoptosis.

The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules.

The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules. Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and are important in vivo for microtubule function. Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating the control of normal growth and differentiation processes in the colonic epithelium.

FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells.

TcRzeta/CD3 ligation initiates a signaling cascade involving CD4/CD8-p56(lck), p59(fyn), and ZAP-70, as well as lymphoid downstream proteins VAV, SLP-76, and FYB/SLAP. A current question concerns the nature of the downstream binding partner(s) of FYB in T cells. In this study, using a two-hybrid screen with FYB as bait, we have identified eight clones, four of which correspond to the recently published lymphoid protein SKAP55, and two which correspond to a related protein with some 44% homology to SKAP55 (termed SKAP55-related protein, SKAP55R). The SKAP55 clones showed only minor differences (two substitutions and one residue deletion) from SKAP55. SKAP55R has the same overall structure as SKAP55 except for the presence of a unique N terminus with a well-defined coiled-coil domain. Both SKAP55 and SKAP55R were found to bind FYB through their SH3 domains and to act as substrates for the FYN kinase in T cells. Furthermore, immunofluorescence confocal microscopy showed that FYB and SKAP55 colocalize in the perinuclear region of cells. SKAP55 also colocalizes with another FYB binding protein, SLP-76. Taken together, these observations demonstrate that FYB is part of an interactive matrix with SKAP55 and a SKAP55-related protein.

Mutational evidence for control of cell adhesion through integrin diffusion/clustering, independent of ligand binding.

Previous studies have shown that integrin alpha chain tails make strong positive contributions to integrin-mediated cell adhesion. We now show here that integrin alpha4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the alpha4 cytoplasmic domain caused a severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/ or antibodies. Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon alpha4 tail deletion. Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.

Association of the mammalian helicase MAH with the pre-mRNA splicing complex.

Conversion of pre-mRNAs into mature mRNAs includes several consecutive enzymatic modification steps that are carried out in the spliceosomes. Helicases have been shown to contribute to these catalytic processes both in yeast and in mammalian cells. Our results identify the mammalian protein MAH (matrix-associated helicase) as a new helicase present in the spliceosome complex. Sequence comparison describes MAH as the first higher eukaryotic member of the helicase superfamily I, with demonstrated enzymatic activity. Because MAH does not bind small nuclear ribonucleoproteins (snRNPs), it appears to be a non-snRNP binding factor of the splicing complex. In conclusion, our data suggest the involvement of MAH in processing of pre-mRNAs in mammalian cells.

Measurement of apoptosis in heterogenous cell populations.

The increasing interest in programmed cell death has created the need to measure apoptosis in complex cell systems. We have combined the use of fluorescent antibodies with the Hoechst 33342/propidium iodide system in order to quantitate programmed cell death in fractions of heterogenous cell populations. Here we describe the analysis of T-cell apoptosis after ligation of the T-cell antigen receptor by superantigen in vitro and ex vivo. This technique can separate cells according to seven parameters, fluorescence caused by FITC, PE, allophycocyanin, incorporation of Hoechst 33342, PI, forward scatter, and side scatter, and it allows determination of elevated Hoechst 33342 uptake in less than 10% of the cell population.

Mouse hepatitis virus infection induces an early, transient calcium influx in mouse astrocytoma cells.

Mouse hepatitis virus (MHV), a murine coronavirus, utilizes murine carcinoembryonic antigens as receptors. The events that follow virus-receptor binding and eventually lead to virus entry are poorly understood. We studied the possible effects of MHV infection on intracellular calcium in a mouse astrocytoma cell line. Using the calcium-sensitive dye fluo-3 and confocal laser scanning microscopy, we found that MHV strain JHM induced an immediate (within 20 s) and transient (lasting no longer than 2 min) calcium increase in about 5% of the infected cells. The calcium increase was blocked by antibodies against the viral spike protein, suggesting that it was specifically triggered by the interaction of the viral spikes with cells. It was also inhibited by L-type calcium channel blockers and was not detected in calcium-free medium, suggesting that the calcium increase was caused by calcium influx from the extracellular medium. Studies of the kinetics of viral replication by immunofluorescence staining of the viral nucleocapsid protein revealed that at 3 h postinfection there was roughly the same percentage of cells (5%) that produced the viral protein as the percentage of cells that had responded with a calcium signal. This finding and the virus dilution studies together suggest that calcium responders may represent cells that had been infected with multiple viruses and undergone rapid viral replication. Furthermore, calcium channel blockers, including verapamil and cadmium chloride, and the calcium chelator EGTA inhibited virus infection. Therefore, the transient intracellular calcium increase reported here may be an early signaling event associated with virus infection.