Human Salivary Gland Cell Line Research Articles

Inflammatory caspases are critical for enhanced cell death in the target tissue of Sjögren's syndrome before disease onset

To date, little is known about why exocrine glands are subject to immune cell infiltrations in Sjögren's syndrome (SjS). Studies with SjS-prone C57BL/6.NOD-Aec1Aec2 mice showed altered glandular homeostasis in the submandibular glands (SMX) at 8 weeks before disease onset and suggested the potential involvement of inflammatory caspases (caspase-11 and -1). To determine whether inflammatory caspases are critical for the increased epithelial cell death before SjS-like disease, we investigated molecular events involving caspase-11/caspase-1 axis. Our results revealed concurrent upregulation of caspase-11 in macrophages, STAT-1 activity, caspase-1 activity and apoptotic epithelial cells in the SMX of C57BL/6.NOD-Aec1Aec2 at 8 weeks. Caspase-1, a critical factor for interleukin (IL)-1beta and IL-18 secretion, resulted in an elevated level of IL-18 in saliva. Interestingly, TUNEL-positive cells in the SMX of C57BL/6.NOD-Aec1Aec2 were not colocalized with caspase-11, indicating that caspase-11 functions in a noncell autonomous manner. Increased apoptosis of a human salivary gland (HSG) cell line occurred only in the presence of lipopolysaccharide (LPS-) and interferon (IFN)-gamma-stimulated human monocytic THP-1 cells, which was reversed when caspase-1 in THP-1 cells was targeted by siRNA. Taken together, our study discovered that inflammatory caspases are essential in promoting a pro-inflammatory microenvironment and influencing increased epithelial cell death in the target tissues of SjS before disease onset.

Isoflavones and gamma irradiation inhibit cell growth in human salivary gland cells

We studied the effects of isoflavones and irradiation on cell cycle in a human salivary gland cell line (HSG). Genistein and a soy isoflavone conjugate (NS) inhibited DNA synthesis. Cells deconjugated the glucoside form of isoflavones in NS to the aglycones genistein and daidzein. NS, genistein and IR increased phosphorylation of p53 and p21 CIP1 at serine 15 (phos-p53). Irradiation and NS also increased levels of p21 CIP1. In a cologenic survival assay, cells in log phase growth had high radio-sensitivity with 2 Gy causing a reduction in survival (SF 2 = 0.45). Conclusion: isoflavones and radiation may interact to sensitize cancer cells to radiation.

Fcγ receptors mediate internalization of anti‐Ro and anti‐La autoantibodies from Sjögren’s syndrome and apoptosis in human salivary gland cell line A‐253

The presence of serum anti-Ro and anti-La autoantibodies directed against the ribonucleoproteins Ro and La has been associated with Sjögren's syndrome (SS), an autoimmune rheumatic disease that targets salivary and lachrymal glands. There is increasing evidence of the direct involvement of autoantibodies in the pathogenesis of tissue injury and correlation of their presence with clinical manifestations in SS. The focus of this work was to explore the cellular apoptotic pathway triggered by binding and penetration of anti-Ro and anti-La autoantibodies in human salivary gland cell line A-253 and to identify the membrane receptors through which anti-Ro and anti-La could exert their effect. Anti-Ro and anti-La autoantibodies were purified from IgG fractions, obtained from eleven healthy volunteers and patients with primary Sjögren's syndrome, using Sepharose 4B-Ro and Sepharose 4B-La affinity columns. Flow cytometry, RT-PCR, western blot and confocal microscopy analysis were used to visualize the FCgammaRI, FCgammaRII and FCgammaRIII receptors on the A-253 cell membrane. DNA laddering and western blot analysis of caspases activation were studied to evaluate in A-253 cells treated with anti-Ro and anti-La autoantibodies. The results yeilded the evidence of the presence of members of the Fcgamma receptors (FcgammaRs) family on the cell membrane of the human salivary gland cell line A-253. Furthermore, we demonstrated that, in the A-253 cell line, anti-Ro and anti-La autoantibodies can access the cells probably through Fcgamma receptors, and trigger apoptotis. We conclude that anti-Ro and anti-La autoantibodies have pathogenic effects that could depend on binding to Fcgamma receptors.

Autoantibodies from Sjögren's Syndrome Trigger Apoptosis in Salivary Gland Cell Line

The presence of serum autoantibodies has been associated with Sjögren's syndrome (SS), an autoimmune rheumatic disease that targets salivary and lachrymal glands. The association of apoptosis with autoantibodies production seems to play a role in the pathogenesis of glandular damage. The best-defined antibodies in SS are those reacting with the ribonucleoprotein antigens SS-A (Ro) and SS-B (La). Anti-Ro antibodies are found in about 70-90%, and anti-La in approximately the same frequency, of patients with primary SS. The objective of this work was to explore whether anti-Ro and anti-La autoantibodies purified from Sjögren IgG fractions are able to trigger apoptotic process in the human salivary gland cell line A-253. Anti-Ro and anti-La autoantibodies were purified on protein G Sepharose affinity column and used for the A-253 cell treatment. Apoptosis induced by autoantibodies was revealed by FACS analysis, and the active caspase-3 and the cleaved caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was demonstrated by colorimetric assay and Western blot. This report shows that anti-Ro and anti-La autoantibodies, but not healthy IgG, activate the caspase-3 and determine the cleavage of PARP in A-253 cells. Apoptosis triggered by Sjögren autoantibodies could be responsible for the impairment of the secretory function in the salivary glands.

Matrigel‐induced acinar differentiation is followed by apoptosis in HSG cells

It has been shown that a human salivary gland cell line (HSG) is capable of differentiation into gland-like structures, though little is known of how morphological features are formed or controlled. Here we investigated the changes in cell proliferation and apoptosis upon terminal differentiation of HSG cells in Matrigel, an extracellular matrix derivative. Changes in the expression of survivin, a prominent anti-apoptotic factor, and caspase-3, a key apoptotic factor were also measured. In order to better understand the involvement of key signal transduction pathways in this system we pharmacologically blocked the activity of tyrosine kinase, nuclear factor kappa B(NF kappa B), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) and matrix metalloproteases (MMP). Results of these studies demonstrate that cytodifferentiation of HSG cells to an acinar phenotype is accompanied first by a decrease of cell proliferation and then by a massive programmed cell death, affected by multiple signal transduction pathways. Thus, Matrigel alone is insufficient for the full maturation and long term survival of the newly formed acini: the presence of other factors is necessary to complete the acinar differentiation of HSG cells.

Human parotid saliva contains soluble toll-like receptor (TLR) 2 and modulates TLR2-mediated interleukin-8 production by monocytic cells

Toll-like receptor (TLR) family members are pattern-recognition receptors and very important molecules in innate immunity. Although TLRs are originally type I transmembrane receptors, soluble forms of TLRs are detected in human plasma and milk. This study showed that soluble TLR2 (sTLR2) is detected in human parotid saliva. Western blotting with anti-TLR2 antibodies (Abs) showed that three polypeptides are detected as sTLR2 with molecular weights of 55, 40 and 27 kDa, respectively. Parotid saliva neutralized the binding of anti-TLR2 polyclonal Ab to cell-surface TLR2 on THP-1, a human monocytic cell line. Immunohistochemical analysis revealed that TLR2 is expressed in serous and interlobular ductal cells of human salivary gland. Human salivary gland cell lines, AZA3 and HSY, constitutively expressed TLR2. Parotid saliva augmented IL-8 production of THP-1 cells stimulated with a synthetic TLR2 ligand, Pam 3Cys-Ser-(Lys) 4 (Pam 3CSK 4). Depletion of sCD14 from parotid saliva by immunoprecipitation eliminated the augmentation of IL-8 production, indicating that the augmentable effects depended on sCD14 in parotid saliva. On the other hand, preincubation of Pam 3CSK 4 with parotid saliva abrogated the augmentation of IL-8 production, indicating that sTLR2 in saliva bound to Pam 3CSK 4 and neutralized its function. These results suggest that parotid saliva modulates the TLR2-mediated immune responses with binary mechanisms via sTLR2 and sCD14 in the oral cavity.

Interferon-gamma sensitizes the human salivary gland cell line, HSG, to tumor necrosis factor-alpha induced activation of dual apoptotic pathways

Activated immune cells secrete proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma) and Fas ligand (FasL) and these cytokines have been reported to induce apoptosis in numerous cell types. Apoptotic cell death has been associated with the progression of numerous autoimmune diseases. Proinflammatory cytokines are reportedly involved in apoptosis in the salivary glands of patients with Sjögren's syndrome (SS); an autoimmune disorder characterized by the destruction of salivary and lachrymal glands. In this study, we used the HSG cell line to determine if exposure to proinflammatory cytokines induces apoptosis in human salivary gland cells. In addition, we identified the mediators controlling the apoptotic process in response to TNF alpha and IFN gamma. TNF-alpha and IFN-gamma induced apoptosis in HSG cells and resulted in the activation of caspase 8 and the "death receptor" pathway. We further determined that caspase 9 and the "mitochondrial" pathway was also activated. Induction of the intrinsic and extrinsic pathways in HSG cells resulted in substrate cleavage by effector caspases, in particular the cleavage of alpha II spectrin, an autoantigen in Sjögren's syndrome. Our results suggest that HSG cells provide a model system to study processes regulating proinflammatory cytokine-induced apoptotic cell death.

Autoantibodies from Sjögren's syndrome induce activation of both the intrinsic and extrinsic apoptotic pathways in human salivary gland cell line A-253

Sjögren's syndrome (SS) is an autoimmune rheumatic disease that targets salivary and lachrymal glands, characterized by a high concentration of serum autoantibodies directed against nuclear and cytoplasmic antigens. It is known that autoantibodies can enter viable cells and this phenomenon has functional consequences including activation of apoptotic process. The objective of this work was to explore whether autoantibodies contained in IgG purified from Sjögren sera trigger apoptotic process in an experimental model represented by the human salivary gland cell line A-253. To define if the intrinsic or extrinsic pathways are activated, we examined which caspases are critical for inducing cell death. The results have demonstrated that morphological changes and DNA laddering, consistent with apoptotic cell death, occurred in A-253 cells treated with IgG from Sjögren sera. Sjögren IgG induced cleavage and activation of the effector caspase-3 and degradation of the caspase-3 substrate poly(ADP-ribose)polymerase. Both the intrinsic and extrinsic apoptotic pathways were activated, since both caspase-8 and caspase-9 cleavages occurred. In conclusion, autoantibodies contained in IgG purified from Sjögren sera mediate apoptosis of the A-253 cell line in a caspase-dependent manner.

Small Rho GTPases are important for acinus formation in a human salivary gland cell line

Rho GTPases participate in a wide variety of signal transduction pathways regulating the actin cytoskeleton, gene expression, cellular migration and proliferation. The aim of this study was to evaluate the role of Rho GTPases in signal transduction pathways during acinus formation in a human salivary gland (HSG) cell line initiated by extracellular matrix (ECM; Matrigel) alone or in combination with epidermal growth factor, basic fibroblast growth factor and lysophosphatidic acid (LPA). Immunohistochemical and Western blotting analyses showed that HSG cells contained RhoA, RhoB, Rac1 and Cdc42 proteins. All growth factors enhanced the effects of ECM on acinus formation, in a pathway dependent on PI3-kinase and Rho GTPases. The role of ROCK, a major RhoA effector, seemed limited to cortical actin polymerization. LPA stimulated cell migration and acinus formation in a PI3-kinase-independent pathway. The results suggest that Rho proteins are important for epithelial-mesenchymal interactions during salivary gland development.

Effect of γ-secretase inhibitors on muscarinic receptor-mediated calcium signaling in human salivary epithelial cells

Altered intracellular Ca(2+) signaling has been observed in cells derived from Alzheimer's disease patients, and a possible link between gamma-secretase activity and the content of intracellular Ca(2+) stores has been suggested. To test this hypothesis we studied the effects of several gamma-secretase inhibitors on muscarinic receptor-mediated intracellular calcium release in the human salivary gland cell line HSG. Although several inhibitors in the peptide aldehyde class partially blocked carbachol-induced Ca(2+) transients, these effects did not appear to be due to gamma-secretase inhibition, and overall we found no evidence that inhibition of gamma-secretase activity had any significant effect on agonist-induced intracellular calcium release in HSG cells. In complementary experiments with presenilin-null cells we found that the reconstitution of gamma-secretase activity by transfection with wild-type presenilin 1 likewise had no significant effect on thapsigargin-induced Ca(2+) release. In a test of the specific hypothesis that the level of APP intracellular domain (AICD), the intracellular fragment of the beta-amyloid precursor protein (APP) resulting from gamma-secretase cleavage, can modulate the Ca(2+) content of the endoplasmic reticulum, we were unable to demonstrate any effect of APP small interfering RNA on the magnitude of carbachol-induced intracellular calcium release in HSG cells. Together our data cast considerable doubt on the hypothesis that there is a direct link between gamma-secretase activity and the content of intracellular Ca(2+) stores.

Detection of apoptosis-specific autoantibodies directed against granzyme B-induced cleavage fragments of the SS-B (La) autoantigen in sera from patients with primary Sjögren's syndrome

The objective of this study was to detect autoantibodies against granzyme B cleavage products in sera from patients with primary Sjögren's syndrome (SS). Cell lysates derived from human salivary gland (HSG) cell lines were incubated with granzyme B. The susceptibility to the generation of cleavage fragments of SS autoantigens was assayed by immunoblotting using sera from 57 primary SS patients, 17 primary SS patients with malignant lymphoma (ML), 28 systemic lupus erythematosus (SLE) patients, and 20 healthy controls. A 27 kD protein was recognized by serum autoantibodies in 8 (14.0%) of 57 primary SS patients, 5 (29.4%) of 17 SS patients with ML, 2 (7.1%) of 28 SLE patients, but not in 20 normal subjects. This protein was recognized by anti-SSB (La) monoclonal antibodies. Granzyme B-treated recombinant La protein was also shown to migrate as a discrete 27 kD protein by SDS PAGE. Blocking studies demonstrated the existence of an apoptosis-specific B cell epitope present in sera from 2 of 8 primary SS patients and in 2 of 5 primary SS patients with ML which recognized the 27 kD protein. Granzyme B-induced La fragments are generated during cytotoxicity in vitro. This is the first report describing autoantibodies in sera from primary SS patients that specifically recognize fragments of the La protein that are produced by the granzyme B protease.

β-Adrenergic-responsive activation of extracellular signal-regulated protein kinases in salivary cells: role of epidermal growth factor receptor and cAMP

The beta-adrenergic receptor agonist isoproterenol exerts growth-promoting effects on salivary glands. In this study, activation of ERKs, members of the mitogen-activated protein kinase family, by isoproterenol was examined in a human salivary gland cell line (HSY). Immunoblot analysis indicated that isoproterenol (10(-5) M) induced transient activation of ERK1/2 (4.4-fold relative to basal at 10 min) similar to that caused by EGF (6.7 fold). Isoproterenol, like EGF, also induced phosphorylation of the EGF receptor. However, inhibition of EGF receptor phosphorylation by the tyrphostin AG-1478 only partially attenuated isoproterenol-induced ERK phosphorylation, whereas EGF-responsive ERK activation was completely blocked. The G(i) inhibitor pertussis toxin also caused partial inhibition of isoproterenol-stimulated ERK activation. The cAMP analog 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and the cAMP-elevating agents IBMX and cholera toxin produced transient ERK1/2 activation, similar to the effect of isoproterenol, in HSY cells. The stimulatory effects of isoproterenol and cAMP on ERK phosphorylation were not reduced by the PKA inhibitor H-89, whereas the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidase (PP2) and transfection of a dominant-negative Src construct diminished isoproterenol-induced ERK activation. Isoproterenol induced marked overexpression of the cell growth-related adhesion molecule CD44, and this effect of isoproterenol was abolished by the ERK pathway inhibitor PD-98059. In summary, we show a dual mechanism of isoproterenol-induced ERK phosphorylation in HSY cells-one pathway mediated by EGF receptor transactivation and the other by an EGF receptor-independent pathway possibly mediated by cAMP. Our results also suggest that isoproterenol-induced growth of salivary tissue may involve ERK-mediated CD44 expression.

Subcellular redistribution and surface exposure of the Ro52, Ro60 and La48 autoantigens during apoptosis in human ductal epithelial cells: a possible mechanism in the pathogenesis of Sjögren's syndrome.

The Ro52, Ro60 and La48 autoantigens are associated with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The mechanisms behind tolerance breakdown of these self-peptides remain unclear; however, apoptosis has been proposed to cause their presentation to the immune system. We have examined the localization of transiently expressed enhanced green fluorescent protein (EGFP)-tagged Ro52, Ro60 and La48 autoantigens in a human salivary gland (HSG) cell line by laser confocal microscopy under normal growth conditions and during apoptosis. Surface exposure of Ro52, Ro60 and La48 was demonstrated on nonfixed apoptotic cells with monoclonal antibodies (MoAbs) or with primary SS patient antisera. Laser scanning cytometry determined the apoptotic frequency. EGFP alone was studied as control. We found that Ro52 mainly is cytoplasmic, Ro60 both nuclear and cytoplasmic, while La48 only resides in the nucleus under normal conditions. During early apoptosis, La48 is dramatically redistributed to the cytoplasm, while the localization of Ro52 and Ro60 is maintained. All three autoantigens filled apoptotic blebs and covered TUNEL (terminal-deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick end labelling)-positive apoptotic bodies. Identical results were obtained in COS-7 cells. We have developed a transfection system to study the intracellular localization of the three autoantigens Ro52, Ro60 and La48, without antibody detection. During apoptosis, there is an intracellular redistribution of endogenous and EGFP-tagged Ro52, Ro60 and La48, leading to surface exposure. These findings may indicate a role for apoptosis in the induction and facilitation of humoral responses to Ro52, Ro60 and La48 in the autoimmune exocrinopathy of SS.

Autoantigens and Sjögren syndrome.

To review recent experimental evidence on the involvement of autoantigens and autoantibodies in the pathogenesis of organ-specific autoimmunity in Sjögren syndrome (SS). Among candidate autoantigens in SS, we investigated the role of alpha-fodrin in the lacrimal gland using a mouse model and a lacrimal gland cell line established in p53 knockout mice. We also tried to identify a novel organ-specific autoantigen by screening the human salivary gland cell line (HSG) against sera from SS patients. The specificity and sensitivity of this autoantigen to SS patient sera, its cellular localization, and the gene encoding the protein were analyzed. In addition to the previously identified 120-kd alpha-fodrin in the salivary gland, a shorter fragment was detected, indicating that there may be a distinct apoptosis-related protease that cleaves alpha-fodrin in the lacrimal gland. A novel salivary gland-specific autoantibody was detected in 50.9% of sera from SS patients. The antigen recognized by this antibody may be a 45-kd nucleus protein not recognized in its native form. The precise roles of autoantigens in organ-specific autoimmunity are still unclear, although accumulated evidence suggests that they may be associated with disease progression. Further studies of alpha-fodrin and the 45-kd antigen may contribute to the understanding of the pathogenesis of SS and may provide a new strategy for organ-specific therapy, such as vaccination with analogue peptides.

Regulation of apoptotic cell death by cytokines in a human salivary gland cell line: Distinct and synergistic mechanisms in apoptosis induced by tumor necrosis factor α and interferon γ

We examined the mechanisms of apoptosis in a human salivary gland (HSG) cell line induced by tumor necrosis factor (TNF) α and interferon (IFN) γ. DNA fragmentation and the activation of caspase-3 were determined in HSG cells cultured with TNF-α or IFN-γ. Mitochondrial dysfunction also appeared to be involved in the process because a disruption of mitochondrial transmembrane potential with the activation of caspase-9 was demonstrated in TNF-α– and IFN-γ–stimulated HSG cells. Activation of caspase-8 was thought to be essential in TNF-α–induced apoptosis of HSG cells; however, the activation of caspase-8 was not involved in IFN-γ–induced apoptosis of HSG cells. In contrast, Bcl-2 appeared to be an indispensable regulatory molecule in IFN-γ–induced, but not in TNF-α–induced, apoptosis of HSG cells because its expression was inhibited in IFN-γ–stimulated, but not in TNF-α–stimulated, cells. The inhibitory effect of IFN-γ in Bcl-2 expression was enhanced by coadministration of TNF-α and, interestingly, apoptosis of HSG cells, as assessed by DNA fragmentation and the activation of caspase-9 and caspase-3, and disruption of mitochondrial transmembrane potential was also synergistically augmented by TNF-α and IFN-γ. Our results suggest that cytokines expressed in the salivary glands of patients with Sjögren syndrome play an important role in regulating apoptosis of acinar-ductal epithelial cells through distinct and synergistic mechanisms, thereby modulating salivary gland function in patients with Sjögren syndrome. (J Lab Clin Med 2002;139:13-9)

Distinct mechanisms of [Ca2+]i oscillations in HSY and HSG cells: role of Ca2+ influx and internal Ca2+ store recycling.

This study examined [Ca2+]i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca2+]i and KCa currents in both cell types. Low IP3, but not thapsigargin (Tg), induced [Ca2+]i oscillations, whereas Tg blocked CCh-stimulated [Ca2+]i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca2+ from HSY cells (i) did not affect CCh-stimulated [Ca2+]i oscillations or internal Ca2+ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca2+]i into oscillations. CCh- or thapsigargin-induced Ca2+ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic reticulum Ca2+ pump (SERCA) and inositoltrisphosphate receptors (IP3Rs) than HSG cells. These data demonstrate that [Ca2+]i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca2+ from, and release of Ca2+ into, the cytosol by the SERCA and IP3R activities, respectively. In HSY cells, Ca2+ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca2+]i. In HSG cells, [Ca2+]i oscillations directly depend on Ca2+ influx; Ca2+ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences in the mechanism of [Ca2+]i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca2+ stores.

Regulation of insulin-like growth factor-I receptor gene expression by tumor necrosis factor-α and interferon-γ

The insulin-like growth factor-I receptor (IGF-l-R) plays a critical role in normal and pathological growth processes. The expression of the IGF-l-R gene is regulated by various stimuli, including hormones and growth factors. We have investigated the molecular mechanisms by which two inhibitory cytokines, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), regulate IGF-l-R gene expression. TNF-α and IFN-γ reduced the proliferation rates of the osteogenic sarcoma cell line, Saos-2, and the human salivary gland cell line, HSG, in a dose- and time-dependent fashion. This effect was associated with significant reductions in the levels of IGF-l-R mRNA and protein, and with inhibition of IGF-l-R promoter activity, suggesting that TNF-α and IFN-γ affect IGF-l-R gene expression at the transcriptional level. In addition, TNF-α significantly decreased IGF-l-R mRNA stability. Combined cytokine treatment inhibited cellular proliferation and promoter activity in an additive manner. Taken together, these results suggest that a novel potential mechanism by which TNF-α and IFN-γ affect cellular proliferation involves suppression of IGF-l-R promoter activity, as well as destabilization of IGF-l-R transcripts.

Amplification of extracellular matrix and oncogenes in tat-transfected human salivary gland cell lines with expression of laminin, fibronectin, collagens I, III, IV, c- myc and p53

Considerable progress has been made in the transfer of foreign genes into salivary glands in vivo using adenovirus vectors in rats. In an attempt to avoid the transient expression inherent, when using these vectors, retroviral vectors and human cell lines where used here in attempt to develop an in vitro model of HIV-associated salivary gland disease. The HIV-1– tat protein is increasingly implicated in the pathogenesis of the AIDS through altering the expression of strategic cellular genes. The purpose of this study was to transfect human salivary gland (HSG) cell lines in vitro, with the pHIV-1/LTR–tat plasmid, and examine the effect of tat on expression of matrix and basement membrane genes known to be important in the pathogenesis of salivary gland disease. HSG cells were transfected with HIV-1–tat plasmid by the lipofection method. Transfection was confirmed by polymerase chain reaction (PCR) and Southern blot, which verified that tat-specific DNA was present. Tat-mRNA was analysed by Northern blotting and quantified by reverse transcriptase polymerase chain reaction (RT-PCR) to demonstrate its expression. Numerous clones were found to contain integrated tat DNA sequences and analysis of mRNA showed stable expression of tat-specific RNA. Further analysis of mRNA expression for various marker proteins important in HIV pathogenesis showed that the HSG cell line transfected with HIV-1–tat, was associated with significant induction of mRNA expression for extracellular matrix protein. Tat-amplified transcription of the major basement membrane protein laminin, as well as of fibronectin, collagen I and III, and c -myc oncogene was demonstrated. Conversely, expression of p53 suppressor gene mRNA was reduced. Post-transfection expression of collagen IV was erratic and inconclusive. It was concluded that the presence of HIV- tat in this in vitro model of salivary ductal epithelial cell model alters the mRNA expression of several matrix, basement membrane and oncoproteins known to be involved in HIV pathogenesis. These cell lines provide a useful system for studying the role of tat in the immunopathogenesis of HIV-associated salivary gland disease.

Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cells.

The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis.

Expression and Function of X Chromosome-Linked Inhibitor of Apoptosis Protein in Sjögren's Syndrome

Apoptotic cell death in acinar and ductal epithelial cells is thought to play an important role in the development of salivary gland dysfunction in patients with Sjögren's syndrome (SS). We examined the expression of anti-apoptotic molecules in salivary glands from patients with SS. The labial salivary glands from six human T-cell leukemia virus (HTLV)–I–seronegative and eleven HTLV-I–seropositive SS patients were analyzed by immunohistochemistry. In vitro experiments were performed with a human salivary gland cell line (HSG cells). Immunohistologic analyses revealed that Bcl-2 and Bcl-x were preferentially expressed in salivary infiltrating mononuclear cells more than acinar and ductal epithelial cells. In contrast, strong X chromosome-linked inhibitor of apoptosis protein (XIAP) expression was evident in both acinar and ductal epithelial cells. The pattern of expression of these anti-apoptotic molecules was similar in both HTLV-I–seropositive and HTLV-I–seronegative SS patients. Western blot analysis confirmed expression of XIAP in cultured HSG cells. The expression of XIAP in HSG cells was increased by IL-1β, TGF-β1, or IL-10. However, XIAP expression was down-regulated by TNF-α, which induced apoptotic cell death of HSG cells with an increase in caspase-3 activity. These effects of TNF-α in HSG cells were antagonized by IL-1β, TGF-β1, or IL-10. Our results suggest that XIAP is important in regulating apoptotic cell death of acinar and ductal epithelial cells in patients with SS.