Laminin-6 and laminin-5 are recognized by autoantibodies in a subset of cicatricial pemphigoid

To aid treatment choice in early stage of Hodgkin's disease, we analysed patients registered in the IDHD Database with clinical stages I or II Hodgkin's disease who were not staged with laparotomy and whose initial treatment was with radiotherapy alone. The factors analysed for outcome after first relapse included initial stage, age, sex, histology, number of involved areas, mediastinal involvement, E-lesions, B-symptoms, erythrocyte sedimentation rate, alkaline phosphatase, serum albumin and haemoglobin. As well as presentation variables, we analysed the disease-free interval after initial radiotherapy and the extent of disease at relapse. A total of 1364 patients with clinical stage I or II Hodgkin's disease were treated with initial radiotherapy, of whom 473 relapsed. The probability of survival 10 years after relapse was 63%. For cause-specific survival (CSS), both multivariate and univariate analysis identified the importance of age at presentation and histological subtypes. When all causes of death were considered, the multivariate analysis identified age as the only significant factor. The length of initial disease-free interval had no influence on prognosis after relapse, but the 169 patients with nodal relapse had a higher cause-specific survival than those with an extranodal component of relapse (74% versus 51% at 10 years, P < 0.005). Thus, the important factors for outcome after initial treatment with radiotherapy are those factors predicting the risk of relapse after initial treatment together with those predicting outcome after relapse, namely age, histologic subtype and extent of disease at relapse.     

Characterization and localization of P2 receptors in rat submandibular gland acinar and duct cells

[Ca2+]i and the Cl- current were measured in isolated submandibular gland acinar and duct cells to characterize and localize the purinergic receptors expressed in these cells. In both cell types 2'-3'-benzoylbenzoyl (Bz)-ATP and ATP increased [Ca2+]i mainly by activation of Ca2+ influx. UTP had only minimal effect on [Ca2+]i at concentrations between 0.1 and 1 mM. However, a whole cell current recording showed that all nucleotides effectively activated Cl- currents. Inhibition of signal transduction through G proteins by guanyl-5'-beta-thiophosphate revealed that the effect of ATP on Cl- current was mediated in part by activation of a G protein-coupled and in part by a G protein-independent receptor. BzATP activated exclusively the G protein-independent portion, whereas UTP activated only the G protein-dependent portion of the Cl- current. Measurement of [Ca2+]i in the microperfused duct showed that ATP stimulated a [Ca2+]i increase when applied to the luminal or the basolateral sides. BzATP increased [Ca2+]i only when applied to the luminal side, whereas UTP at 100 microM increased -Ca2+-i only when applied to the basolateral side. The combined results suggest that duct and possibly acinar cells express P2z receptors in the luminal and P2u receptors in the basolateral membrane.     

Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.     

EGF-related peptides are involved in the proliferation and survival of MDA-MB-468 human breast carcinoma cells

A majority of human breast carcinomas co-express the epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGF-alpha). MDA-MB-468 breast carcinoma cells express CR, AR and TGFalpha, while SK-BR-3 cells express CR and TGF-alpha. Anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against either CR or TGF-alpha inhibit the proliferation of both cell lines. A 40-50% growth inhibition was observed at a 2-microM concentration of each AS S-oligo. Treatment with the AR AS S-oligo also resulted in a significant inhibition of MDA-MB-468 anchorage dependent growth (ADG). No significant growth inhibition was observed when MDA-MB-468 or SK-BR-3 cells were treated with a mis-sense S-oligo. The AS S-oligos inhibited the expression of AR, CR or TGF-alpha proteins and mRNAs, as assessed by immuno-cytochemistry and semi-quantitative RT-PCR. An additive growth-inhibitory effect was observed when MDA-MB-468 cells were treated with a combination of EGF-related AS S-oligos. Indeed, treatment of MDA-MB-468 cells with a combination of AR, CR and TGF-alpha AS S-oligos resulted in about 70% growth inhibition at a concentration of 0.7 microM each. Finally, treatment of MDA-MB-468 cells with a combination either of the 3 AS S-oligos or of an EGF receptor-blocking antibody (MAb 225) and either CR, AR or TGFalpha AS S-oligos resulted in a significant increase in DNA fragmentation. Our data suggest that the EGF-related peptides are involved in the proliferation and survival of breast carcinoma cells.     

MUC4 is a major component of salivary mucin MG1 secreted by the human submandibular gland

High molecular weight salivary mucin (MG1) is an important component of saliva, contributing to the lubricative and tissue-protective functions of this biological fluid. We have shown previously that the human mucin gene MUC5B is expressed at high levels in sublingual gland and is a significant constituent of MG1. Since many epithelia express multiple mucin genes, it seemed likely that MG1 in salivary secretions is also a heterogeneous mixture of mucin gene products. The aim of this study was to determine whether MUC4, a mucin shown in Northern blotting experiments to be expressed in salivary glands, was a significant protein component of MG1 in salivary secretions. Two cDNA clones containing MUC4 tandem repeats were isolated from a human submandibular gland cDNA library. In addition, recombinant MUC4 produced in a bacterial expression system cross-reacted with an antibody directed against deglycosylated MG1. This shows conclusively that human salivary mucin MG1 contains both MUC5B and MUC4 gene products suggesting that each mucin may perform distinct functions in the oral cavity.     

Alterations in Ca2+ storage and mobilization in submandibular acinar cells of reserpine-treated rats

For cosmetic reasons, hand prostheses are provided with cosmetic gloves. Their pleasing appearance, however, is accompanied by poor mechanical behavior, resulting in a negative influence on prosthesis operation. Glove stiffness is high and nonlinear, and internal friction in the glove material causes energy dissipation (hysteresis). In this article, two methods for reducing hysteresis in cosmetic gloves are proposed, that may be applied independently or in combination. Glove modification. Altering the mechanical properties of the glove itself is the first method that is presented. It was found possible to reduce both stiffness and hysteresis about 50% by forming grooves into the inside of the glove. Together with the evaluation of this method, several properties of the cosmetic glove were determined. Motion optimization. Additionally, a second method for reducing hysteresis was developed. The amount of hysteresis is influenced by the way the glove is forced to deform. The prosthesis mechanism, determining this deformation, was designed for minimum hysteresis and maximum cosmesis. For the prosthesis-glove combination used in this study, thumb motion optimization reduced hysteresis by about 65%.     

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT

In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher L-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.     

Expression and localization of human kallistatin in rat submandibular gland after intracapsular gene injection

Gene delivery into rat submandibular gland in vivo by direct intracapsular injection has been studied. After the administration of adenovirus constructs, Ad-RSV-LacZ and Ad-CMV-LacZ, beta-galactosidase expression was localized in the granular convoluted tubular and striated duct cells of rat submandibular gland by in situ enzyme histochemistry. Adenovirus-mediated delivery of the human kallistatin gene (Ad-RSV-HKBP) into rat submandibular gland results in the expression of human kallistatin in a time-dependent manner. The expression of immunoreactive kallistatin in submandibular gland was detected 1 day after the Ad-RSV-HKBP injection and it reached a plateau (1-2 ng/mg protein) 2 days after gene delivery. Higher levels of human kallistatin were found in the submandibular gland of 6-month-old rats than in one-month-old rats. After direct gene injection, human kallistatin was localized mainly in cells of the granular convoluted tubules and striated ducts of rat submandibular gland using a specific monoclonal antibody to human kallistatin. The results indicate that direct intracapsular gene delivery into the submandibular gland provides a simple and reliable method for introducing foreign genes into the gland. This method can be used for studying gene regulation in vivo and may have potential for gene therapy in oral diseases.     

Tamoxifen induces Na+ -dependent uridine transport and dome formation in a human breast tumor cell line

PURPOSE: To correlate changes in uridine transport and colony morphology with differentiation of human breast cancer cells by tamoxifen and related agents. MATERIALS AND METHODS: Cultures of MCF-7 human breast cancer were treated with estradiol or the antiestrogen derivatives tamoxifen, hydroxytamoxifen, and ICI 164, 384. Initial rates of uridine transport and equilibrium concentrations were determined and morphological characteristics of the cultures evaluated. RESULTS: Tamoxifen causes an early induction of a Na+ -dependent transport of uridine characteristic of normal epithelial cells but absent in normal MCF-7 cultures and most human neoplasms examined. The pure antiestrogen ICI 164,384 and the more potent 4-hydroxytamoxifen also induced concentrative uridine transport; estradiol could prevent the expression of this transporter. Associated with induction of transport was a dramatic increase in dome formation in the cultures, a measure of unidirectional inorganic ion transport characteristic of the differentiated state. CONCLUSIONS: The induction of a concentrative transport of uridine is a concomitant of cellular differentiation of breast tumor cells. These findings give added weight to evidence that uridine may play a regulatory role in the transition to the neoplastic state. The absence of the transporter and low intracellular uridine concentrations in the undifferentiated state may relate to 5-FU sensitivity of breast tumors. Induction of the transporter by tamoxifen and the consequent major increase in intracellular concentrations of free uridine suggests a potentially negative effect of tamoxifen on regimens containing 5-FU.     

Establishment of a diploid cell line of human lung (PHuE-1)

The obtention of a diploid cell line from human lung of great concern for the virological diagnosis and research is reported. Certain aspects about its application and characterization are discussed.     

Kappa1-opioid binding sites are the dominant opioid binding sites in surgical specimens of human pheochromocytomas and in a human pheochromocytoma (KAT45) cell line

The adrenal medulla produces opioids which exert paracrine effects on adrenal cortical and chromaffin cells and on adrenal splanchnic nerves, via specific binding sites. The opioid binding sites in the adrenals are detectable mainly in the medullary part of it and differ in type between species. Thus, the bovine adrenal medulla contains mostly kappa-opioid binding sites and fewer delta- and mu-opioid binding sites while primate adrenals contain mainly delta sites and few kappa-opioid binding sites. Most chromaffin cell tumors, the pheochromocytomas, produce opioids which suppress catecholamine production by the tumor. The aim of the present work was to identify the types of opioid binding sites in human pheochromocytomas. For this purpose, we characterized the opioid binding sites on crude membrane fractions prepared from 14 surgically excised pheohromocytomas and on whole KAT45 cells, a recently characterized human pheochromocytoma cell line. Our data showed that human pheohromocytomas are heterogeneous, as expected, with regard to the production of catecholamines and the distribution and profile of their opioid binding sites. Indeed, only one out of the 14 pheochromocytomas expressed exclusively delta and mu opioid sites, while in the remaining 13 tumors kappa-type binding sites were dominant. The KAT45 cell line possessed a significant number of kappa1 binding sites, fewer kappa2-opioid binding sites and kappa3-opioid binding sites, and minimal binding capacity for delta- and mu-opioid receptor agonists sites. More specifically, the kappa1 sites/cell were approximately 18,000, the kappa2 4500/cell and the kappa3 sites 2000/cell. Our findings for the surgical specimens and the cell line combined with previously published pharmacological data obtained from KAT45 cells suggest that kappa sites appear to be the most prevalent opioid binding sites in pheochromocytomas. Finally, in normal bovine adrenals the profile of opioid binding sites differs in adrenaline and noradrenaline producing chromaffin cells. To test the hypothesis that the type of catecholamine produced by a pheochromocytoma depends on its cell of origin, we compared our binding data with the catecholamine content of each pheochromocytoma examined. We found no correlation between the type of the predominant catecholamine produced and the opioid binding profile of each tumor suggesting that this hypothesis may not be valid.     

Modulation of stress proteins by Cd2+ in a human T cell line

We previously showed in a human T cell line (CEM-C12 cells) that Cd2+ induced gene expression of stress proteins, metallothionein-IIA and heat shock protein 70 in a time- and dose-dependent manner. In the present study, CEM-C12 cells were pretreated for 24 h with 1 microM Cd2+ and then challenged with toxic concentrations of this metal. We found that maximal expression of the metallothionein-IIA and heat shock protein 70 genes was increased and this maximal level occurred at higher Cd2+ toxic concentrations. Actinomycin D chase experiments indicated that Cd2+ pretreatment did not modify metallothionein-IIA mRNA stability. The modulatory effect of Cd2+ pretreatment was dose-dependent from 100 pM to 1 microM. Such pretreatment also enhanced resistance to Cd2+ toxicity. Finally, verapamil, a calcium/potassium channel blocker displaced the dose-response curve for Cd2+ toxicity as well as metallothionein-IIA and heat shock protein 70 gene expression to higher Cd2+ concentrations.     

Characterization and chemosensitivity of a human epithelioid sarcoma cell line (SARCCR 2)

A new cell line was derived from the epithelioid sarcoma of a Caucasian woman who had previously received chemotherapy. The cells grew as an adherent monolayer, with a doubling time of 28 hr and had mainly epithelial morphology, but with areas of mesenchymal-like cytoplasmic extensions. The cells were tumorigenic in nude mice, with a short growth time, and a doubling time of 8 days. The cell line showed over-expression of P-glycoprotein by Western blot analysis, and its sensitivity to doxorubicin and vincristine was low. This sensitivity could be enhanced by reversants of multidrug resistance (MDR), such as cyclosporin or verapamil. This cell line constitutes an excellent model for studying compounds able to reverse MDR.     

Diameters of the main excretory ducts of the adult human submandibular and parotid gland: a histologic study

When glycosaminoglycan (GAG)-degrading enzymes were measured in normal human stool suspensions, all 5 tested different stools degraded titrable heparin and acharan sulfate. GAG-degrading bacteria were screened from the isolates of human stools. Among them, HJ-15 had the most potent activities of heparinases (GAGs-degrading enzymes). However, HJ-15 produced the enzyme even if in the media without heparin. Acharan sulfate lyase was induced by acharan sulfate and heparin. Heparinase production was also induced by these GAGs. These enzymes, acharan sulfate lyase and heparinase, were produced in exponential and stationary phase of HJ-15 growth, respectively. Optimal pHs of the acharan sulfate lyase and heparinase activities were 7.2 and 7.5, respectively. The biochemical properties of HJ-15 was similar to those of B. stercoris. However, difference from B. stercoris was utilization of raffinose. This HJ-15 also degraded chondroitin sulfates A and C.     

Changes in heregulin beta1 (HRGbeta1) signaling after inhibition of ErbB-2 expression in a human breast cancer cell line

Separation conditions for antibodies, glycoproteins and peptides were optimized to fully realize the potential of automated imaged capillary isoelectric focusing (imaged cIEF) for protein analysis. Two commercially available capillary coatings, polyacrylamide and fluorocarbon, were found to provide reproducible results for cIEF separations. Both coatings could last more than 100 runs under normal cIEF conditions. Up to 30 mM salts (Na+) could be added to samples to prevent protein precipitation before and during isoelectric focusing performed under imaged cIEF. Short analysis time of the imaged cIEF also aided in the prevention of protein precipitation. High current at the beginning of the focusing for samples in salt could be avoided by applying a voltage gradient. Additions of up to 6 M urea and 20% glycerol could enhance solubility of proteins and peptide. Imaged cIEF was applied to the quantitation of monoclonal antibodies.     

Identification and synthesis of altered peptides modulating T cell recognition of a synthetic peptide antigen

In studies of T cell responses to synthetic peptides we have observed agonist and antagonist activities associated with contaminants identified within the parent synthesis. The synthesis of two candidate analogues implied by a peptide contaminant formed during the synthesis of La 51-58 (IMIKFNRL) has been carried out. The peptide contaminant was 17-18 Da smaller than the parent peptide consistent with a modified asparagine residue at position 6 and so we synthesised both an aspartimide and a nitrile analogue, representing cyclisation or dehydration of the asparagine residue. The candidate aspartimide and nitrile analogues both bound empty MHC class I molecules to form allo determinants recognised by monoclonal antibodies. These results demonstrate that altered synthetic peptides can bind class I MHC molecules and prompt caution in the use of synthetic peptides as a source of immunising antigen.     

Ca2+-dependent inactivation of a store-operated Ca2+ current in human submandibular gland cells. Role of a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump

Stimulation of human submandibular gland cells with carbachol, inositol trisphosphate (IP3), thapsigargin, or tert-butylhydroxyquinone induced an inward current that was sensitive to external Ca2+ concentration ([Ca2+]e) and was also carried by external Na+ or Ba2+ (in a Ca2+-free medium) with amplitudes in the order Ca2+ > Ba2+ > Na+. All cation currents were blocked by La3+ and Gd3+ but not by Zn2+. The IP3-stimulated current with 10 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-triphosphate and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette solution, showed 50% inactivation in <5 min and >5 min with 10 and 1 mM [Ca2+]e, respectively. The Na+ current was not inactivated, whereas the Ba2+ current inactivated at a slower rate. The protein kinase inhibitor, staurosporine, delayed the inactivation and increased the amplitude of the current, whereas the protein Ser/Thr phosphatase inhibitor, calyculin A, reduced the current. Thapsigargin- and tert-butylhydroxyquinone-stimulated Ca2+ currents inactivated faster. Importantly, these agents accelerated the inactivation of the IP3-stimulated current. The data demonstrate that internal Ca2+ store depletion-activated Ca2+ current (ISOC) in this salivary cell line is regulated by a Ca2+-dependent feedback mechanism involving a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump. We suggest that the Ca2+ pump modulates ISOC by regulating [Ca2+]i in the region of Ca2+ influx.     

p53 modulation of Fas/Apo-1 mediated apoptosis in a human renal cell carcinoma cell line

The mutant p53 gene was transfected into ACHN, a wild-type p53-containing human renal cell carcinoma (RCC) cell line. The colony forming efficiency in soft agar in the mutant-type p53-transfected cell line (ACHN/MP) was significantly higher than that in the vector-only transfected control cell line (ACHN/C). The anti-Fas monoclonal antibody (CH11) induced apoptosis in the ACHN/C cells in a dose-dependent manner, whereas the effect of CH11 on the ACHN/ MP cells was markedly suppressed. In addition, the cytotoxic effect of CH11 on the ACHN/MP cells was augmented by the pretreatment with interferon- , but the corresponding effect on ACHN/C cells was not. These findings suggest that Fas-mediated therapy could be a novel approach to RCC, if interferon- treatment is added according to the p53 gene status.     

Loss of heterozygosity analysis in a human fibrosarcoma cell line

Loss of heterozygosity (LOH) is an important event in tumor formation. We have used polymorphic microsatellite repeat markers to identify and characterize LOH in spontaneous mutants of a human cell line, MR12-1, that is heterozygous for the adenine phosphoribosyltransferase gene (APRT+/-) located on chromosome 16q24.3. Initially, clones without extensive LOH (which are likely derived as a consequence of intragenic point mutations) and clones with multilocus LOH (which are likely due to major chromosome alterations) were identified. Clones with major regions of LOH were further characterized by assaying additional informative microsatellite markers. Analysis of 20 spontaneously-arising, independent APRT-/- clones from MR12-1 demonstrated that nine of the mutants retained both copies of APRT and 11 had undergone multilocus genetic alterations. The nature of LOH in four of the latter clones has been examined in detail by karyotype and fluorescence in situ hybridization analysis (Shao et al., 1996). These data demonstrate that LOH of chromosome 16 may be due to mitotic recombination, interstitial or partial deletion, or to more complex mechanisms. LOH in these clones may be a consequence of events similar to those observed in many tumors.