Human tumor cells grown in fetal calf serum and human serum: influences on the tests for lymphocyte cytotoxicity, serum blocking and serum arming effects

Peripheral blood lymphoid cells (PBL) from cancer patients and normal donors were tested against three melanoma cell lines grown in either 10% fetal calf serum (FCS) or 2.5-5% human AB serum in order to determine if the heterologous membrane (HM) antigen or other FCS antigens acquired from the bovine serum supplement could influence lymphoid cell-mediated cytotoxicity in vitro. FCS-grown melanoma cells were more susceptible than the AB serum-grown subline to lymphocyte cytotoxic effects. Arming effects by autologous sera on normal donor lymphocytes and to a lesser extent on lymphocytes of cancer patients were more pronounced on the FCS-grown M12 melanoma cells. This effect was abrogated when the cells were grown in human AB serum for at least 8 weeks. The non-HM tumor-associated antigen remained at the same original low level. Blocking effects were more evident on the AB-grown M14 melanoma line. These data suggest that the FCS antigens on the cell surface may have been responsible for the augmented PBL cytotoxicity. The anti-FCS antibody present in normal and cancer patients' blood induced an antibody-dependent cellular cytotoxicity (ADCC). Elimination of arming activity against HM or other FCS antigens from AB-grown cells may have made the serum blocking factors more apparent. However, cytotoxicity against tumor cells by PBL from normal donors was still apparent even on the human serum-grown cells, suggesting that a different antigen-antibody system was also responsible for this "non-specific" activity.     

Effects of propentofylline on tumor necrosis factor-alpha and cytokine-induced neutrophil chemoattractant production in rats with cerulein-induced pancreatitis and endotoxemia

Using rabbit polyclonal antiurinary protein 1 antibody to study the female prostate (Skene's gland) and the male prostate, characteristic localizations patterns appeared in single cells and groups of cells. The majority correspond to cells positive for neuroendocrine markers. In the cytoplasm, cells positive for protein 1 were most frequently found in the epithelial lining of the female urethra, in the pars prostatica of the male urethra, and in the ducts of the female and male prostate where the lining consisted of pseudostratified columnar epithelium. Their occurrence rate was far lower among secretory and basal cells of the male and female prostate glands. The cells with protein 1 corresponded to those displaying positivity for chromogranin A, silver staining by the Grimelius and less by the Sevier-Munger method, and by neuron specific enolase. Using the Masson-Hamperl argentaffin method, positive cells were only exceptionally found. The cells positive for protein 1, and particularly chromogranin A, and characterized by Grimelius positivity, contained different amounts of neuroendocrine granules and varied in size and shape. The majority of these cells had contact with the lumen of male and female prostatic ducts (open type of neuroendocrine cells). In some cases of the male and female urethra and of the great paraurethral ducts, a remarkably high number of cells containing protein 1 corresponded to cells only containing neuron-specific enolase but not chromogranin A and other neuroendocrine markers. These cells can be considered stem cells responsible for the renewal of the uroepithelium of the urethra and prostatic ducts. Protein 1 may thus be a further, though presumably not specific marker for the identification of cells of the neuroendocrine system in the prostate of the male and female. This marker could well be used to study uroepithelium maturation. The corresponding immunohistochemical distribution of human protein 1 in neuroendocrine and other cells of the male and the female prostate provides another analogous functional and morphological parameter of prostatic tissue in both sexes and further evidence supporting the non-vestigial concept of the prostate in the female.     

Biochemical diagnosis of neuroendocrine GEP tumor

Neuroendocrine gut and pancreatic tumors are known to contain and secret different peptide hormones and amines. During the last two decades, many radioimmunoassays and Elizas have been developed to analyze these substances in blood and urine, which has enabled clinicians to improve the diagnosis and monitoring of patients with various neuroendocrine tumors. Due to cost constraints in medical care, it is important to try to define the most useful biochemical markers from the clinical point of view. The glycoprotein chromogranin A has been shown to be a useful marker for diagnosing various neuroendocrine tumors, both by histopathology and circulating tumor markers. In patients with demonstrable endocrine tumors, about 90 percent of the patients present high circulating levels of chromogranin A. A hundred-fold increase of plasma chromogranin is seen in patients with midgut carcinoid tumors and liver metastases. The plasma levels of chromogranin A reflect the tumor mass and can be used for monitoring the patient during treatment and follow-up, although the day-to-day variation might be 30-40 percent. High circulating levels of the chromogranin A might be an indicator of bad prognosis in patients with malignant carcinoid tumors. Besides analyzing plasma chromogranin A, specific analyses such as urinary 5-HIAA in midgut carcinoid patients, serum gastrin in patients with Zollinger-Ellison syndrome and insulin/proinsulin in patients with hypoglycemia should be performed. In patients with small tumor masses or intermittent symptoms, provocative tests such as a meal stimulation test, secretin test or pentagastrin stimulation of tachykinin release can supplement the basal measurements of peptides and amines. To fully evaluate the growth potential in neuroendocrine tumors, traditional biochemical markers should be supplemented with indicators of growth proliferation (Ki-67, PCNA) and immunohistochemical staining for the adhesion molecule CD44 and the PDGF-alpha receptor. Finally, analysis of somatostatin receptor subtypes and induction of the enzymes 2-5A syntethase and PKR are of clinical value.     

Prostasomes are neuroendocrine-like vesicles in human semen

BACKGROUND: Prostasomes are prostate-derived organelles that exist extracellularly in human seminal plasma. METHODS: In this study, we have investigated and characterized human prostasomes with regard to their contents of synaptophysin, members of the chromogranin family, and some neuropeptides. RESULTS: By radioimmunoassay measurement and electron microscopy we show the presence of the neuroendocrine markers chromogranin B, neuropeptide Y, and vasoactive intestinal polypeptide in about equimolar amount in human prostasomes and chromogranin A in about 2% of that amount. To our knowledge, such a high ratio of chromogranin B to chromogranin A has never before been observed. The membrane-bound protein synaptophysin, a well-established immunocytochemical marker for neuroendocrine cells and neurones, was also detected. Hence, we show that synaptophysin could be used as a marker for intact prostasomes. CONCLUSIONS: The presence of synaptophysin has recently been shown in the serotonincontaining vesicles in platelets. A protein with a similar structure denoted granulophysin has been found in granulocytes and prostasomes. It is suggested that synaptophysin and granulophysin molecules are members of a family of proteins, maybe expressed in all cells that have regulated release of granule content. Our presented data indicate a neurotransmittor function of the prostasomes. The target cells are however not known but could be either the spermatozoa, the epithelial mucous cells of the uterus or tubas or perhaps the ovum.     

Generation of reactive oxygen species and activity of platelet-activating factor acetylhydrolase in human monocyte-derived macrophages

Monocytes were prepared from healthy human volunteers and were allowed to differentiate into macrophages by adhesion to plastic surface and cultured over 7 days in presence of either 10% fetal calf serum (FCS), human control serum or serum from hyperlipaemic patients. Hyperlipaemic serum stimulated the differentiation (measured as an increase in cellular protein and DNA content) to a higher extent when compared to control serum and FCS. With all sera a marked increase of the cellular activity of the enzyme platelet-activating factor acetylhydrolase (PAF-AH) and a tremendous decrease in the capacity of cells to generate reactive oxygen species (ROS) was observed. After seven days of culture the increase in PAH-AH activity was about 19-fold with hyperlipaemic serum, 11-fold with control serum and 6-fold with FCS. During the same period of time ROS generation measured as zymosan-induced chemiluminescence decreased by about 98% and no significant differences between the three types of serum were found. The results indicate that the activity of PAF-AH and the capacity of ROS generation which are both assumed to play an important role in the oxidation of low-density lipoproteins (LDL) and thus in the development of atherosclerosis, change in opposite direction during the differentiation of blood monocytes into macrophages, and that hyperlipaemic serum stimulates PAF-AH activity but not ROS generation.     

Effects of lactoferrin and its peptides on proliferation of rat intestinal epithelial cell line, IEC-18, in the presence of epidermal growth factor

The cell growth-stimulating activity of lactoferrin (LF) in combination with epidermal growth factor (EGF) was evaluated by using a rat intestinal epithelial cell line, IEC-18. LF was found to be more effective than EGF for inducing an increase in cell numbers when cultured for over 6 days using a medium containing 0.2% fetal calf serum (FCS), although the 3H-thymidine incorporation-stimulating activity of EGF was more potent than that of LF. A synergistic effect of LF and EGF was observed in both cell proliferation and DNA synthesis assays. The increase in cell numbers when stimulated with LF plus EGF corresponded to about 5 times that of the control. Iron was not required for manifestation of these effects of LF. On the other hand, iron-saturated transferrin (TF) had cell-growth-stimulating activity, but iron-free TF did not, either in the presence or absence of EGF. These results indicate that LF induces cell proliferation by a mechanism distinct from that of TF. A pepsin-generated hydrolysate of LF (LFH) had an activity similar to that of undigested LF, and a peptide with cell-growth-stimulating activity from bovine LFH was isolated by monitoring its effects in combination with EGF on DNA synthesis in IEC-18 cells. Sequence analysis indicated that the peptide has the structure Ala-Glu-Ile-Tyr-Gly-Thr-Lys-Glu-Ser-Pro-Gln-Thr-His-Tyr-Tyr, corresponding to residues 79-93 of bovine LF.     

Neuroendocrine properties of adrenocortical cells

Recent data suggest that adrenocortical cells under pathological as well as under physiological conditions show neuroendocrine properties. Within the normal adrenal, this neuroendocrine differentiation seems to be restricted to cells of the zona glomerulosa and might be important for an autocrine regulation of adrenocortical function. In addition, such neuroendocrine differentiation is a common phenomenon in adrenocortical carcinomas and is therefore of clinical importance. In our studies, the expression of neuronal cell adhesion molecule (NCAM) could be shown in the zona glomerulosa of the normal human adrenal and in the human adrenocortical cell line NCI-H295 that also produces synaptophysin, a synaptic vesicle associated protein. In this chapter, data on neuroendocrine characteristics of adrenocortical cells are summarized and discussed.     

Signal transduction for proliferation of glioma cells in vitro occurs predominantly through a protein kinase C-mediated pathway

Previous work has demonstrated that glioma cells have very high protein kinase C (PKC) enzyme activity when compared to non-malignant glia, and that their PKC activity correlates with their proliferation rate. The purpose of this study was to determine whether the elevated PKC activity in glioma is secondary to an autonomously active PKC isoform implying oncogenic transformation, or whether this activity is driven by upstream ligand-receptor tyrosine kinase interactions. We treated established human glioma cell lines A172, U563 or U251 with either the highly selective PKC inhibitor CGP 41 251, or with genistein, a tyrosine kinase inhibitor. The proliferation rate and PKC activity of all the glioma lines was reduced by CGP 41 251; the IC50 values for inhibiting cell proliferation corresponded to the IC50v values for inhibition of PKC activity. Genistein also inhibited cell proliferation, with IC50 proliferation values approximating those for inhibition of tyrosine kinase activity in cell free protein extracts. Importantly, in genistein-treated cells, downstream PKC enzyme activity was dose dependently reduced such that the correlation coefficient for effects of genistein on proliferation rate and PKC activity was 0.92. These findings suggest that upstream tyrosine kinase linked events, rather than an autonomously functioning PKC, result in the high PKC activity observed in glioma. Finally, fetal calf serum (FCS) evoked a strong mitogenic effect on glioma cell lines. This mitogenic activity was completely blocked by CGP 41 251, suggesting that although the many mitogens in FCS for glioma cells signal initially through genistein-inhibitable tyrosine kinases, they ultimately channel through a PKC-dependent pathway. We conclude that proliferative signal transduction in glioma cells occurs through a predominantly PKC-dependent pathway and that selectively targeting this enzyme provides an approach to glioma therapy.     

Effects of fetal calf serum and disruption of cadherin function on the formation of bile canaliculi between hepatocytes

The polarization of hepatocytes to form a connected network of bile canaliculi (BC) is necessary for the function of the liver. Hepatocyte polarization may be controlled by soluble factors and/or physical interactions between cells. Monolayer cultures of embryonic chicken hepatocytes in DMEM supplemented with ornithine, dexamethasone, and insulin express BC-specific antigens for at least 7 days. However, BC-specific antigen expression is lost within 3 days of culture initiation in DMEM containing 10% fetal calf serum. The dedifferentiating effects of fetal calf serum (FCS) can be reversed. Furthermore, cultures in medium containing ornithine, dexamethasone, insulin, and 10% FCS appear identical to cultures grown in 10% FCS alone. Thus FCS contains a soluble inhibitor of hepatocyte polarization. Aggregate cultures grown in suspension maintain hepatocyte polarization for 10-12 days. This may be due to the increased cell-cell contact between hepatocytes in aggregate culture or to more normal contact with the extracellular matrix. We have evaluated the role of cadherin-mediated interactions on hepatocyte polarization. Anti-E-cadherin Fab' fragments disrupted the formation of long networks of BC in monolayer cultures but did not stop polarized expression of BC-specific antigens. The BC antigens in anti-E-cadherin-treated cells were concentrated in small areas between cells and were present at lower levels uniformly on the cell surface. These results indicate that E-cadherin is required for the formation of extended BC networks, but that other factors are responsible for maintaining the synthesis and localization of BC-specific antigens.     

Photocytotoxic effect of pseudohypericin versus hypericin

Pseudohypericin and hypericin, the major photosensitizing constituents of Hypericum perforatum, are believed to cause hypericism. Since hypericin has been proposed as a photosensitizer for photodynamic cancer therapy, the photocytotoxicity of its congener pseudohypericin has been investigated. The presence of foetal calf serum (FCS) or albumin extensively inhibits the photocytotoxic effect of pseudohypericin against A431 tumour cells, and is associated with a large decrease in cellular uptake of the compound. These results suggest that pseudohypericin, in contrast to hypericin, interacts strongly with constituents of FCS, lowering its interaction with cells. Since pseudohypericin is two to three times more abundant in Hypericum than hypericin and the bioavailabilities of pseudohypericin and hypericin after oral administration are similar, these results suggest that hypericin, and not pseudohypericin, is likely to be the constituent responsible for hypericism. Moreover, the dramatic decrease of photosensitizing activity of pseudohypericin in the presence of serum may restrict its applicability in clinical situations.     

Functional analysis of the C2A domain of synaptotagmin 1: implications for calcium-regulated secretion

Synaptotagmin 1 is proposed to function as a low affinity calcium sensor for calcium-triggered exocytosis from neural and neuroendocrine cells. Because of the calcium-binding properties of the C2A domain of synaptotagmin 1, calcium-dependent interactions through this domain may modulate neurotransmitter release. We addressed this question by using alanine-scanning mutagenesis to generate a series of mutations within the C2A domain of synaptotagmin 1. The effects of these mutations on synaptotagmin 1 C2A function were analyzed for (1) calcium-dependent phospholipid binding, (2) calcium-dependent binding to syntaxin 1A, a plasma membrane protein critical for vesicle docking or fusion, and (3) calcium-regulated secretion after microinjection into neuroendocrine pheochromocytoma (PC12) cells. Our analyses reveal that a polylysine motif at residues 189-192 confers an inhibitory effect on secretion by recombinant synaptotagmin C2A fragments. The synaptotagmin 1 C2A polylysine motif functions independently of calcium-mediated interactions with phospholipids and syntaxin 1A. Furthermore, alpha-latrotoxin reverses the inhibitory effect of injected recombinant C2A fragments, suggesting that they perturb the cellular calcium-sensing machinery by interfering with synaptotagmin 1 activity in vivo. Our results indicate that novel calcium-independent interactions mediated through the C2A polylysine motif of synaptotagmin 1 function to modulate neurotransmitter release.     

Novel autocrine feedback control of catecholamine release. A discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist

Catecholamine secretory vesicle core proteins (chromogranins) contain an activity that inhibits catecholamine release, but the identity of the responsible peptide has been elusive. Size-fractionated chromogranins antagonized nicotinic cholinergic-stimulated catecholamine secretion; the inhibitor was enriched in processed chromogranin fragments, and was liberated from purified chromogranin A. Of 15 synthetic peptides spanning approximately 80% of chromogranin A, one (bovine chromogranin A344-364 [RSMRLSFRARGYGFRGPGLQL], or catestatin) was a potent, dose-dependent (IC50 approximately 200 nM), reversible secretory inhibitor on pheochromocytoma and adrenal chromaffin cells, as well as noradrenergic neurites. An antibody directed against this peptide blocked the inhibitory effect of chromogranin A proteolytic fragments on nicotinic-stimulated catecholamine secretion. This region of chromogranin A is extensively processed within chromaffin vesicles in vivo. The inhibitory effect was specific for nicotinic cholinergic stimulation of catecholamine release, and was shared by this chromogranin A region from several species. Nicotinic cationic (Na+, Ca2+) signal transduction was specifically disrupted by catestatin. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. This small domain within chromogranin A may contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release from chromaffin cells and neurons.     

Vascular malformations of the jaw bones. Report on nine patients

Partial complementary DNA (cDNA) for thymidine phosphorylase (dThdPase) was cloned by means of a polymerase chain reaction. There was complete sequence identity between the amino acid sequence deduced from the nucleotide sequence of a clone (288 nucleotides) and the residues of platelet-derived endothelial cell growth factor (PD-ECGF). The amino acid sequence of all four peptide fragments from purified human dThdPase could be aligned with that of PD-ECGF. Our data indicate that residues 125-244 of PD-ECGF are identical to the sequence of human dThdPase. The molecular weights of human dThdPase and recombinant PD-ECGF (rPD-ECGF) that lacks 10 amino acids at the amino terminal were 55 and 52 kDa, respectively. Anti-PD-ECGF antibody recognized dThdPase, and anti-dThdPase antibody recognized rPD-ECGF. rPD-ECGF had dThdPase activity and its specific activity was similar to that of purified human dThdPase. dThdPase activity and molecules were detected in COS cells transfected with human PD-ECGF cDNA, but not in nontransfected cells. The sizes of PD-ECGF and dThdPase in the transfected COS cells were identical. These data suggest that human dThdPase is identical to PD-ECGF.     

Phosphorylation of vitronectin by casein kinase II. Identification of the sites and their promotion of cell adhesion and spreading

The cell adhesion protein vitronectin (Vn) was previously shown to be the major target in human blood for an extracellular protein kinase A, which is released from platelets upon their physiological stimulation with thrombin and also prevails as an ectoenzyme in several other types of blood cells. Because plasma Vn was shown to have only one protein kinase A phosphorylation site (Ser378) but to contain approximately 3 mol of covalently bound phosphate, and because human serum and blood cells were shown to contain also a casein kinase II (CKII) on their surface, we studied the phosphorylation of Vn by CKII attempting to find out whether such phosphorylation modulates Vn function, an acid test for its having a physiological relevance. Here we show (i) that the CKII phosphorylation of Vn has a Km of 0.5-2 microM (lower than the Vn concentration in blood, 3-6 microM), (ii) that it is targeted to Thr50 and Thr57, which are vicinal to the RGD site of Vn, and (iii) that the phosphorylation of Thr57 facilitates the phosphorylation of Thr50. The maximal stoichiometry of the CKII phosphorylation of plasma Vn was found to be low, which, in principle, could be due to its partial prephosphorylation in vivo. However, for the detection of a functional modulation, we needed a comparison between a fully phosphorylated Vn (at Thr57 and Thr50) and a nonphosphorylated Vn. Therefore, we expressed Vn in a baculovirus system and show (i) that the CKII phosphorylation of wt-Vn enhances the adhesion of bovine aorta endothelial cells; (ii) that the double mutant T50E/T57E (in which the neutral Thr residues are replaced by the negatively charged Glu residues considered analogs of Thr-P) has a significantly enhanced capacity to promote cell adhesion and to accelerate cell spreading when compared with either wild-type Vn or to the neutral T50A/T57A mutant; and (iii) that, at least in the case of bovine aorta endothelial cells, the T50E/T57E mutant exhibits an enhanced adhesion, which seems to be due to an increased affinity toward the alphav beta3 Vn receptors.     

Not just scaffolding: plectin regulates actin dynamics in cultured cells

Plectin, a major linker and scaffolding protein of the cytoskeleton, has been shown to be essential for the mechanical integrity of skin, skeletal muscle, and heart. Studying fibroblast and astroglial cell cultures derived from plectin (-/-) mice, we found that their actin cytoskeleton, including focal adhesion contacts, was developed more extensively than in wild-type cells. Also it failed to show characteristic short-term rearrangments in response to extracellular stimuli activating the Rho/Rac/Cdc42 signaling cascades. As a consequence, cell motility, adherence, and shear stress resistance were altered, and morphogenic processes were delayed. Furthermore, we show that plectin interacts with G-actin in vitro in a phosphatidylinositol-4,5-biphosphate-dependent manner and associates with actin stress fibers in living cells. The actin stress fiber phenotype of plectin-deficient fibroblasts could be reversed to a large degree by transient transfection of full-length plectin or plectin fragments containing the amino-terminal actin-binding domain (ABD). These results reveal a novel role of plectin as regulator of cellular processes involving actin filament dynamics that goes beyond its proposed role in scaffolding and mechanical stabilization of cells.     

Immunochemical analysis of UMP kinase from Escherichia coli

Mono- and polyclonal antibodies directed against UMP kinase from Escherichia coli were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in whole E. coli cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.     

Dominant-negative effect of the lymphocyte function-associated antigen-1 beta (CD18) cytoplasmic domain on leukocyte adhesion to ICAM-1 and fibronectin

The cytoplasmic domains of LFA-1 (CD11a/CD18) are thought to play an important role in the regulation of LFA-1 function. To further elucidate the role of the LFA-1 cytoplasmic domains, we transfected chimeric proteins consisting of the extracellular domain of CD4 fused with the transmembrane and cytoplasmic domains of LFA-1 into T and B cell lines, EL-4 and A20, respectively, and examined their effects on LFA-1-mediated cell adhesion. The CD4/18, but not CD4/11a, chimera profoundly inhibited LFA-1-mediated cell adhesion to ICAM-1, as well as cell spreading following cell adhesion. Unexpectedly, cell adhesion to fibronectin was also inhibited by the CD4/18 chimera. The CD4/18 chimera did not affect the expression of endogenous LFA-1 or the association of CD11a and CD18. Truncation of the carboxyl-terminal 13 amino acid residues of the CD18 cytoplasmic domain of the chimera completely abrogated the inhibitory effect on LFA-1. Among these amino acid residues, the carboxyl-terminal six residues were dispensable for the inhibitory effect in EL-4 cells, whereas it significantly reduced the inhibitory activity of CD4/18 in A20 cells. A larger truncation of the CD18 cytoplasmic domain was needed to fully abrogate the inhibitory effects of CD4/18 on the adhesion to fibronectin. These results show that 1) the CD4/18 chimera has dominant-negative effects on cell adhesion mediated by LFA-1 as well as fibronectin receptors, and 2) amino acid residues of the CD18 cytoplasmic domain involved in the inhibition of LFA-1 seem to be different from those for fibronectin receptors.