Effects of bovine beta-casein (1-28) and its chemically synthesized partial fragments on proliferative responses and immunoglobulin production in mouse spleen cell cultures.

Effects of bovine beta-casein (1-28) having a phosphoserine-rich region (Glu14-SerP-Leu-SerP-SerP-SerP-Glu-Glu21) and its chemically synthesized partial fragments on proliferation of lymphocytes and immunoglobulin production were investigated in mouse spleen cell cultures. The parent fragment 1-28 and all fragments containing SerP-Leu-SerP and/or SerP-SerP-SerP had a significant mitogenic effect, stimulated proliferation of lymphocytes induced by lipopolysaccharide, phytohemagglutinin, or concanavalin A, and increased immunoglobulin (IgG + IgM + IgA) or IgA levels in the cell cultures. In contrast, dephosphorylated beta-casein (14-21) and SerP-SerP amide had hardly any immunoregulatory activity. On the other hand, SerP-Leu-SerP amide reacted little with antibodies specific to bovine beta-casein (1-28), but beta-casein (14-21), and SerP-SerP-SerP amide obviously reacted with the antibody. These results confirm that the immunoregulatory activity of casein phosphopeptides is attributable to SerP-X-SerP, which may well be available as a non-allergic food ingredient having an adjuvant activity for mucosal IgA responses.     

CD8+ CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+ CD28- T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+ CD28- T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+ CD25+ T regulatory lymphocytes associate with CD8+ CD28- T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.     

A 45-kDa human T-cell membrane glycoprotein functions in the regulation of cell proliferative responses

A 45-kDa human T cell surface glycoprotein which is tightly bound in the membrane of the resting T cell is released into the cell medium in soluble form after cell growth activation by phytohemagglutinin or neuraminidase/galactose oxidase treatments. In limited proteolysis by Staphylococcus aureus V8 protease, two major 35-kDa and 27-kDa peptide fragments of the surface-iodinated 45-kDa protein are common to the membrane-bound and the released forms, but a third 18-kDa fragment is observed exclusively with the released protein. The apparent molecular masses of the deglycosylated peptide backbones of the membrane-bound and the released molecule are 30 +/- 1 kDa, although a small size difference cannot be excluded. A polyclonal rabbit anti-(T cell membrane protein) antiserum precipitates the 45-kDa protein. A monoclonal anti-(45-kDa protein) antibody precipitates the membrane-bound 45-kDa protein solubilized with octyl glucoside, but does not precipitate the released protein. In cell culture assays, the monoclonal anti-(45-kDa protein) antibody specifically enhances the cell proliferative responses in phytohemagglutinin-treated and mixed lymphocyte cultures. These observations suggest that the 45-kDa protein has a specific receptor function in the regulation of cell proliferative responses.     

Somatostatin 28(1-12) in a somatostatin-secreting human medullary thyroid carcinoma cell line

We have identified a system, the TT human medullary thyroid carcinoma cell line, which we found to contain 31.3 +/- 27.7 ng of somatostatin 28(1-12) immunoreactivity/mg protein. Radioimmunoassay of gel filtration fractions showed that the major form of immunoreactive somatostatin 28(1-12) had a molecular weight of 1,500 daltons. During reversed-phase high pressure liquid chromatography, this 1,500-dalton species coeluted with synthetic somatostatin 28(1-12). Somatostatin 28(1-12) containing forms larger than 7,000 daltons were also observed. Further studies will be required to elucidate the route of processing of prosomatostatin. The fact that the products of prosomatostatin processing in these cells are similar to those in normal tissues indicates that the TT medullary thyroid carcinoma cell line constitutes a useful model for human somatostatin gene expression.     

Platelet-endothelial cell adhesion molecule-1 (CD31) recycles and induces cell growth inhibition on human tumor cell lines

CD31 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, and most leukocytes. This report demonstrates by Western Blot and immunofluorescence that some human melanoma and adenocarcinoma cell lines express CD31 on the cell surface. The surface expression of CD31 was regulated by cell-cell contact, being higher on sparse and spontaneously detached cells. Indeed, fixing and permeabilizing tumor cells revealed a cytoplasmic pool, which was confirmed by confocal microscopy. Some of the plasma surface molecule is endocytosed following mAb binding. Engagement of CD31 on tumor cells via domain-3 inhibited proliferation by inducing cell apoptosis. On the other hand, apoptosis does not increase CD31 expression. Overall, these results indicate that there is an intracellular pool of CD31 on some tumor cells, which modulates CD31 surface expression and its role in cancer cell growth and metastasis. Thus, the expression of CD31 and its role in cell survival in some tumor cells appears to differ from endothelial cells.     

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene restored subclonal cell line (? = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG(-/-) cells were radioresistant relative to the transgene-restored subclonal cell line (? = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2(-/-) cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.     

Secondary conformations and temperature effect on structural transformation of amyloid beta (1-28), (1-40) and (1-42) peptides

Secondary structure of three amyloid b-peptides [A beta(1-28), A beta(1-40) and A beta(1-42)] in the solid state was respectively determined by Fourier transform infrared (FT-IR) microspectroscopy. Their thermal-dependent structural transformation were also investigated by FT-IR microspectroscopy equipped with a thermal analyzer. The present result demonstrates that the solid-state A beta(1-28), A beta(1-40) and A beta(1-42) peptides showed a significant IR spectral difference in the amide I and II bands. The secondary conformation of A beta(1-28) peptide was the combination of major beta-sheet and minor alpha-helix with little random coil structures, but A beta(1-40) peptide showed the co-existence of major beta-sheet and minor random coil with little alpha-helix structures. A beta(1-42) peptide mainly consisted of the predominant b-sheet structure. Although the intact A beta(1-28), A beta(1-40) or A beta(1-42) peptide exhibits a different secondary structure, a similar beta-conformation may form after thermal treatment. A thermal-dependent transition was found for solid A beta(1-28) and A beta(1-40) peptides near 40 degrees C and 45 degrees C, respectively. There was no transition temperature for solid A beta(1-42) peptide, however, due to only a very little level of alpha-helix and random coil structure containing in the solid A beta(1-42) peptide. The thermal denaturation plays an important role in the structural transformation from alpha-helix/random coil to beta-sheet.     

Effect of polylysine-bound laminin on human retinoblastoma cell lines

Summary We have studied the responses of three human retinoblastoma cell lines (GM1232, Y79, and WERI-Rb1) to substratum-bound laminin (LN), fibronectin (FN), or collagen (CN) in serum-containing medium. About 95% of the cells attached to poly-d-lysine (PN)-pretreated plastic surfaces either unbound or protein-bound within 1 h after plating. With PN-bound LN, GM1232 cells showed an outgrowth of processes and cell spread within 1 d, but very little was seen on PN-bound FN, CN, or unbound PN even by Day 4. Both the percentage of cells with processes and the number of processes/cell were dependent on the amount of LN bound to the surfaces at Day 4. On LN surfaces without PN pretreatment, by Day 2 most cells had formed floating aggregates and were not attached to the surfaces. By Day 4 only a part of the peripheral cells of attached aggregates displayed process outgrowth and cell spreading. Dibutyryl cyclic AMP (dbcAMP) maintained these effects of PN-bound LN, and promoted process branching, cell spreading, and elongation with concomitant inhibition of cell growth. The percentage of cells with processes was 83%. Y79 and WERI-Rb1 cells, passed for several years, showed little and weak response to PN-bound LN either in the absence or presence of dbcAMP. These results indicate that the morphologic differentiation of GM1232 cells is elicited specifically by PN-bound LN, and that dbcAMP maintains and promotes this differentiated status.     

Effect of recombinant human TNF on human ovarian cancer cell lines

Ovarian cancer is the second most common gynecologic malignancy. Standard therapeutic approaches to this disease, surgery followed by chemotherapy, have produced response rates of up to 80%. However, the five-year survival rate remains around 30%. Recently, Tumor Necrosis Factor (TNF) has received attention as either an alternative or an associated agent for chemotherapy of ovarian cancer. TNF is known to have direct cytotoxic and cytostatic effects on a variety of transformed cell lines "in vitro". Furthermore, TNF is known to enhance significantly the "in vitro" effects of a class of chemotherapeutic agents, specifically those targeted at DNA topoisomerase II. In this work we have investigated TNF-induced cytotoxicity in four established human epithelial ovarian cancer cell lines: A-2774; SV-626; SKOV-3 and Pa-1. TNF mediated cytotoxic activity was observed in a range of concentrations between 1 U/ml and 10-3 U/ml. A-2774 and SV-626 were the two most sensitive lines, especially when exposed to high concentrations of TNF.     

Different effects of three kinds of somatostatin (15-28, 1-14, 1-28) on rabbit's platelet aggregation

We studied the different effects of three kinds of somatostatin (somatostatin 1-14, somatostatin 15-28, somatostatin 1-28) on the aggregation of rabbit's platelets. It was clarified that somatostatin 15-28 had inhibitory effects on rabbit's platelet aggregation stronger than somatostatin 1-14 did, and that somatostatin 1-28 did not have any such effects. These anti-aggregatory effects of somatostatin were stronger when induced by collagen than induced by ADP.     

Expression of the marker(X)(q28) in lymphoblastoid cell lines

The marker(X)(q28) chromosome associated with one type of X-linked mental retardation has been demonstrated in lymphoblastoid cell lines established from affected individuals. The mar(x) can reliably and repeatedly be seen by the addition of FUdR to the cultures for 24 hrs prior to harvest. This simple technique provides an excellent in vitro experimental test system for investigation of the mar(X).     

Effect of B cell differentiation factor (BCDF) on biosynthesis and secretion of immunoglobulin molecules in human B cell lines

Stimulation of human B lymphoblastoid cell lines, CESS and SKW6-CL4 with BCDF induced an increase in IgG- and IgM-secreting cells, as well as a slight increase in IgG and IgM expression on their respective surfaces. Biosynthetic labeling demonstrated that BCDF stimulation induced an increase in synthesis of secretory gamma- and mu-chains, as well as their precursors. A slight but significant increase in synthesis of membrane-bound gamma- and mu-chains and their precursors was also observed by BCDF stimulation. However, an increase in synthesis of secretory heavy chains was much higher than that in membrane-bound heavy chains in both cell lines. Pulse-chase experiments showed that increased synthesis of secretory heavy chains was not due to a decrease in degradation. BCDF stimulation induced a preferential increase in mRNA specific for secretory gamma- and mu-chains in CESS and SKW6-CL4 cells, respectively. These results suggest that BCDF induces an increase in the level of mRNA specific for secretory heavy chains, and then induces final maturation of B cells into immunoglobulin-secreting cells.     

Effect of suplatast tosilate (IPD-1151T) on cytokine production by allergen-specific human Th1 and Th2 cell lines.

Suplatast tosilate (IPD-1151T) is an antiallergic agent that suppresses airway eosinophil infiltration in asthma. We investigated the effects of IPD-1151T on proliferative response and cytokine production by human antigen-specific T cell lines. Purified protein derivatives (PPD)-specific T helper 1 (Th1) cell lines and Dermatophagoides farinae (Der f)-specific T helper 2 (Th2) cell lines were established from patients with asthma sensitized with house dust mite. Stimulation of PPD-specific and Der f-specific T cell lines with relevant antigens resulted in production of mostly interferon (IFN)-gamma and of interleukin (IL)-4 and IL-5, respectively. IPD-1151T did not inhibit the proliferative responses of either the Th1 or Th2 cell line to antigens. Although IPD-1151T did not inhibit IFN-gamma production by PPD-specific Th1 cell lines, it did inhibit IL-4 and IL-5 production by antigen-stimulated Der f-specific Th2 cell lines in a dose-dependent manner. IPD-1151T directly inhibited cytokine production by Der f-specific Th2 cell lines stimulated with immobilized anti-CD3 antibodies. Although IPD-1151T did not inhibit the clonal expansion of memory T cells among PBMCs into PPD-specific Th1 and Th2 cell lines, it did inhibit IL-4 and IL-5 production by Der f-specific Th2 cell lines but not IFN-gamma production by PPD-specific Th1 cell lines. These results suggest that IPD-1151T selectively inhibits Th2-type cytokine production.     

Programmed death (PD)-1:PD-ligand 1/PD-ligand 2 pathway inhibits T cell effector functions during human tuberculosis

Protective immunity against Mycobacterium tuberculosis requires the generation of cell-mediated immunity. We investigated the expression and role of programmed death 1 (PD-1) and its ligands, molecules known to modulate T cell activation, in the regulation of IFN-gamma production and lytic degranulation during human tuberculosis. We demonstrated that specific Ag-stimulation increased CD3+PD-1+ lymphocytes in peripheral blood and pleural fluid from tuberculosis patients in direct correlation with IFN-gamma production from these individuals. Moreover, M. tuberculosis-induced IFN-gamma participated in the up-regulation of PD-1 expression. Blockage of PD-1 or PD-1 and its ligands (PD-Ls: PD-L1, PD-L2) enhanced the specific degranulation of CD8+ T cells and the percentage of specific IFN-gamma-producing lymphocytes against the pathogen, demonstrating that the PD-1:PD-Ls pathway inhibits T cell effector functions during active M. tuberculosis infection. Furthermore, the simultaneous blockage of the inhibitory receptor PD-1 together with the activation of the costimulatory protein signaling lymphocytic activation molecule led to the promotion of protective IFN-gamma responses to M. tuberculosis, even in patients with weak cell-mediated immunity against the bacteria. Together, we demonstrated that PD-1 interferes with T cell effector functions against M. tuberculosis, suggesting that PD-1 has a key regulatory role during the immune response of the host to the pathogen.     

Microwave assisted synthesis and in vitro cytotoxicities of substituted (Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)methylene-1-methyl-1H-imidazol-4(5H)-ones against human tumor cell lines

The synthesis of several novel substituted (Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)methylene-1-methyl-1H-imidazol-4(5H)-ones structurally related to aplysinopsin have been carried out under microwave irradiation and conventional heating methods. The analogs 3a, 3b, 3d–3g, 3k and 3l were evaluated for their in vitro cytotoxic activity against an NCI 60 human tumor cell line panel. Compound 3f exhibited good growth inhibitory properties against all but four of the human cancer cell lines examined, and afforded LC₅₀ values <10 μM for 30% of the cell lines in the panel. Compound 3e was an effective inhibitor of leukemia, CNS, melanoma, and breast cancer cell growth, but generally less effective as a cytotoxic agent. Thus, the aplysinopsin analog 3f was regarded as a useful lead compound for further structural optimization.     

The effect of culture conditions on colony morphology and proliferative capacity in human prostate cancer cell lines

Primary cultures and cell lines form three types of colonies, termed holoclones, meroclones and paraclones by Barrandon and Green (Proc Natl Acad Sci U S A 84:2302-2306, 1987). They suggested that the three types correspond to colonies derived from stem, transit-amplifying and terminally differentiated cells. We determined the effect of culture conditions (seeding density, serum concentration, type of medium and substrate) on the proportion of each colony type and the cell number of individual colonies, using three prostate cancer cell lines, DU145, LNCaP and PC-3. In less favourable culture conditions, stem cell (SC) colonies tended to be lost; but in more favourable conditions, only modest increases in the proportion of SC colonies were observed. Under some conditions, cell number, but not colony-forming ability, was altered, indicating that colony cell number is controlled, at least in part, by different factors to colony formation. Colony-forming ability of individual cell lines is remarkably stable and there is little evidence for clonal evolution in culture, which might be expected and would result in more aggressive, faster-growing cells. Better understanding of how colony-forming efficiency is controlled could lead to the identification of drug targets that control SC growth and modify the progression of cancer.     

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds induce apoptotic patterns in cultures of human tumor cell lines

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds were extensively studied as anti-gestational agents and some of these molecules were also described as inhibitors of ornithine decarboxylase. Polyamine depletion has been frequently related to the induction of apoptosis and consequently we investigated DL-111-IT and analogs for this effect in myeloid (HL60), neuroblastic (SK-N-MC) and epithelial (BeWo) human tumor cell lines, by means of electron microscopy and DNA electrophoresis. HL60 and SK-N-MC appeared notably sensitive to apoptosis, whereas BeWo responsiveness was variable and frequently associated with necrosis. Our results indicate that the contragestational effect of DL-111-IT and analogs is associated with apoptotic deletion of chorionic tissue and that these molecules, due to their effect on human tumor cell lines, can be considered as antiblastic lead compounds.     

Effect of sodium butyrate on human breast cancer cell lines

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 m m . The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G₂M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.     

Effect of lycopene on cell viability and cell cycle progression in human cancer cell lines

ABSTRACT: BACKGROUND: Lycopene, a major carotenoid component of tomato, has a potential anticancer activity in many types of cancer. Epidemiological and clinical trials rarely provide evidence for mechanisms of the compound's action, and studies on its effect on cancer of different cell origins are now being done. The aim of the present study was to determine the effect of lycopene on cell cycle and cell viability in eight human cancer cell lines. METHODS: Human cell lines were treated with lycopene (1-5 uM) for 48 and 96 h. Cell viability was monitored using the method of MTT. The cell cycle was analyzed by flow cytometry, and apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling (TUNEL) and by DAPI. RESULTS: Our data showed a significant decrease in the number of viable cells in three cancer cells lines (HT-29, T84 and MCF-7) after 48 h treatment with lycopene, and changes in the fraction of cells retained in different cell cycle phases. Lycopene promoted also cell cycle arrest followed by decreased cell viability in majority of cell lines after 96 h, as compared to controls. Furthermore, an increase in apoptosis was observed in four cell lines (T-84, HT-29, MCF-7 and DU145) when cells were treated with lycopene. CONCLUSIONS: Our findings show the capacity of lycopene to inhibit cell proliferation, arrest cell cycle in different phases and increase apoptosis, mainly in breast, colon and prostate lines after 96 h. These observations suggest that lycopene may alter cell cycle regulatory proteins depending on the type of cancer and the dose of lycopene administration. Taken together, these data indicated that the antiproliferative effect of lycopene was cellular type, time and dose-dependent. KEY WORDS: lycopene, cancer, bioactive compounds, cell cycle.