Aggregation kinetics of well and poorly differentiated human prostate cancer cells

Aggregation of attachment-dependent animal cells represents a series of motility, collision, and adhesion events applicable to such diverse fields as tissue engineering, bioseparations, and drug testing. Aggregation of human prostate cancer cells in liquid-overlay culture was modeled using Smoluchowski's collision theory. Using well (LNCaP) and poorly differentiated (DU 145 and PC 3) cell lines, the biological relevance of the model was assessed by comparing aggregation rates with diffusive and adhesive properties. Diffusion coefficients ranged from 5 to 90 microm(2)/min for single LNCaP and PC 3 cells, respectively. Similar diffusivities were predicted by the persistent random walk model and Einstein relation, indicating random motion. LNCaP cells were the most adhesive in our study with reduced cell shedding, 100% adhesion probability, and enhanced expression of E-cadherin. There was an increase in DU 145 cells staining positive for E-cadherin from nearly 20% of single cells to uniform staining across the surface of all aggregates; under 30% of PC 3 aggregates stained positive. Aggregation rates were more consistent with adhesive properties than with motilities, suggesting that aggregation in our study was reaction-controlled. Relative to other assays employed here, aggregation rates were more sensitive to phenotypic differences in cell lines and described size-dependent changes in aggregation at a finer resolution. In particular, model results suggest similar aggregation rates for two-dimensional DU 145 and PC 3 aggregates and upwards of 4-fold higher rates for larger three-dimensional DU 145 spheroids, consistent with expression of E-cadherin. The kinetic model has application to spheroid production, to cell flocculation and as an adhesion assay.     

Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type–specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1–5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators. J. Cell. Biochem. 66:9–15, 1997. © 1997 Wiley-Liss, Inc.     

B cell chemoattractant CXCL13 is preferentially expressed by human Th17 cell clones

Th 17 cells represent a novel subset of CD4+ T cells that have a protective effect against extracellular microbes, while they are also responsible for autoimmune disorders in mice. However, the protein expression profile of Th17 cells remains to be clarified. In this study, we report an effective method to establish human allo-reactive Th17 cell clones and demonstrate that human Th17, but not Th1 or Th2, cells express B cell chemoattractant CXCL13, by using DNA chips, RT-PCR, and ELISA. Such a pattern was also the case in Candida albicans-specific Th17 clones and synovial fluid specimens obtained from patients with rheumatoid arthritis. The biological implication of this finding is discussed.     

Effects of growth stimulatory factors on mitogenicity and c-myc expression in poorly differentiated and well differentiated human colon carcinoma cells

We demonstrate the differential sensitivity of poorly differentiated and well differentiated human colon carcinoma cells to nutrients alone or to nutrients and polypeptide growth factors under completely serum-free conditions. 3H-Thymidine incorporation into trichloroacetic acid precipitable material and autoradiographic analysis indicated that nutrient replenishment alone was sufficient to initiate DNA synthesis in quiescent poorly differentiated cells, whereas defined polypeptide growth factors produced no additional effect. In contrast, well differentiated cells were mitogenically stimulated to a much greater extent by growth factors (epidermal growth factor + insulin + transferrin), than by nutrient replenishment alone. Expression of the c-myc protooncogene was increased approximately 5-fold after growth factor addition to the well differentiated cells. Maximal expression of c-myc occurred at 4 h post stimulation. In contrast, nutrients resulted in only a slight up-regulation of c-myc (1.8-fold) at approximately 90 min after addition. Addition of nutrients and/or growth factors to the poorly differentiated colon carcinoma cells resulted in an initial decline in c-myc expression (90 min), presumably due to removal of endogenous growth stimulators. Expression of c-myc returned to baseline levels by 24 h after additions. The results indicate that differential sensitivity to polypeptide growth factors is related to differentiation status in this model system and suggest that the insensitivity of poorly differentiated cells to exogenous growth factors may be due to a greater production of autocrine growth stimulators.     

A human B lymphocyte antigen (P-76) shared by B-cell chronic lymphocytic leukemia cells and hairy cell leukemia cells

A membrane antigen with an apparent specificity to B lymphocytes was detected with immunochemical techniques and its properties were analyzed. Anti-B-CLL serum was raised in a rabbit by immunization with B-cell chronic lymphocytic leukemia (B-CLL) cells. This anti-B-CLL serum was absorbed with erythrocytes, liver homogenate and insolubilized immunoglobulins. After further absorption with T-CLL cells, chronic myelocytic leukemia (CML) cells and acute myelocytic leukemia (AML) cells, the anti-B-CLL serum still reacted with peripheral blood B lymphocytes, B-CLL cells and hairy cell leukemia (HCL) cells. In contrast, no reactivity was seen with peripheral blood T lymphocyte or monocytes, or leukemia cells of non-B cell origin. An immunoprecipitation of radiolabeled cell surface proteins was attempted using the anti-B-CLL serum in the presence of Staphylococcus Aureus Cowan 1 (SaCl), and the precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A membrane antigen with an apparent molecular weight of 76,000 daltons (P-76) was immunoprecipitated with the anti-B-CLL serum from the lysates of normal B lymphocyte, B-CLL cells and HCL cells. The antigen (P-76) is not composed of disulfide-linked subunits and has no structural relationship with HLA-DR (Ia-like) antigens or other known antigens. These results suggest that this antigen is B-lymphocyte specific, and favour the B-lymphocyte nature of HCL cells.     

Antigen-induced acceleration of shedding and replacement of B cell antigen receptors requires mature T cells

To determine which early and intermediate events in the response of antigen-binding B cells to a T-dependent antigen (sheep erythrocytes [SRC]) require T help, the antigen-induced changes in receptor turnover and surface IgD loss in BALB/c athymic nu/nu mice were compared with that of nu/+ littermates and +/+ BALB/c mice. Nonimmune SRC antigen-binding spleen B cells (ABC) from +/+, nu/+, and nu/nu BALB/c mice coexpressed IgM and IgD, and 85 to 95% retained receptors well when incubated for 2.5 hr in 100 micrograms/ml cycloheximide (which prevents receptor replacement). Also they were able to regain their ability to bind antigen by 18 hr after pronase treatment, but not by 2 hr. However, 5 days after in vivo immunization, 1) the proportion of ABC expressing surface IgD declined from around 90% to less than 50% in +/+ mice and nu/+ mice but not in nu/nu mice; 2) substantial recovery of antigen-binding occurred by 2 hr after pronase treatment in +/+ and nu/+ ABC but not in nu/nu ABC; and 3) when spleen cells were incubated in cycloheximide, uncompensated receptor shedding reduced +/+ and nu/+ ABC by around 80% but produced only about a 10% reduction in nu/nu ABC. Thus, although the ABC in nonimmune nu/nu mice appeared normal with respect to their surface Ig turnover and expression, they failed to undergo the normal antigen-induced loss of IgD or acceleration of surface Ig shedding and replacement, suggesting that these intermediate activation events require interaction with mature T cells. To determine whether this interaction had to occur during B cell development, during the development of the immune response, or during receptor shedding or replacement itself, cell transfer experiments were carried our wherein nu/+ T cells were transferred i.v. to nu/nu littermates 1 day before immunization with SRC. In the transfer recipients, pronase-treated day 5 ABC were then able to replace and shed their receptors at the accelerated rate, like ABC from +/+ and nu/+ mice. In contrast, the co-incubation of 5-day immune nu/+ T cells with nu/nu B cells did not alter the rate of shedding or replacement.     

A unique antigen on mature B cells defined by a monoclonal antibody

A novel 42,000 dalton antigen (MB-1) expressed by mature human B cells in blood and tonsil was identified and characterized by utilizing a hybridoma monoclonal antibody. A comparison of MB-1 with other known B cell antigens suggests that the MB-1 antigen has not been previously identified. From one-and two-color immunofluorescence studies, it appears that the MB-1 antigen is found on all normal immunoglobulin (Ig)-expressing cells, but not on T cells, thymocytes, granulocytes, or platelets. Studies of malignant B cell tumors reveal that the antigen is expressed by virtually all Ig-expressing B cell tumors but only 10% of SIg- B-lineage leukemias. Data from these studies suggest that the MB-1 antigen is expressed late in B cell ontogeny before the expression of SIg.     

Characterization of an antigen expressed on activated human T cells and platelets

We have identified a protein complex on the surface of activated human T cells and platelets. This Ag, detected by the murine mAb 1B3, is not expressed on resting human T cells or platelets or on other hematopoeitic cells. The majority of T cells express the Ag after stimulation with PHA, Con A, or allogeneic cells. Platelets express the Ag after activation with 1 U/ml thrombin. Immunoprecipitation of 125I-labeled activated T cells with antibody 1B3 shows a heterodimer of 180,000 and 155,000 Mr. An additional protein of 130,000 Mr is very faintly seen depending on the cell type. On platelets, the 180,000 Mr protein is the predominant protein immunoprecipitated under both nonreducing and reducing conditions. Antibody 1B3 may prove useful in identification of activated T cells and platelets as well as definition of specific mechanisms of T cell and platelet activation.     

B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis

Human endothelial cells (ECs) provide costimulatory signals sufficient to activate resting memory T cells to produce IL-2 and IFN-gamma, at least in part through CD58-CD2 interactions. Recently, the B7-like molecule, B7-H1 (PD-L1), was described and shown to regulate T cell activation; however, there are conflicting reports on whether it stimulates or inhibits T cell cytokine synthesis. B7-H1 is not expressed constitutively by ECs; however, it is rapidly induced by IFN-gamma, and synergistically by IFN-gamma and TNF. In inflamed skin, B7-H1 is expressed by a subset of microvessels, and by keratinocytes, but is barely detectable in normal skin. Blocking the interaction of EC-expressed B7-H1 with its T cell ligand, programmed death-1 (PD-1), using a PD-1-Fc fusion protein, or by blocking B7-H1 expression with morpholino antisense oligonucleotides, augments expression of IL-2 and IFN-gamma, implicating B7-H1 as a negative regulator of cytokine synthesis. However, signaling through PD-1 does not affect induction of the activation markers CD25 or CD69 on T cells, suggesting that its effects are specific to cytokine synthesis. The suppressive effects of B7-H1 on cytokine expression are proportional to the strength of the primary stimulus, allowing for B7-H1 to determine the level of T cell activation in response to ECs. Our results demonstrate that B7-H1 negatively regulates cytokine synthesis in T cells activated by ECs.     

B lymphoblast antigen (BB-1) expressed on Epstein-Barr virus-activated B cell blasts, B lymphoblastoid cell lines, and Burkitt's lymphomas

Two new cell surface antigens expressed on B lymphoblastoid cell lines (B-LCL) were defined with cytotoxic mouse monoclonal antibodies. One marker, BB-1 (for B lymphoblast antigen-1), was detected on human and nonhuman primate B-LCL, Epstein-Barr virus (EBV)-activated B cell blasts, most Burkitt's lymphomas, and Ia+ B lymphoblast-like myelomas. Polyclonal B cell activators such as pokeweed mitogen (PWM) and lipopolysaccharide (LPS) also induced the expression of BB-1 on immunoglobulin (Ig)-positive cells. In contrast, BB-1 could not be detected on normal lymphoid tissues by complement-dependent cytotoxicity and immunofluorescence (IF) assays or by analysis with a fluorescence-activated cell sorter (FACS). T cell blasts, T cell leukemias, and pre-B cell or erythroblastic leukemia cell lines were also BB-1 negative. Of particular interest was the finding that BB-1 was expressed on the Jijoye lymphoma but only marginally on a subline of Jijoye, P3HR-1, that lacks receptors for EBV and produces a defective virus incapable of transforming lymphocytes. A second lymphoblast antigen (LB-1) unlike BB-1, was present on both T and B cell blasts and virus-transformed T- and B-LCL but not on normal lymphoid tissues.     

A novel glycoprotein expressed on sheep T and B lymphocytes is involved in a T cell activation pathway

We have produced a new mouse mAb that identifies a sheep T cell activation Ag. The mAb B5-5 is specific for low m.w. components on nearly all sheep thymocytes and peripheral T and B lymphocytes but does not label immature B cells in Peyer's patches or germinal centers. After cross-linking of target structures either directly by plastic-bound mAb or indirectly using anti-Ig reagents, peripheral T cells, but not thymocytes or peripheral B cells, were activated. IL-2 was secreted by T cells after cross-linking and activation was strongly augmented in the presence of PMA. The addition of soluble B5-5 mAb to mitogen-stimulated cultures of sheep lymphocytes resulted in a suppression of PHA responses and augmentation of PWM responses and had a variable effect on Con A responses but had no effect on LPS- or protein A-induced proliferation. When added to alloantigen-stimulated cultures, B5-5 augmented the proliferative response. The B5-5 membrane component consists of 14- to 19-kDa glycoproteins but the banding patterns obtained during SDS-PAGE analysis of 125I-labeled Ag differed between thymocytes, peripheral T cells, and peripheral B cells. On the basis of its range of expression on lymphoid cells and known biochemical and functional properties, we conclude that the B5-5 component on sheep lymphocytes is different from T cell activation Ag in other species.     

Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix

Two highly related 70K heat shock proteins, encoded by the hsc70 and hsp70 genes, are located in the nucleocytoplasmic compartment of mammalian cells. In contrast to rodent cell lines, which express Hsp70 only when stressed, many human cell lines constitutively express Hsp70. The degree to which this reflects constitutive expression of Hsp70 in normal human tissues has not been extensively examined. In this study, we show by immunoblotting that human Hsp70 is constitutively expressed in the ovary, cervix, and endometrium and, by immunohistochemical analysis using Hsp70- and Hsc70-specific antibodies, that Hsp70 and Hsc70 are expressed in distinctive and predominantly overlapping patterns in the cervix and endometrium. In these two tissues, the highest levels of both proteins are seen in differentiated, non-proliferating epithelial cells, which is surprising in light of previous studies suggesting growth stimulation of hsp70 gene expression. These observations sugest the possibility that in certain human tissues, basal expression of the hsp70 and hsc70 genes is coregulated.     

A novel cell surface antigen, 4C8, is expressed on human eosinophils

BACKGROUND: A novel monoclonal antibody, anti-4C8, reacted with human peripheral lymphocytes and monocytes but not with neutrophils. In this study, we investigated whether the 4C8 antigen is expressed on human peripheral eosinophils. METHODS: Expression of the 4C8 antigen on eosinophils was analyzed by flow cytometry and molecular analysis of the antigen was performed with eosinophils by Western blotting. RESULTS: Among human peripheral granulocytes, the 4C8 antigen was expressed on CD16-negative cells but not on CD16-positive cells. The 4C8 antigen also appeared to be expressed on eosinophils. To confirm the latter finding, eosinophils were purified from peripheral blood. On flow cytometric analysis, anti-4C8 antibody reacted with purified eosinophils. On Western blotting analysis, anti-4C8 reacted with a single band of 80 kDa in lysates from purified eosinophils. The correlation between the percentage of eosinophils determined by May-Giemsa staining and the percentage of 4C8-positive/CD16-negative cells among granulocytes was good (r = 0.91, P < 0.0001). CONCLUSIONS: Only a few cell surface antigens are available to distinguish human peripheral eosinophils from neutrophils. The novel cell surface antigen, 4C8, is a useful new marker of human eosinophils.     

Degradation of NF-kappa B in T cells by gangliosides expressed on renal cell carcinomas

T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which in renal cell carcinomas includes defective NF-kappaB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-kappaB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.     

A novel pathway of human T cell activation via a 103 kD T cell activation antigen

A novel triggering signal for human proliferating and cytotoxic T lymphocytes defined by a 103 kD T cell-specific activation antigen (Tp103) is described. Tp103 is expressed on all proliferating normal T cells but is not present, or present only in low amounts, on resting peripheral blood T lymphocytes. Cross-linking of T cell and Fc receptor-positive accessory or target cells by an antibody against Tp103 leads to activation of the T cell. The proliferative response is due to an autocrine IL 2-dependent mechanism and can be inhibited by antibodies against the IL 2 receptor or by Cyclosporin A. Resting Tp103-positive T cells also respond to anti-Tp103. Although Tp103 is not linked to the antigen receptor/T3 complex, triggering via Tp103 can be inhibited by modulation of the T3 molecule. Thus, Tp103 defines a new antigen-independent pathway of T cell activation that can be regulated via other T cell surface structures.     

3C8 antigen is a novel protein expressed by human follicular dendritic cells

Although the pivotal role of follicular dendritic cells (FDCs) in humoral immune responses has been demonstrated, little is known at the molecular level of how FDCs contribute to the organogenesis, B cell differentiation, and regulation of T cell functions in the germinal center. By immunizing with FDC-like cells, we have developed a monoclonal antibody (MAb), which stains the germinal centers in tonsil section. In the current study, the target cell of MAb 3C8 was identified as FDC by confocal scanning fluorescence microscopy. Unlike other MAbs against FDC, MAb 3C8 does not cross-react with bone marrow-derived blood cells. Amino acid sequencing of NH(2)-terminal region of immunoprecipitated 3C8 Ag reveals that 3C8 is a novel FDC protein. Further studies of 3C8 molecule will shed light on the cellular origin of FDC and reveal unknown functions of FDC.     

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.     

Identification of a novel type I cytokine receptor CRL2 preferentially expressed by human dendritic cells and activated monocytes

From a human dendritic cell (DC) cDNA library, we identified a novel type I cytokine receptor, designated as cytokine receptor-like molecule 2 (CRL2). CRL2 cDNA encoded a 371-residue type I transmembrane protein with an extracellular domain of 210 residues and an intracellular domain of 119 residues. Its extracellular domain contains conserved cysteine residues and WAS-like motif in place of the hallmark of WSXWS motif present in other type I cytokine receptors. The intracellular domain contained a membrane-proximal "box 1" motif and conserved tyrosine residue potentially as a binding site for signal transducing molecules. CRL2 protein shares significant homology with common cytokine receptor (gammac) and interleukin-13 receptor alpha1 chain. Northern blot analysis showed that CRL2 was restrictedly expressed by spleen and peripheral blood leukocytes, and abundantly expressed by HL-60 cells. RT-PCR analysis demonstrated that CRL2 was preferentially expressed by DC and monocytes. Interestingly, CRL2 expression was up-regulated when monocytes were activated by LPS. These indicate that CRL2 may be involved in the biological functions of DC and monocytes. The Ba/F3 transfectants of CRL2 was retrovirally established with the expressed FLAG-tagged CRL2 in the size of approximately 48 kD, which could be efficiently immunoprecipitated. We also prepared a CRL2Ig fusion protein. The identification of its ligand and involvement of signal transduction will help to elucidate its potential function.     

Identification of a novel high molecular weight protein preferentially expressed by sinusoidal endothelial cells in normal human tissues

Mouse mAb MS-1, raised against human spleen, detects an endothelial cell antigen abundantly expressed by the sinusoidal endothelia of spleen, lymph node, liver, and adrenal cortex, but absent from nonsinusoidal continuous endothelia in these organs. Immunoelectron microscopy of splenic tissue demonstrates that the MS-1 antigen is predominantly deposited at zones of intercellular contact between adjacent sinusoidal endothelial cells. mAb MS-1 also reacts with a variable proportion of high endothelial venules in tonsil, but not in other lymphoid tissues, and with an interstitial dendritic cell population most abundant in placenta. mAb MS-1 does not react with cultured resting or mediator- activated human umbilical vein endothelial cells, dermal fibroblasts, peripheral blood mononuclear cells, or the cell lines U937, HL-60, K562 or Mo7E; it does react with the primitive myeloid cell line KG-1. mAb MS-1 immunoprecipitates a major protein of 215 kD and minor proteins of 320 and 120 kD from splenic extracts as analyzed by SDS-PAGE with reduction. These proteins are soluble in aqueous buffers. Immunoprecipitation from KG-1 cell lysates detects four proteins of 280, 300, 205, and 120 kD; the 300-, 205-, and 120-kD species, presumably corresponding to the 320-, 215-, and 120-kD species in spleen, respectively, are secreted into the media. Under nonreducing conditions, immunoprecipitates from KG-1 cell lysates or conditioned media contain one predominant 300-kD species; upon isolation and reduction, this 300-kD species separates into the previously observed 300-, 205-, and 120-kD species. Pulse-chase experiments and limited proteolysis peptide mapping suggest that the 280-kD species is a precursor of the mature 300-kD species which may be subsequently cleaved to yield the 205- and 120-kD species. Because of its size, solubility and expression pattern, the antigen recognized by mAb MS-1 is likely to be an extracellular matrix protein utilized by endothelial cells of contorted, large caliber, or leaky microvessels that lack a well-formed basement membrane.