Heterogeneity in antigenic expression and radiosensitivity in human colon carcinoma cell lines

Summary A panel of human colon carcinoma cell lines were characterized regarding both antigenic heterogeneity and variations in radiosensitivity. Monoclonal antibodies were used to study the expression of carcinoembryonic antigen (CEA), gastrointestinal cancer antigen (GICA or CA 19-9) and carcinoma-associated antigen (CA-50). Radiosensitivity was studied with the clonogenic survival technique. Three cell lines, LS 174T, HCTC, and SW 1116 stained positive for all three antigens. HT-29 was positive for CA 19-9 and CA-50 whereas Caco-2 was positive for CEA and CA 19-9. The cell lines SW 620 and LIM 1215 only stained positive for one of the antigens, CA-50 and CEA, respectively. In nearly all positive cases the stainings were very heterogeneous with mixtures of positive and negative cells. One exception was the HCTC cells which stained homogeneously for the CA 19-9 and CA-50 antigens. The neuroendocrinelike COLO 320 cells were negative in all cases. The radiosensitivity varied strongly between the cell lines with D_q-values between 0.8 and 1.9, extrapolation numbers between 2.0 and 4.7, D_o-values between 1.1 and 2.8. The surviving fraction at 2 Gy varied between 0.3 and 0.7 with HCTC as the most radiosensitive and HT-29 as the most radioresistant cell line. Thus, there were differences in antigenic expression and intrinsic radiosensitivity between the cell-lines and antigenic heterogeneities within each cell line. The analyzed panel of cell lines will be valuable in studies of dose-effect relations for monoclonal antibodies labeled with toxic radionuclides simulating both antigenic heterogeneity and variations in radiosensitivity.     

Monoclonal antibodies for carcinoembryonic antigen (CEA) as a model system: identification of two novel CEA-related antigens in meconium and colorectal carcinoma tissue by Western blots and differential immunoaffinity chromatography

In a previous study, five monoclonal antibodies against the carcinoembryonic antigen (CEA) with different epitope specificities were delineated. One of these antibodies which exhibits a high affinity for CEA binds to different carcinoma tissues, to liver tissue, and to granulocytes. This antibody was selected for the immunoaffinity purification of CEA and related antigens from colorectal carcinoma tissue, from spleen tissues, from bile, and from meconium. After elution from the immunosorbent, the antigens were separated by SDS-PAGE, were transferred to nitrocellulose, and were incubated with the five different antibodies. Antibody T84.1 bound to the following antigens: 177 kD and 128 kD from colonic carcinoma, 81 kD from bile, 49 kD from spleen, as well as 165 kD and 100 kD from meconium. Two additional antibodies showed a similar binding pattern. The fourth antibody (CEA.11) bound to the 165 kD meconium antigen and to the two colorectal carcinoma antigens. The fifth antibody (T84.66) showed a strong reaction with the 177 kD colorectal carcinoma antigen and a faint reaction with a 183 kD antigen in meconium. As judged from m.w. and immunochemical properties, the 128 kD colorectal carcinoma antigen and the 100 kD meconium antigen are two novel CEA-related antigens. Because antibody CEA.11 did not bind to the 100 kD meconium antigen in Western blots, the 165 kD antigen could be eluted from a CEA.11 immunosorbent without contamination by the 100 kD antigen. Similarly, as predicted from the binding pattern in the Western blots, the two colorectal carcinoma antigens were separated from each other by a T84.66 immunosorbent.     

Effects of131I-labeled TNT-1 radioimmunotherapy on HT-29 human colon adenocarcinoma spheroids

Summary Radiolabeled murine monoclonal antibody TNT-1, directed against the nuclear histones of degenerating cells, was used to treat human colon adenocarcinoma HT-29 spheroids in vitro. The therapeutic effects of¹³¹I-TNT-1 were investigated as a function of the radioactive dose, treatment time, and number of treatments. Efficacy of treatment was assessed by TNT-1 antibody uptake, spheroid growth delay, and morphological examination using light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). From these studies, it was determined that the therapeutic effect increased with the number of doses and the duration of treatment. Spheroids treated for 24 h showed approximately two to four times more cell death than those with a 2-h treatment. As previously shown in animal models, additonal treatment with radiolabeled TNT-1 produced an expanding number of TNT-1 targets, and subsequent treatments were more effective as shown by antibody uptake studies. Microscopic examinations demonstrated that morphological changes consistent with spheroid destruction correlated well with antibody uptake data and increased gradually with dose, treatment time, and frequency of treatments. At the ultrastructural level, destruction of cells in the treated spheroids included the formation of porous cell membranes, crater-like holes (SEM), blebbing, and dissolution of cytoplasmic organelles (TEM). With continued culture, the injured spheroids were found to disaggregate after intensive¹³¹I-TNT-1 therapy (e.g. 50 µCi/ml or 100 µCi/ml with two or three 24-h treatments). These findings suggest that tumor spheroids can be used as an in vitro model to evaluate monoclonal antibody therapy using TNT-1 and other candidate mAbs directed against intracellular antigens exposed in degenerating cells of tumors.     

Influence of ionizing radiation on oxygen profiles in different types of multicellular spheroids

Human glioma (U-118 MG and U-138 MG), human colorectal adenocarcinoma (HT-29), human thyroid carcinoma (HTh 7), and hamster embryonic lung (V79-379A) spheroids were irradiated with either single doses of 16 or 40 Gy or fractionated doses of eight times 5 Gy. Oxygen profiles in the spheroids were measured with microelectrodes at different times following irradiation, and these profiles were then compared with the oxygen profiles measured in parallel cultured nonirradiated spheroids. No significant radiation-induced changes in the oxygen profiles were seen in any of the spheroids within the first few days after irradiation. The glioma spheroids did not show any significant increase in oxygen tension even after longer times; however, they were growth inhibited, and the number of S-phase cells was strongly suppressed. Increases in oxygen tension did occur in the HT-29 and V79-379A spheroids but only appeared more than a week after irradiation, when degeneration had started. Histological changes and decrease in diameter were seen in the spheroids that started to degenerate about 5 days after irradiation. Thus radiation doses in the therapeutic range did not, for the spheroids studied, produce rapid increases in the oxygen tension. When a change occurred, it appeared rather late and was probably a consequence of cell degeneration.     

Effect of glutaraldehyde treatment of human melanoma cells on the expression of melanoma-associated antigens

Summary Human melanoma cells were treated with different concentrations of glutaraldehyde, and retention of serological reactivity with antisera against melanoma-associated antigens, HLA antigen, and 2-microglobulin was assessed by quantitative absorption analysis in mixed hemadsorption microassays. Glutaraldehyde concentrations of 0.025% or greater significantly impaired binding to melanoma cells of antibody against melanoma-associated antigens. At a concentration of 0.0025% antibody binding was not decreased although plating efficiency was reduced to less than 1%. Glutaraldehyde concentrations of 0.25% or greater significantly reduced binding to the same melanoma cells of antisera against HLA antigen and 2-microglobulin. Glutaraldehyde treatment (up to 2.5%) of HT-29 colon carcinoma cells failed to reduce reactivity of antisera against CEA and blood group A isoantigen, which are present on these cells. These studies indicate that the effect of glutaraldehyde treatment of cells on retention of surface antigens is critically dependent on the concentration of glutaraldehyde used and the type of antigens involved. Abbreviations used in this paper: MAA, melanoma-associated antigens; GA, glutaraldehyde; FCS, fetal calf serum; RPMI, Roswell Park Memorial Institute; 2M, 2-microglobulin; CEA, carcinoembryonic antigen; PBS, phosphate-buffered saline; NGP, normal glycoprotein cross-reacting with CEA; SRBC, sheep red blood cells     

Effects of alpha, beta and gamma recombinant interferons on CEA production from HT-29 human colon adenocarcinoma cell line

The human colon adenocarcinoma derived cell line HT-29 is a good in vitro model for the study of CEA production and release under various experimental conditions. Many studies indicate that CEA secretion is correlated with cell proliferation and seems to depend on the growth conditions and differentiation characteristics induced by the culture medium. The present study demonstrates that recombinant interferons alpha, beta and gamma (rIFN alpha, rIFN beta, rIFN gamma) can modify CEA production and release by HT-29 cell-line. rIFN gamma in particular causes an enhancement of CEA production and release in the culture medium. This dose-depending effect is in some way correlated to cell growth inhibition since the enhancement of CEA expression in the interferon treated cells is evident in the presence of a reduction in cell proliferation. The activity of rIFN alpha and rIFN beta on CEA release is much less remarkable than that demonstrated by rIFN gamma, and is probably only due to the fact that HT-29 colon adenocarcinoma cells respond poorly to the effects of rIFN alpha and rIFN beta at the doses we used. These findings suggest that CEA production, expression and release can be modulated in a variety of ways under the influence of different rIFN treatment and this situation must be taken into account in immunodiagnostic and immunotherapeutic applications of anti-CEA monoclonal antibodies in the cancer patient.     

Penetration of substances into tumor tissue—A methodological study on cellular spheroids

Summary The penetration of [³H]thymidine, [³H]d-leucine, [¹²⁵I]albumin, and the drugs [³H]5-fluorouracil and [³H]vinblastine into human glioma spheroids (in vitro tumor models) was studied by a method based on rapid freezing, freeze drying, vapor fixation, wax embedding, dry sectioning, and contact autoradiography. No significant disturbances in the distribution of water soluble substances were observed. Thymidine andd-leucine penetrated the whole spheroids relatively fast, whereas albumin showed reduced penetration. the concentration of albumin was highest at the periphery of the spheroids, but only smaller amounts were detected in the deeper regions. A significant difference between the penetration patterns of the drugs studied was also observed. Fluorouracil penetrated rather freely, but the penetration of vinblastine was limited. The work was supported financially by Lennanders Foundation, OE and Edla Johanssons Foundation, and the Swedish Cancer Society.     

Quantitative immunocytochemical characterization of mononuclear phagocytes. I. Monoblasts, promonocytes, monocytes, and peritoneal and alveolar macrophages

The purpose of the present study was to compare the phenotype of tissue macrophages with that of their precursors in the bone marrow and blood. The phenotype was determined on the basis of the quantitative binding of monoclonal antibodies to cell-surface antigens (antigen F4/80, complement receptor III, Fc receptor II, Ia antigen, common leukocyte antigen, and Mac-2 and Mac-3 antigens) on individual mononuclear phagocytes. Monoclonal antibody binding to cells, detected by the biotin-avidin immunoperoxidase procedure, was quantitated by cytophotometric determination of the amount of enzyme reaction product on cells. The results of this quantitation are expressed as the median of the specific absorbance per unit of cell-surface area (0.25 micron2) and per cell. Shortly after collection of the mononuclear phagocytes, binding of all monoclonal antibodies except those directed against the common leukocyte and Mac-2 antigens to peritoneal macrophages was enhanced compared with binding to blood monocytes; for alveolar macrophages we found reduced binding of monoclonal antibodies F4/80 and M1/70 (complement receptor III) and enhanced binding of monoclonal antibodies with specificity for the common leukocyte antigen and Mac-2 and Mac-3 antigens. The results obtained with cultured mononuclear phagocytes show that during the development from monoblast to tissue macrophages, monoclonal antibody binding to the various types of mononuclear phagocyte, expressed per unit of cell-surface area, was not significantly altered except that of M3/38 (Mac-2 antigen) to peritoneal macrophages and that of F4/80 and M1/70 (complement receptor III) to alveolar macrophages. Expressed on a per cell basis, the results show an increase in the binding of all monoclonal antibodies except those directed against the Fc receptor II and Mac-3 antigen during the development from promonocytes to peritoneal macrophages; binding of most monoclonal antibodies to alveolar macrophages was considerably lower than that to blood monocytes. It is concluded that the expression of the various cell-surface antigens alters during mononuclear phagocyte differentiation. The expression changed also during culture, although distinct patterns of alteration could not be distinguished.     

Monoclonal antibodies PMN 6, PMN 29, and PM-81 bind differently to glycolipids containing a sugar sequence occurring in lacto-N-fucopentaose III

Three monoclonal antibodies, PMN 6, PMN 29, and PM-81, bind myeloid cells. Antibodies PMN 6 and PMN 29 bind specifically to granulocytes but differ in their ability to bind some other cell lines [E. D. Ball, R. F. Graziano, L. Shen, and M. W. Fanger (1982) Proc. Natl. Acad. Sci. USA 79, 5374-5378]. Antibody PM-81, in addition to granulocytes, also binds to eosinophils, monocytes, and most acute myelocytic leukemia cells [E. D. Ball, R. F. Graziano, and M. W. Fanger (1983) J. Immunol. 130, 2937-2941]. Despite these differences, the binding of all three antibodies to cells was inhibited by the oligosaccharide, lacto-N-fucopentaose III [Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc]. Solid-phase radioimmunoassays using purified glycolipids containing sugar sequences found in lacto-N-fucopentaose III demonstrated different binding characteristics for each antibody. PM-81 bound lower concentrations of glycolipids than PMN 29, while PMN 6 required the highest concentration of glycolipids for binding. Autoradiography of thin-layer chromatograms of glycolipid antigens supported these results. The binding of these monoclonal antibodies to cells probably depends on the density of antigens on the cell surface, each antibody requiring a different density. Thus, cells containing antigen below a certain threshold concentration may not bind low-affinity antibodies.     

Imaging proteolysis by living human glioma cells

Degradation of basement membrane is an essential step for tumor invasion. In order to study degradation in real time as well as localize the site of proteolysis, we have established an assay with living human cancer cells in which we image cleavage of quenched-fluorescent basement membrane type IV collagen (DQ-collagen IV). Accumulation of fluorescent products is imaged with a confocal microscope and localized by optically sectioning both the cells and the matrix on which they are growing. For the studies described here, we seeded U87 human glioma cells as either monolayers or spheroids on a 3-dimensional gelatin matrix in which DQ-collagen IV had been embedded. As early as 24 hours after plating as monolayers, U87 cells were present throughout the 3-dimensional matrix. Cells at all levels had accumulated fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Similar observations were made for U87 spheroids and the individual cells migrating from the spheroids into the gelatin matrix. Both the migrating cells and those within the spheroid contained fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Thus, glioma cells like breast cancer cells are able to degrade type IV collagen intracellularly, suggesting that this is an important pathway for matrix degradation.     

Antigen forks: bispecific reagents that inhibit cell growth by binding selected pairs of tumor antigens

Bispecific antibodies of a new category, termed antigen forks, were constructed by crosslinking antibodies that recognized pairs of distinct tumor cell surface antigens. At concentrations of 1–100 nM, several such forks inhibited the growth of human tumor cell lines bearing both relevant antigens. The same cells were not inhibited by unconjugated component antibodies, and the active conjugates did not inhibit the growth of human cell lines that expressed lower levels of relevant antigens. The three most active antigen forks all contained monoclonal antibody 454A12, which recognizes human transferrin receptor. This antibody was conjugated respectively to antibodies 113F1 (against a tumor-associated glycoprotein complex), 317G5 (against a 42-kDa tumor-associated glycoprotein), or 520C9 (against the c-erbB-2 protooncogene product). The 317G5-454A12 fork strongly inhibited the HT-29 and SW948 human colorectal cancer cell lines, while the 113F1-454A12 fork was also effective against SW948. By designing forks against antigens of incompatible function that are co-expressed at high levels on tumor cells but not on normal tissues, it may be possible to generate reagents that inhibit tumor growth with enhanced selectivity.     

Human intestinal epithelial cells express a novel receptor for IgA

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.     

Immunological characteristics of monoclonal antibodies against human carcinoembryonic antigen (CEA)

Four hybridomas secreting monoclonal antibodies (MAbs) of the IgG1 subclass against human carcinoembryonic antigen (CEA) were obtained from fusion of P3-NS1/1-Ag4 myeloma cells with splenic cells from mice immunized with purified CEA. None of the MAbs showed cross-reactivity to perchloric acid extractable antigens from the normal human colon by an inhibition radioimmunoassay. However, MAb C27 showed the highest affinity to CEA. The intensity of immunofluorescence staining of human colorectal cancer cells with MAb C27 correlates well to the cellular CEA content of cancer cells. LS174T showed the highest intensity of fluorescence (95%) while COLO320DM and COLO320HRS were the lowest (0.5%). None of the normal human organs - colon, lungs, liver, spleen or kidneys-showed positive staining by immunoperoxidase anti-peroxidase (PA) techniques, while tissues from colorectal carcinoma (CRC), gastric carcinoma, hepatoma and lung cancer gave a positive rate of 100% (30/30), 96.6% (28/29), 32.1% (9/28) and 82.1% (69/84) respectively. Results suggest that MAb C27 can be used in immunodetection and radiolocalization of colorectal carcinoma.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epitope specificities of monoclonal antibodies

A systematic approach for the determination of epitope specificities of monoclonal antibodies to a complex antigen system is described. After initial screening to identify antigen-binding monoclonal antibodies, one or more of the clones are isolated by limiting dilution cloning, grown in ascites, and the resulting antibodies secreted into the ascitic fluid are affinity purified on Sepharose-bound protein A, radiolabeled, and cross-compared with antibodies from other clones by a solid-phase competitive immunoassay. In this work, BALB/c mice were immunized with either purified carcinoembryonic antigen (CEA) or the CEA-producing cell line HC 84S. Spleen cells were fused with the mouse myeloma cell line Sp2/0-Ag14. The supernatants from 25 hybrids showed a significant binding of 125I-CEA (greater than or equal to 15%). Nine hybrids were cloned, resulting in 33 different clones. The antibodies produced by the different cloned hybrids and the remaining uncloned hybrids recognized a total of five different epitopes on CEA. All of the epitopes reside on the protein moiety of the molecule as determined by antibody binding to deglycosylated CEA. The monoclonal antibodies with five different epitope specificities were reacted with tissue sections of normal and cancerous tissues and with peripheral blood smears. Each of the five monoclonal antibodies reacted with tissue sections from colonic, gastric, lung, and mammary carcinomas, as well as from a benign colonic polyp and a resection margin from a colonic carcinoma. Four monoclonals reacted with normal liver tissue. Granulocytes in peripheral blood smears bound three antibodies strongly and one antibody weakly, and one antibody was not bound. One monoclonal antibody that reacted with normal liver tissue was not bound by granulocytes. The ability of these five monoclonal antibodies to differentially detect three different CEA-related antigens in normal and malignant tissues may have clinical utility.     

In vitro characterization of a recombinant 32P-phosphorylated anti-(carcinoembryonic antigen) single-chain antibody

 The major limitations of monoclonal antibody conjugates as therapeutic agents have been their poor tumour targeting, inadequate tumour penetration and immunogenicity. More even and deeper tissue penetration has been demonstrated with smaller antibody fragments. The smaller size and absence of an Fc segment may contribute to a lowered immunogenicity with single-chain antibodies (scFv) and also permit their recombinant engineering and bacterial expression. We describe the successful engineering, expression and pre-clinical characterisation of a phosphorylatable “kemptide” (Leu-Arg-Arg-Ala-Ser-Gly) anti-carcinoembryonic antigen (anti-CEA) scFv (PKS-scFv), for use as a radioimmunotherapeutic agent. Specifically, a yield of 6 mg/l induced culture was obtained. Site-specific phosphorylation was demonstrated without loss of specificity. In vitro assays revealed a selective cytotoxicity of ³²P-PKS-scFv for high-CEA-expressing LS-174T cells compared to the low-CEA-expressing HT-29 cells, with a rapid internalisation rate. Received: 20 March 1997 / Accepted: 5 February 1998     

Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity,bivalency, and stability

Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CD66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells

Neutrophil adherence to cytokine-activated endothelial cell (EC) monolayers depends on the expression of the endothelial leukocyte adhesion molecule-1 (ELAM-1). The ligand for ELAM-1 is the sialylated Lewis-x antigen (SLe(x)) structure. The selectin LAM-1 (or LECAM-1) has been described as one of the SLe(x)-presenting glycoproteins involved in neutrophil binding to ELAM-1. Other presenter molecules have not yet been described. Our data demonstrate that the carcinoembryonic antigen (CEA)-like surface molecules on neutrophils--known as the nonspecific cross-reacting antigens (NCAs)--are involved in neutrophil adherence to monolayers of IL-1-beta-activated EC. The NCAs are recognized by CD66 (NCA-160 and NCA-90) and CD67 (NCA-95). Because NCA-95 and NCA-90 have previously been found to be phosphatidylinositol (PI)-linked, paroxysmal nocturnal hemoglobinuria (PNH) neutrophils (which lack PI-linked surface proteins) were tested as well. PNH neutrophils showed a diminished binding to activated EC. CD66 (on PNH cells still recognizing the transmembrane NCA-160 form) still inhibited the adherence of PNH cells to IL-1-beta-activated EC, but to a limited extent. Soluble CEA(-related) antigens inhibited normal neutrophil adherence as well, whereas neutrophil transmigration was unaffected. Sialidase-treatment as well as CD66 preclearing abolished the inhibitory capacity of the CEA(-related) antigens. The binding of soluble CEA antigens to IL-1-beta-pretreated EC was blocked by anti-ELAM-1. These soluble antigens, as well as the neutrophil NCA-160 and NCA-90, both recognized by CD66 antibodies, presented the SLe(x) determinant. Together, these findings indicate that the CD66 antigens (i.e., NCA-160/NCA-90) function as presenter molecules of the SLe(x) oligosaccharide structures on neutrophils that bind to ELAM-1 on EC.     

Intranuclear localization of a new snRNP-related antigen.

The intranuclear distribution of a new antigen (F78) associated with U snRNPs (small nuclear RNA-protein complexes) was compared with that of the RNP and Sm protein antigens previously identified on individual snRNP particles. Human and rat cells were double stained with human autoantisera and mouse monoclonal antibodies. The binding of the human and mouse antibodies was detected with secondary antibodies conjugated with fluorescein and rhodamine, respectively. The resulting immunofluorescence patterns were compared by digital image analysis. The F78, RNP, and Sm antigens show speckled fluorescence patterns which overlap to a great extent. The F78 pattern, however, also contains two classes of structural elements not present in the RNP pattern. Furthermore, during mitosis expression of the F78 antigen is completely suppressed from early prophase to telophase, while the RNP and Sm antigens are found evenly distributed throughout the cytoplasm of the dividing cells.     

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.     

Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies.

Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.