Cell surface glycoproteins of 13762NF mammary adenocarcinoma clones of differing metastatic potentials

Rat 13762NF mammary adenocarcinoma cell surface glycoproteins from s.c. tumor- or lung metastases-derived cell clones of differing spontaneous metastatic potentials were examined for their relationship to metastasis. After treatment with neuraminidase, lectin-binding assays showed that highly metastatic clone MTLn3 cells express approximately twice the quantity of peanut agglutinin (PNA) binding sites (approximately 2.3 X 10(8) sites/cell) than clones of lower metastatic potential. However, the number of wheat germ agglutinin (WGA)-binding sites on the various cell clones decreased slightly as the metastatic potential of the clones increased. The quantities of concanavalin A (conA)-binding sites were similar (approximately 1.7 X 10(8) sites/cell) in all cell clones and growth conditions. Glycoprotein analysis was performed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) and subsequent staining with 125I-labeled lectins. SDS-PAGE gels stained with 125I-labeled conA revealed mainly one glycoprotein (Mr approximately 150 kD), and the amounts of this glycoprotein did not correlate with metastasis. Differences in WGA-binding glycoproteins were detected between s.c. tumor- and lung metastases-derived cell clones. Several desialylated glycoproteins were detected with 125I-labeled PNA after SDS-PAGE, and the labeling intensity of one (Mr approximately 580 kD) correlated with the metastatic potentials of the various cell clones. This high Mr galactoprotein was further analyzed by [3H]glucosamine metabolic labeling, solubilization, sequential gel filtration, and chondroitinase ABC treatment prior to SDS-PAGE. The 580 kD galactoprotein was expressed in increased amounts on the more highly metastatic clones. Chemical labeling of cell surface sialic acid residues using periodate treatment followed by [3H]borohydride reduction showed an additional change in a major sialoglycoprotein (Mr approximately 80 kD), which decreased in labeling intensity on clones of increasing metastatic potential. The results suggest quantitative changes in cell surface glycoproteins rather than major qualitative alterations are associated with differences in the metastatic behavior of 13762NF tumor cell clones.     

A 6-thioguanine-resistant variant of the rat mammary adenocarcinoma 13762 that is more immunogenic

Summary A 6-thioguanine-resistant (TgR) variant of the metastatic mammary tumor 13762 was found to be very immunogenic. This TgR variant was nontumorigenic and nonmetastatic, whereas the parent 13762 cell line is very tumorigenic and metastatic in normal syngeneic animals. The TgR variant was tumorigenic in irradiated animals. The mechanism of the hosts' immune rejection of this TgR variant was investigated. A ⁵¹Cr-release cytotoxic cell assay was used to assess lymphocyte cell-mediated cytotoxicity (CMC) of tumor-draining lymph nodes and spleens from animals injected with tumor cells. In a secondary CMC response of splenic T cells from animals injected with TgR cells, there was a much stronger response as compared to animals injected with 13762 cells. This strong cytotoxic T cell response was short-term and correlated to the host rejection of TgR cells. Previously, we selected revertant cell lines (TgRrev, TgRrevM) from the TgR variant line that were more metastatic and tumorigenic. The revertant cell lines induced a lower CMC response than the TgR line, but a higher response compared to the parent 13762 line. The poor CMC response from 13762 tumorbearing animals was investigated and appeared to be due to a suppressor T cell response.     

Chemotactic response of rat mammary adenocarcinoma cell clones to tumor-derived cytokines

A cytokine with an apparent molecular weight of 53,000 daltons was isolated from serum-free medium conditioned by MTLn3 cells or from homogenates of MTLn3 cells, a highly metastatic variant of the rat 13762NF mammary adenocarcinoma. The chemotactic responses of MTLn3 and the low metastatic variant MTLn2 cells to this cytokine were tested in vitro using modified Boyden chambers. Both the chemotactic and chemokinetic movements of MTLn3 cells were stimulated by the MTLn3-derived cytokine. In addition, the MTLn3-derived cytokine stimulated a relatively small, but significant chemotactic migration of MTLn2 tumor cells, while these cells did not respond to medium conditioned by MTLn2 cells. MTLn3 cells themselves did not respond chemotactically to type I collagen or medium conditioned by MTLn2 cells. These results suggest that the chemotactic response may be a function of metastatic potential of the invading tumor cells. The production of tumor cytokines that enhance tumor cell motility may thus represent a phenotypic difference between 13762NF tumor cell subpopulations of high and low metastatic potential.     

Sialoglycoprotein differences between xenotransplantable and nonxenotransplantable ascites sublines of the 13762 rat mammary adenocarcinoma

MAT-B1 and MAT-CI rat ascites mammary adenocarcinoma cells differ in morphology, lectin receptor mobility, and xenotransplantability. Since these properties may be related to cell surface organization, the predominant sialoglycoproteins of these sublines have been investigated by chemical labeling, proteolysis, and alkaline borohydride elimination. Treatment of both sublines with periodate and tritiated borohydride labels one major sialoglycoprotein (ASGP-1) with a low electrophoretic mobility on polyacrylamide gels in dodecyl sulfate. Treatment of labeled or unlabeled cells with trypsin releases about 30% of the total cell sialic acid without significant decrease in cell viability. Gel filtration in pyridine-acetate buffer or in dodecyl sulfate indicates that the released materials are very heterogeneous, and that most of the MAT-C1 sialoglycopeptides are larger than sialoglycopeptides of MAT-B1. Amino acid compositions are quite similar for the released material from the two sublines, but they differ substantially in sialic acid. Further degradation of trypsin-released material with Pronase gives products which are included in a column of mixed Bio-Gel P-10 and P-30 and which also indicate a larger average size for MAT-C1 sialoglyco-peptides. Oligosaccharides from the sialoglycopeptides were obtained by alkaline borohydride treatment of trypsin-released, labeled material and fractionated by chromatography on Bio-Gel P-2. The oligosaccharide(s) comprising the major peak from MAT-C1 cells was larger in size than most of the material from MAT-B1 cells and contained galactosaminitol, galactose, glucosamine, sialic acid, and fucose. These results suggest that MAT-C1 ASGP-1 has more complex oligosaccharides than MAT-B1 ASGP-1, a difference which may play an important role in the differences in cell behavior between the sublines, including transplantability. Regardless of whether the ASGP-1 plays a role in transplantation, investigations of the sialoglycoproteins of these sublines provide a potentially valuable tool for understanding some of the mechanisms by which tumor cells control their cell surface properties.     

Sulfation of the tumor cell surface sialomucin of the 13762 rat mammary adenocarcinoma

ASGP-1, the major cell surface sialomucin of the 13762 ascites rat mammary adenocarcinoma, is at least 0.5% of the total ascites cell protein and has sulfate on 20% of its O-linked oligosaccharide chains. We have used this system to investigate the O-glycosylation pathway in these cells and to determine the temporal relationship between sulfation and sialylation. The two major sulfated oligosaccharides (S-1 and S-2) were isolated as their oligosaccharitols by alkaline borohydride elimination, anion exchange HPLC, and ion-suppression HPLC. From structural analyses S-1 is proposed to be a branched, sulfated trisaccharide -O4S-GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and S-2 its sialylated derivative -O4S-GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNac. Pulse labeling with sulfate indicated that sulfation occurred primarily on a form of ASGP-1 intermediate in size between immature and mature sialomucin. Pulse-chase analyses showed that the intermediate could be chased into mature ASGP-1. The concomitant conversion of S-1 into S-2 had a half-time of less than 5 min. Monensin treatment of the tumor cells led to a 95% inhibition of sulfation with the accumulation of unsulfated trisaccharide GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and sialylated derivative GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNAc. These data suggest that sulfation of ASGP-1 is an intermediate synthetic step, which competes with beta-1,4-galactosylation for the trisaccharide intermediate and thus occurs in the same compartment as beta-1,4-galactosylation. Moreover, sulfation precedes sialylation, but the two are rapidly successive kinetic events in the oligosaccharide assembly of ASGP-1.     

Helper and suppressor functions of panned populations of immune peritoneal T cells with reactivity to the rat mammary adenocarcinoma 13762A

The phenotype of glass-adherence-depleted tumor-immune peritoneal exudate lymphocytes (PEL) which generated anti-tumor reactivity against the rat mammary adenocarcinoma 13762A in vitro was examined by indirect panning. Monoclonal antibodies W3/25 and OX8, directed against the CD4 and CD8 differentiation antigens, respectively, were used to separate immune PEL into subsets of functionally different T cells. The panned populations of immune PEL were examined for anti-tumor reactivity in three different in vitro assays. Tumor-specific proliferation, tumor-specific induction of the helper lymphokine interleukin 2 (IL-2), and tumor-specific induction of an antiproliferative tumor-induced suppressor lymphokine (TISL) were determined. Panning experiments together with indirect immunofluorescence analysis of unpanned immune PEL indirectly indicated that a proportion of cells (15-20%) coexpressed CD4 and CD8 antigens. Strong tumor-specific proliferation and IL-2 and TISL production were generated from these double-positive cells, as determined by double-panning experiments. Significant tumor-specific proliferation and IL-2 production were produced also from CD4+CD8- cells, but TISL production was minimal, and could be accounted for by contaminating CD4+CD8+ cells. CD4-CD8+ cells produced negligible responses against the tumors as measured by these three assays, and no synergistic responses were demonstrated when CD4-CD8+ cells were incubated together with CD4+CD8- cells. These data demonstrated that CD4+CD8+ T cells exist in primed populations of rat peripheral lymphocytes, and that both helper and suppressor functions were generated from these double-positive cells.     

Shedding of the major plasma membrane sialoglycoprotein from the surface of 13762 rat ascites mammary adenocarcinoma cells

ASGP-1 (ascites Sialoglycoprotein 1) the major sialoglycoprotein of 13762 rat ascites mammary adenocarcinoma cells, is shed from MAT-B1 (nonxenotransplantable) and MAT-C1 (xenotransplantable) sublines when incubated in vitro after labeling in vivo with [³H]glucosamine. The rates of shedding of label in both particulate and soluble form are similar for the two sublines, but the turnover of label in the cells is 80% greater for MAT-C1 cells ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ 2.4 days) than for MAT-B1 cells ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ 4.1 days). Shed soluble ASGP-1 was smaller than ASGP-1 in the particulate fraction by gel filtration in dodecyl sulfate. By CsCl density gradient centrifugation, gel filtration, and sucrose density gradient centrifugation, all in 4 m guanidine hydrochloride, the shed soluble ASGP-1 was found to be slightly more dense and smaller than ASGP-1 purified from membranes. No differences in sialic acid or oligosaccharides released by alkaline borohydride treatment were found between the shed soluble ASGP-1 and purified ASGP-1. These results suggest that the shed soluble ASGP-1 is released from the membrane by a proteolytic cleavage. This mechanism is supported by the inhibition of the release of soluble shed ASGP-1 by aprotinin, a protease inhibitor. Soluble ASGP-1 in ascites fluid is also smaller by gel filtration, but is more heterogeneous, suggesting a similar release mechanism in vivo followed by more extensive degradation in the ascites fluid.     

Cell surface properties of ascites sublines of the 13762 rat mammary adenocarcinoma : Relationship of the major sialoglycoprotein to xenotransplantability

The relationship between cell surface sialoglycoprotein and xenotransplantation has been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma. Two of the five sublines (MAT-C and MAT-C1) can be transplanted into mice. These two sublines also have the greatest amounts of total, trypsin-releasable and neuraminidase-releasable sialic acid. Chemical labeling using periodate treatment followed by [³H]borohydride reduction indicates that most of the protein-bound sialic acid is associated with a single major sialoglycoprotein (or family of glycoproteins) with a low mobility on polyacrylamide gels in dodecyl sulfate (SDS). This glycoprotein, denoted ASGP-1, is also labeled by lactoperoxidase and ¹²⁵I, indicating its presence at the cell surface. Metabolic labeling with [³H]glucosamine shows that ASGP-1 is the major glycosylated protein in both xenotransplantable (MAT-C1) and non-xenotransplantable (MAT-B1) sublines, representing >70% of the protein-bound label in each. The labeling studies indicate that the non-xenotransplantable subline does not have a substantially greater amount of ASGP-1 on its cell surface. Likewise cationized ferritin labeling and transmission electron microscopy (TEM) do not show substantially greater amounts of negatively charged groups distributed along the cell surfaces of MAT-C1 than of MAT-B1 cells. The results indicate that the transplantation differences between these sublines cannot be explained solely by the presence of a major sialoglycoprotein at the cell surface.     

Intrinsic defects explain altered proliferative responses of T lymphocytes and HVS-derived T-cell lines in gastric adenocarcinoma

We have taken advantage of a recently described technique of transformation and immortalization of T lymphocytes using the lymphotropic Herpesvirus saimiri, to achieve long-lasting T-cell lines from gastric cancer patients and healthy volunteers. Blood samples were drawn and T lymphocytes were transformed. Once sustained growth was observed, lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Cytofluorometric analysis revealed that CD3 and CD45 were found at lower proportion in primary cells from patients than from control individuals (54% vs 75%, p<0.001, 90% vs 96%, p<0.05, respectively), and in HVS-derived T-cell lines (90% vs 98%, p<0.05, 97% vs 100%, p<0.05, respectively). Proliferative analyses showed that primary isolated cells were unable to respond adequately to CD3-, CD2-, and PHA-mediated stimulation, as compared to controls. Similarly, T-cell lines from patients proliferated to a lesser extent when CD3- and CD2-mediated stimuli were considered, especially when simultaneous stimulation via CD3 and CD2 molecules was carried out (47,824 counts per minute [cpm] vs 121,478 cpm, p<0.05). Altogether these results show that the defects reported in T cells from patients with cancer are not exclusively due to tumour-derived factors, since the alterations persist in long-lasting, HVS-transformed, T-cell lines, suggesting that this model seems a suitable one to disclose them.APV and MP-B should be considered as joint first authors     

Poly(U) binding to ascites cells of the 13762A rat mammary adenocarcinoma: Evidence for a protein receptor on the cell surface

Ascites cells of the 13762 rat mammary adenocarcinoma bind poly(U) in a reaction that is complete within 5 min at 0°C. Poly(U) binding is saturable; the capacity of these cells is 5×10⁷ UMP residues/cell (approx. 2×10⁵ chains/cell). Most [³H]poly(U) bound in the rapid reaction can be recovered in an undergraded state. However, it is rapidly degraded by low concentrations of exogenous pancreatic ribonuclease. The magnitude of binding is independent of temperature and ionic conditions, and is unaffected by metabolic inhibitors or concanavalin A (ConA). Radioactivity presented as [³H]poly(U) tends to co-fractionate with 5′-nucleotidase after homogenization of cells in the media of low ionic strength, but is efficiently released from cells exposed to protein denaturants that effectively fix cellular RNA in situ. Cells pretreated with proteolytic enzymes have sharply reduced capacities to bind poly(U). Autoradiography of cells bearing [³H]poly(U) demonstrates a uniform distribution of radioactivity through the cell population and is consistent with binding to the plasma membrane. These and other results imply that binding of poly(U) to 13762 ascites cells is mediated by protein receptors on the cell surface.     

Virus-specific proliferative T-cell responses: Parameters and specificity

The genetic requirements for inducing virus-specific T-cell proliferation were investigated by taking spleen cells from animals primed with vaccinia virus in vivo, then culturing the cells in vitro with vaccinia virus-infected syngeneic peritoneal macrophages, and finally restimulating these cells a second time in vitro with vaccinia virus-infected macrophages from several strains of mice. Under these conditions, T cells proliferated in the tertiary response to virus-specific stimulation, whereas background proliferation caused by allogeneic differences between stimulator and responder cells was minimal. Compatibility between T cells and infected stimulator cells at the K or I regions alone or at I-A or I-A + I-B regions of the major histocompatibility complex (MHC) produced strong proliferative responses, whereas compatibility at D alone often resulted in somewhat weaker responses. However, these responses were rarely as great as in combinations of completely syngeneic stimulator and responder cells. Homology between responding and virus-infected stimulating cells in more than one of the H-2K, D, or I regions resulted in an additive, but not potentiating, effect. Genes coded outside the H-2 region did not seem to play a role in this system. In some rare cases, a weak response occurred across allogeneic barriers, but in general, virus-specific T-cell proliferation was strongly H-2 restricted.     

Biosynthesis of the cell surface sialomucin complex of ascites 13762 rat mammary adenocarcinoma cells from a high molecular weight precursor

Cell surfaces of metastatic 13762 ascites rat mammary adenocarcinoma cells are covered with a sialomucin complex composed of the high Mr sialomucin ASGP-1 (approximately 600,000) and a concanavalin A-binding, integral membrane glycoprotein ASGP-2 (120,000). Antibodies prepared against ASGP-2 and deglycosylated ASGP-1 react on immunoblots of ascites cells or their isolated microvilli with the Mr = 120,000 species and the high Mr sialomucin, respectively. No cross-reactivity was observed. Under complex dissociating conditions, anti-ASGP-2 immunoprecipitated primarily components of Mr = 120,000 and about 400,000 from lysates of cells labeled for 1 h with mannose, glucosamine, and threonine. Under similar conditions, anti-ASGP-1 immunoprecipitated the Mr = 400,000 component and a second major labeled component of about 330,000. Pulse-chase labeling with 35S-labeled amino acids followed by immunoprecipitation with anti-ASGP-2 indicated a precursor-product relationship for the Mr = 400,000 component, designated pSMC-1 (precursor, sialomucin complex), and ASGP-2. Similar pulse-chase analyses of threonine-labeled cells using anti-ASGP-1 showed equivalent amounts of immunoprecipitated pSMC-1 and pSMC-2, both of which disappeared with kinetics similar to those observed for pSMC-1 immunoprecipitated with anti-ASGP-2. A precursor-product relationship of both pSMC-1 and pSMC-2 to ASGP-1 was suggested by combined precipitations with anti-ASGP-1 and peanut agglutinin, which precipitates ASGP-1 specifically. Immunoblot and lectin blot analyses indicated that pSMC-1 and pSMC-2 from the immunoprecipitates bind anti-ASGP-2, anti-ASGP-1, and concanavalin A. Moreover, these three components can also be labeled with mannose; the mannose was removed from 30-min pulse-labeled anti-ASGP-2 immunoprecipitates by incubation with endo-beta-N-acetylglucosaminidase H, indicating the presence of only high mannose N-linked oligosaccharides in pSMC-1. One-dimensional peptide maps of 35S-labeled pSMC-1 and Mr = 120,000 ASGP-2 showed several corresponding bands. These results indicate that both ASGP-1 and ASGP-2 can be synthesized from a common high Mr precursor. We propose that complex is formed from pSMC-1 by proteolytic cleavage to yield Mr = 120,000 ASGP-2 plus the precursor to ASGP-1 early in the transit pathway from the endoplasmic reticulum to the cell surface.     

Isolation and partial characterization of ascites sialoglycoprotein-2 of the cell surface sialomucin complex of 13762 rat mammary adenocarcinoma cells.

Sialomucins are the dominant components of the cell surfaces of some carcinoma ascites cells and have been postulated to inhibit recognition of tumours by the immune system. The sialomucin ASGP-1 (ascites sialoglycoprotein-1) of the 13762 rat mammary adenocarcinoma is associated with the cell surface as a complex with a concanavalin-A-binding glycoprotein called ASGP-2. This sialomucin complex has been purified from ascites cell microvilli by extraction with Triton X-100 and CsCl density-gradient centrifugation. ASGP-1 (which has been purified previously) and ASGP-2 were dissociated in 6 M-guanidine hydrochloride and separated by gel filtration. The molecular mass of the undenatured detergent complex of ASGP-2, estimated by gel filtration and velocity sedimentation in Triton X-100, was 148 kDa. Since the apparent molecular mass by SDS/polyacrylamide-gel electrophoresis was about 120 kDa, ASGP-2 must be a monomer as extracted from the membrane. Studies of its chemical composition indicate that it contains about 45% carbohydrate by weight, including both mannose and galactosamine. Alkaline borohydride treatment of ASGP-2 converted approx. half of the N-acetylgalactosamine to N-acetylgalactosaminitol, demonstrating the presence of O-linked oligosaccharides. Analyses of mannose-labelled Pronase glycopeptides from ASGP-2 by lectin-affinity chromatography on concanavalin A and leucocyte-agglutinating phytohaemagglutinin suggested that 40% of the label was present in high-mannose/hybrid oligosaccharides, 20% in triantennary oligosaccharides substituted on the C-2 and C-4 mannose positions and 40% in tri- or tetra-antennary oligosaccharides substituted on C-2 and C-6. The presence of polylactosamine sequences on these oligosaccharides was suggested by lectin blots and by precipitation from detergent extracts with tomato lectin. From chemical analyses and lectin-affinity studies, we estimate that ASGP-2 contains four high-mannose and 13 complex N-glycosylated oligosaccharides, plus small amounts of polylactosamine and O-linked oligosaccharides. The presence of four different classes of oligosaccharides on this glycoprotein suggests that it will be an interesting model system for biosynthetic comparisons of the different glycosylation pathways.     

Inhibition of in vitro lymphoproliferative responses by in vivo passaged rat 13762 mammary adenocarcinoma cells. I. Characteristics of inhibition and evidence for an infectious agent.

Small numbers of X-irradiated 13762 cells added as third-party cells to mitogen response assays or mixed lymphocyte cultures caused a significant reduction in viability of the cocultivated lymphocytes, and completely inhibited the expected lymphoproliferative responses. Results showed that the factor(s) responsible for the inhibitory effect was preserved after ultrasonic disruption of the tumor cells, could be sedimented by ultracentrifugation, and was sensitive to treatment with ultraviolet light. Further, cytopathic effects could be serially propagated using cell-free supernatants obtained from sonicated 13762 tumor cells. The results suggest that the 13762 adenocarcinoma line, as carried in vivo in this laboratory, harbors an infectious particle which can affect the proliferative responses of lymphocytes in vitro.     

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rat cell line derived from DMBA-induced mammary carcinoma

A new cell line, designated UHKBR-01, was successfully established from a 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumour. DMBA was administered orally at a dose of 4 mg/ml per rat on the first day of the experiment and thereafter at weekly intervals of same dosage, until the rats have reached a weight of around 150-200 g. The tumours grew rapidly after the injection, and were transplanted into nude mice one the harvest size (2.5 x 2 x 1 mm(3)) was reached, it was transplanted onto nude mice. We have developed a cell line from a portion of the DMBA-induced carcinoma of the nude mice. The UHKBR-01 cell exhibited a slow increase in growth rate during the time of culture and was highly tumourigenic in nude mice. The cells have been grown in culture for over 40 passages. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, tumour antigen expression and xenograft implantation into nude mice. These cells exhibit ultrastructural and immunohistochemical features of epithelial cells of mammary origin. The above analyses also demonstrated that UHKBR-01 cells were oestrogen- and progesterone-receptor positive, in likeness to other established breast cancer cell lines such as MDA-MB-231 and MCF-7. The cell line grows as monolayers of oval-shaped cells with large folded nuclei accompanied by a rich supply of mitochondria. This report describes the first in vitro cell line from transplantable DMBA-induced mammary carcinoma of nude mice, which presents unique characteristics that may prove to be a good experimental model for investigating breast cancer biology.     

Structures of the O-linked oligosaccharides of the major cell surface sialoglycoprotein of MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma

Structures of the principal O-glycosides from the major cell surface sialoglycoprotein (ASGP-1) of the MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma have been determined. Oligosaccharitols were released by alkaline borohydride treatments of ASGP-1 and purified by gel filtration, DEAE-Sephadex ion exchange chromatography, and high performance liquid chromatography. On the basis of carbohydrate composition, methylation analysis, periodate oxidation, and exoglycosidase digestion, the five major oligosaccharides released by mild alkaline borohydride were assigned the following structures: Component II-3: (NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)Ga 1 NAcOH(3----1 betaGa 1 3----2 alpha NeuAc) III-2a: (Ga 1 beta 1----4G1cNAc beta 1----6)Ga 1 NAcOH(3----1 beta Ga 1 3----2 alpha NeuAc) III-2c: (Ga 1 alpha 1----3Ga 1 beta 1----4G1cNAc beta 1----6) Ga 1 NAcOH(3----1 beta Ga 1 3----2 alpha NeuAc) IV-1a: (Ga 1 beta 1----4G 1 cNAc beta 1----6)Ga 1 NAcOH(3----1 beta Ga 1) IV-1c: (Ga 1 alpha 1----3Ga 1 beta 1----4G 1 cNAc beta 1----6) Ga 1 NAcOH(3----1 beta Ga 1) Fucosylated derivatives of III-2a, IV-1a, and IV-1c were found in smaller amounts with the fucose tentatively assigned to the 2-position of the lactosamine galactose. Components II-3, III-2a, and the fucosylated derivative of III-2A were found in both MAT-B1 and MAT-C1 sublines. The alpha-galactosides were found in detectable quantities only in subline MAT-B1. Oligosaccharides from MAT-C1 cells were enriched in sialic acid when compared to those from MAT-B1 cells. These results suggest that the 13762 ascites sublines, which bear different oligosaccharides, will provide models useful for the investigation of mechanisms regulating the expression of structures of the larger O-linked oligosaccharides.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.