TA3-Ha Cells Research Articles

Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21.

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

Significant Impact of Immunogen Design on the Diversity of Antibodies Generated by Carbohydrate-Based Anticancer Vaccine.

Development of an effective vaccine targeting tumor associated carbohydrate antigens (TACAs) is an appealing approach toward tumor immunotherapy. While much emphasis has been typically placed on generating high antibody titers against the immunizing antigen, the impact of immunogen design on the diversity of TACA-specific antibodies elicited has been overlooked. Herein, we report that the immunogen structure can significantly impact the breadth and the magnitude of humoral responses. Vaccine constructs that induced diverse TACA-binding antibodies provided much stronger recognition of a variety of Tn positive tumor cells. Optimization of the breadth of the antibody response led to a vaccine construct that demonstrated long lasting efficacy in a mouse tumor model. After challenged with the highly aggressive TA3Ha cells, mice immunized with the new construct exhibited a statistically significant improvement in survival relative to controls (0% vs 50% survival; p < 0.0001). Furthermore, the surviving mice developed long-term immunity against TA3Ha. Thus, both the magnitude and the breadth of antibody reactivity should be considered when designing TACA-based antitumor vaccines.

Induction of caspase-independent apoptotic-like cell death of mouse mammary tumor TA3Ha cells in vitro and reduction of their lethality in vivo by the novel chemotherapeutic agent GIT-27NO

The new chemical entity GIT-27NO was created by the covalent linkage of a NO moiety to the anti-inflammatory isoxazoline VGX-1027. The compound has been shown to possess powerful anticancer effects both in vitro and in vivo. However, its effects on nonsolid and metastatic forms of tumors have not yet been investigated. We have studied the effects of GIT-27NO on the highly invasive mouse mammary TA3Ha cell line in vitro and in vivo. In contrast to the conventional exogenous NO donor sodium nitroprusside, GIT-27NO successfully enhanced intracellular NO concentration in TA3Ha cells. Intracellular accumulation of NO was followed by marked decrease in TA3Ha cell viability accompanied by typical apoptotic features. Interestingly, inverted membrane phosphatidylserine residues, reduced volume of nucleus, condensed chromatin, and terminal fragmentation of DNA were associated with inhibited caspase-3 activity and transcription of the genes encoding caspase-3, -8, and -9. In parallel, GIT-27NO rapidly but transiently prevented the loss of p53 through phosphorylation on Ser 20 and provided the necessary signals for the execution of downstream processes without p53 de novo synthesis. The caspase-independent apoptotic-like death process triggered by GIT-27NO could be mediated by markedly down-regulated expression of the antiapoptotic Bcl-2 molecule observed in TA3Ha cells exposed to GIT-27NO. In agreement with these in vitro data, GIT-27NO efficiently suppressed the growth of the ascites form and associated lethality of tumor induced by TA3Ha cells in mice.

Ligation of tumour-produced mucins to CD22 dramatically impairs splenic marginal zone B-cells

CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to alpha2,6- sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state.

Identification and Expression of Human Epiglycanin/MUC21: a Novel Transmembrane Mucin

The gene for the human orthologue of mouse epiglycanin, a mucin expressed on mammary carcinoma TA3-Ha cells but not TA3-St cells, was identified by homology search to a mouse epiglycanin cDNA fragment identified by representational difference analysis between TA3-Ha and TA3-St cells. The open reading frame of this gene was cloned from human cervical carcinoma ME-180 cells. It consists of a mucin domain with 28 nonidentical tandem repeats of 45 nucleotides each corresponding to a threonine/serine-rich peptide, a stem domain, a transmembrane domain, and a cytoplasmic tail. The cloned cDNA with a FLAG sequence was expressed in K562 cells. A combination of immunoprecipitation with a polyclonal antibody specific for the cytoplasmic tail and Western blotting analysis with an anti-FLAG antibody and lectins revealed a mucin-like component as the gene product. Analysis by the use of tissue cDNA libraries indicated that the gene is expressed in lung, large intestine, thymus, and testis among 16 normal tissues tested. The polyclonal antibody specific for a synthetic peptide from the cytoplasmic tail, when tested for its reactivity with normal lung tissues, reacted with epithelia of bronchi and bronchioli but not with alveoli. All of 24 lung adenocarcinomas specimens tested were reactive with the antibody, whereas reactivity was observed with only 2 out of 24 squamous and none out of 24 small cell lung carcinomas. This is a novel transmembrane mucin and designated as MUC21.

Different Progression of Tumor Xenografts between Mucin-Producing and Mucin–Non-Producing Mammary Adenocarcinoma-Bearing Mice

Previously, we found that MUC2 mucins could activate monocytes/macrophages through a scavenger receptor leading to cyclooxygenase (COX) 2 induction and overproduction of prostaglandin E2 (PGE2). To investigate the role of mucins in the tumor-bearing state, we compared s.c. tumor formation by using mucin-producing (TA3-Ha) and mucin-non-producing (TA3-St) cloned variants of mouse mammary adenocarcinomas. Expression of COX2 mRNA and protein and production of PGE2 were elevated in peritoneal macrophages stimulated with epiglycanin, which is a mucin-like glycoprotein produced by TA3-Ha cells. S.c. tumor tissues comprising TA3-Ha cells grew much faster than tissues comprising TA3-St cells. COX2 protein and vascular endothelial growth factor in TA3-Ha tumor tissues were elevated compared with the TA3-St tumor tissues. Although similar numbers of macrophages were observed immunochemically in the two types of tumor tissues, COX2 was induced prominently in the infiltrating macrophages in TA3-Ha tumor tissues but only faintly in TA3-St tumor tissues. Furthermore, angiogenesis progressed remarkably in TA3-Ha tumor tissues but only slightly in TA3-St tumor tissues. Epiglycanin-induced overproduction of PGE2 down-regulated interleukin-12 production by macrophages. IFN-gamma-producing CD4 T cells in spleens obtained from TA3-Ha tumor-bearing mice were significantly reduced compared with TA3-St tumor-bearing mice, suggesting that mucins cause PGE2-mediated immune suppression. Actually, the tumor growth of a TA3-Ha cell xenograft was suppressed effectively by oral administration of a COX2 inhibitor but that of a TA3-St cell one was not. These results suggest that mucins play an important role in tumor progression through overproduction of PGE2.

Tumor-Host Interactions Regulate Glucocorticoid-Mediated Epiglycanin Expression in TA3Ha Murine Mammary Carcinoma Cells

The mucin-type glycoprotein epiglycanin is highly expressed on the cell surface of TA3Ha mouse mammary carcinoma cells if the cells are maintained by serial intraperitoneal passage in mice. We found that upon transfer of the cells to growth in vitro in medium supplemented with fetal bovine serum, the expression level of epiglycanin slowly diminished to less than 15% of that in vivo. Repassage of the cells in vivo fully restored the expression of epiglycanin. When TA3Ha cells were cultured in media supplemented with ascites fluid obtained from mice bearing these cells, the high expression level of epiglycanin was maintained. Ascites fluid-containing medium also restored the high expression level of epiglycanin in cells cultured for prolonged periods in vitro. Human ascites fluids obtained from patients with mammary and ovary carcinoma were likewise able to induce the expression of epiglycanin, in contrast to ascites fluids obtained from noncarcinoma patients or serum of healthy individuals. The factor responsible for the upregulation of epiglycanin is heat stable and can be removed from ascites fluids by activated charcoal, suggesting that it is a steroid. Indeed, dexamethasone, at a concentration of 10^–7 mol/l, induced maximal epiglycanin expression within 4 days, and upregulation was inhibited by the glucocorticoid receptor antagonist RU486. However, the maximal induction of epiglycanin expression achieved with ascites fluid was higher, while the kinetics of the induction was much slower, but still RU486 sensitive, than upon treatment with dexamethasone. These results suggest that stimulation of epiglycanin expression by ascites also involves a glucocorticoid. However, the induction in vivo is indirect and involves a cascade of events induced by tumor-host interactions that lead to glucocorticoid-mediated induction of epiglycanin.

Biosynthesis and shedding of epiglycanin: a mucin-type glycoprotein of the mouse TA3Ha mammary carcinoma cell

Epiglycanin is a mucin-type glycoprotein present at the surface of TA3Ha mouse mammary tumour cells. It is a long rod-like glycoprotein with a molecular mass of 500 kDa. Its function has not yet been established but its overexpression can affect cell-cell and cell-matrix adhesion. To understand better the biological function of epiglycanin, we have studied the biochemical structure and biosynthesis of epiglycanin in TA3Ha cells. Pulse-chase labelling experiments with [(3)H]threonine revealed an early precursor with a molecular mass of approx. 300 kDa containing approx. 5-10 kDa of N-linked glycans. The precursor was gradually converted into a high-molecular-mass mature form, owing mainly, if not entirely, to O-glycosylation. The mature molecule consists of two major glycoforms that differ in sialylation. Unlike secreted mucins, epiglycanin did not form cysteine-bound multimers, providing further evidence that epiglycanin belongs to the class of membrane-associated mucins. The mature form, but not the precursor form, is shed from the cell surface. The half-life of epiglycanin on the cell surface was found to be approx. 60 h. These results provide the first detailed analysis of the biochemical structure and biosynthesis of epiglycanin.

Biosynthesis and shedding of epiglycanin: a mucin-type glycoprotein of the mouse TA3Ha mammary carcinoma cell

Epiglycanin is a mucin-type glycoprotein present at the surface of TA3Ha mouse mammary tumour cells. It is a long rod-like glycoprotein with a molecular mass of 500kDa. Its function has not yet been established but its overexpression can affect cell–cell and cell–matrix adhesion. To understand better the biological function of epiglycanin, we have studied the biochemical structure and biosynthesis of epiglycanin in TA3Ha cells. Pulse–chase labelling experiments with [3H]threonine revealed an early precursor with a molecular mass of approx. 300kDa containing approx. 5–10kDa of N-linked glycans. The precursor was gradually converted into a high-molecular-mass mature form, owing mainly, if not entirely, to O-glycosylation. The mature molecule consists of two major glycoforms that differ in sialylation. Unlike secreted mucins, epiglycanin did not form cysteine-bound multimers, providing further evidence that epiglycanin belongs to the class of membrane-associated mucins. The mature form, but not the precursor form, is shed from the cell surface. The half-life of epiglycanin on the cell surface was found to be approx. 60h. These results provide the first detailed analysis of the biochemical structure and biosynthesis of epiglycanin.

The potential role of integrin receptor subunits in the formation of local recurrence and distant metastasis by mouse breast cancer cells.

The mechanisms by which surgical injury fosters tumor growth are examined. TA3Ha mouse breast tumor line and its subline (TA3AD) differing in their metastatic abilities as tested by two models were used. In model a, TA3Ha/TA3AD tumors were grown in the mammary fat pads of mice and then surgically removed with a curative intent. In model b, TA3Ha/TA3AD cells were injected intravenously into mice subjected to liver or spleen wedge resection. Frequency of tumor formation at various sites was assessed. Expression of integrin, immunoglobulin, and proteoglycan cell adhesion receptors on TA3Ha and TA3AD cells was examined by flow cytometry. The roles of these receptors in metastasis were examined by blocking them by selected ligands and/or antibodies. Frequencies of local recurrence and axillary metastasis after surgical resection, were 43% (32/74), and 37% (27/74) with TA3Ha tumors and 4% (1/29) at both sites with TA3AD tumors. Tumors at surgically injured spleen and the liver were seen in 75% (141/189) and 45% (107/240) of the mice with TA3Ha cells and in 8% (3/38) and 10% (4/42) of the mice with TA3AD cells. alpha 5 and CD44 receptors were expressed by TA3Ha cells but not by TA3AD cells. Other receptors examined were similarly expressed by both cell lines. Blocking of alpha 5 receptor by fibronectin reduced tumor implantation in a dose-dependent manner. The data suggest a correlation among the ability to implant at surgically injured sites, to form local recurrence, and to express the fibronectin receptor subunit.

Effect of trauma of implantation of metastatic tumor in bone in mice

We studied the influence of surgical trauma to the iliac bone on the implantation of I.V. injected tumor cells, which formed tumor in the surgical wounds of 27/84 mice (32%). None of these mice or nonsurgical mice developed tumor in the opposite or uninjured pelvic bone (P < 0.0001). When different numbers (10(5), 5 x 10(5), and 10 x 10(5)) of TA3Ha cells were injected I.V. immediately after surgery, the frequency of tumor formation showed an increase (respectively, 32%, 63%, 71%). As the interval between induction of trauma and tumor cell injection was increased from 0 to 15 days, the frequency of tumor formation declined from 32% to 0%. These results suggest that the healing wound is a privileged site for experimental metastasis, particularly in the early stages. It is likely that the proteins in the blood clotting cascade are involved in local tumor implantation.

The role of fibronectin in tumor implantation at surgical sites.

Fibronectins are a family of glycoproteins with modular functional domains. They mediate cell-cell and cell-matrix interactions which are important in embryogenesis, wound healing, metastasis and other processes. We present data on the influence of fibronectin on wound implantation of a murine mammary carcinoma line, TA3Ha. Fibronectin used in these studies was derived from bovine plasma, human serum, human foreskin fibroblasts, and mouse embryo cultures. TA3Ha cells rarely form tumors in the liver of syngeneic mice when injected intravenously but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Wound implantation is markedly reduced when the cells are pre-exposed to 200 micrograms/ml bovine plasma fibronectin (13%, P = 0.007), human serum fibronectin (0%, P = 0.02), human cellular fibronectin (0%, P = 0.02), or mouse cellular fibronectin (0%, P = 0.04). Lung colonization is also reduced by these fibronectins. These effects are not due to a cytotoxic action of fibronectin, since intraperitoneally injected fibronectin-treated cells form ascites tumor as effectively as do control untreated cells. Local application of a solution containing 0.25 mg/ml mouse cellular fibronectin to the hepatic wound reduces the frequency of tumor implantation from 45% to 5% (1/21, P = 0.001). No tumor implantation inhibition is seen when only suspending medium or albumin in suspending medium is used. The mechanism by which topical application of fibronectin reduces hepatic wound implantation of tumor cells is unclear, but this finding raises an exciting possibility of preventing local recurrence of cancer.

Inhibition of tumor implantation at sites of trauma by Arg-Gly-Asp containing proteins and peptides

We report on the inhibition of wound implantation by TA3Ha mammary carcinoma cells by Arg-Gly-Asp containing proteins and peptides using a hepatic wedge resection model. Intravenously injected TA3Ha cells rarely form tumor in the liver of syngeneic mice, but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Hepatic wound implantation is significantly (P = 0.01) inhibited by pretreating the cells with whole mouse plasma, but not with fibrinogen-depleted plasma or serum. Tumor inhibition is also achieved by pretreatment of cells with fibrinogen (P = 0.05-0.0004), fibronectin (P = 0.007) and laminin, but not by albumin. The active domain appears to be the RGDS sequence since the deca- and tetrapeptides containing RGDS inhibit wound implantation (P less than 0.05). However, the tetrapeptide Arg-Gly-Glu-Ser has no such activity. None of these agents affects ascites tumor formation by the intraperitoneally injected cells, suggesting that anchorage independent growth of cells is not affected. We propose that proteins and peptides containing RGD occupy the binding sites and prevent the cells from interacting with cell adhesion proteins in healing wounds. Proteins and/or peptides containing RGD may be useful for preventing local recurrence in postsurgical cancer patients.

Inhibition of tumor implantation at sites of trauma by plasminogen activators

The authors report on the influence of plasminogen activators (PA) on implantation of TA3Ha mammary tumor cells in the healing hepatic wounds of syngeneic strain A mice. Intravenously injected TA3Ha cells, although they rarely metastasize to the liver, formed tumors in the hepatic wounds of a significant percent (42%, P less than 0.0001) of mice. The frequency of tumor formation declined as the interval between surgery and tumor cell inoculation was increased. Furthermore, preexposure of cells to fibrinogen, fibronectin, laminin, or peptides containing the arginine-glycine-aspartic acid-serine residues dramatically reduced the frequency of tumor formation in the hepatic wounds. These results indicate that TA3Ha cells interact with fibrinogen-related proteins in the wound to aid their attachment and growth. Because these proteins are susceptible to digestion by plasmin, PA were used in this study to examine whether administration of these drugs to the mice would modulate tumor formation in the liver wounds. Among the PA tested, human plasmin B-chain-streptokinase complex (B-SK) and recombinant tissue plasminogen activator (t-PA) inhibited tumor implantation in a dose-related manner. Administration of 900 units (U) of B-SK or 3300 U of t-PA per mouse reduced the frequency of tumor formation from 42% to 0% (P = 0.02) and 11% (P = 0.02), respectively. The B-SK was complexed with p-nitrophenyl-p-guanidinobenzoate; it did not activate the plasminogen or inhibit tumor formation in the hepatic wounds. Although urokinase activated the plasminogen, it did not inhibit tumor implantation in the hepatic wound. Heparin, an anticoagulant that prevents conversion of fibrinogen to fibrin without being fibrinolytic, had no influence on tumor formation in the hepatic wounds. The PA can generate plasmin that digests the cell attachment proteins in wounds and consequently inhibits tumor cell attachment.

The influence of surgical trauma on experimental metastasis.

Influence of surgical trauma on experimental metastasis in healing wounds is investigated using a transplantable murine mammary carcinoma cell line, TA3Ha. Intravenous injection of 10(5), 10(6), and 2 x 10(6) TA3Ha cells into syngeneic Strain A mice led to liver or kidney tumor development in none of the 96, ten, and ten mice tested, respectively. In contrast, injection of 10(5) cells into mice immediately after hepatic wedge resection performed using milliwatt carbon dioxide laser and electrocautery resulted in tumor formation at the site of trauma in 21/37 (57%) and 25/52 (48%) mice, (P less than 0.001) respectively. Similar results were obtained in mice subjected to partial nephrectomy using the laser (nine of 18) and electrocautery (eight of 13). These results clearly demonstrate that surgical trauma renders a nonprivileged organ susceptible to experimental metastasis formation, and that at least in this model both laser and electrocautery have similar effects. Tumor cell injection 1, 7, and 10 days posthepatic surgery resulted in 36%, 20%, and 0% tumor formation, respectively, indicating that the earlier events in wound healing support tumor implantation and/or growth better than those later on. Frequency of tumor formation at sites of trauma in the peritoneum induced by scalpel blade, laser, and electrocautery were 28%, 50% and 82%, respectively. Peritoneal tumors were seen in 33% of the nonsurgical mice. Skin incisions induced with the three above probes had little influence on experimental metastasis formation. Thus the influence of trauma on tumor formation is not uniform in every organ.

Antitumor and toxicologic properties of the organometallic anticancer agent vanadocene dichloride

We report here on the antineoplastic, toxicologic, and transmembrane transfer properties of vanadocene dichloride (VDC), a representative metallocene dihalide. VDC is cytotoxic to HEp-2 human epidermoid carcinoma cells, in vitro, in a dose dependent manner, with a D o value (dose increment reducing the survival fraction by 1/e) of 0.530 ± 0.005 μg/ml. Under similar experimental conditions, the D o for cisplatin (CDDP) against these cells is 0.46 ± 0.08 μg/ml. In a murine mammary adenocarcinoma (TA3Ha) system, 125 μg/ml VDC inhibits the tumor-forming ability of 10 5 cells upon i.p. inoculation into syngeneic Strain A mice. The transmembrane transfer rate constants for the metal uptake of VDC and CDDP by TA3Ha cells in vitro were found to be 3.3 ± 0.8 × 10 −4 min −1 and 12 ± 2.0 × 10 t-4 min −1, respectively. In vivo studies with TA3Ha cells show that two i.p. treatments of 20, 40, and 60 mg/kg VDC increase the host survival by 30, 50, and 90%, respectively. Under similar conditions, 2, 4, and 6 mg/kg CDDP (equitoxic dose levels) prolong the host survival 50, 75, and 83%, respectively. Morphological, blood urea nitrogen level, and serum creatinine level data for Strain A mice treated with 60 mg/kg VDC give no evidence of renal or small intestinal damage. However, changes in the liver consistent with fatty cell degeneration are observed in these mice.

Factors Influencing the Growth and Metastatic Behavior of Tumors

The factors influencing the growth and metastatic behavior of experimental animal tumors are examined. 10(4) TA3Ha cells were injected intraperitoneally, intravenously, subcutaneously into the flank, and subcutaneously into the tail tissues of syngeneic strain A mice. The tumor takes from these injections were 50/50 (100%), 1/10 (10%), 10/10 (100%), and 7/12 (60%), respectively. The frequency of lung metastasis from these sites was 0, 100, 50, and 100%, respectively. At the time of host death, the flank and tail tumors were, respectively, 2.2 +/- 0.5 cm (geometric mean diameter) and 1.2 +/- 0.3 cm in the TA3Ha and 0.9 +/- 0.1 cm and 0.4 +/- 0.1 cm in the L1210 systems. TA3Ha tumors metastasized regularly to the lymph nodes but the L1210 tumors seldom metastasized to the lymph nodes. Tail implants of TA3Ha tumors behaved similarly in the athymic nude mice and strain A mice. TA3Ha cells inoculated into the Millipore chambers and maintained in mice for greater than 150 days were viable and able to form tumors. The results demonstrate that the anatomic location of the tumor affects the growth and metastatic behavior of the tumors, and that the tumors of different histologic origin metastasize differently even when grown in corresponding locations.

Factors influencing the growth and metastatic behavior of tumors.

The factors influencing the growth and metastatic behavior of experimental animal tumors are examined. 10(4) TA3Ha cells were injected intraperitoneally, intravenously, subcutaneously into the flank, and subcutaneously into the tail tissues of syngeneic strain A mice. The tumor takes from these injections were 50/50 (100%), 1/10 (10%), 10/10 (100%), and 7/12 (60%), respectively. The frequency of lung metastasis from these sites was 0, 100, 50, and 100%, respectively. At the time of host death, the flank and tail tumors were, respectively, 2.2 +/- 0.5 cm (geometric mean diameter) and 1.2 +/- 0.3 cm in the TA3Ha and 0.9 +/- 0.1 cm and 0.4 +/- 0.1 cm in the L1210 systems. TA3Ha tumors metastasized regularly to the lymph nodes but the L1210 tumors seldom metastasized to the lymph nodes. Tail implants of TA3Ha tumors behaved similarly in the athymic nude mice and strain A mice. TA3Ha cells inoculated into the Millipore chambers and maintained in mice for greater than 150 days were viable and able to form tumors. The results demonstrate that the anatomic location of the tumor affects the growth and metastatic behavior of the tumors, and that the tumors of different histologic origin metastasize differently even when grown in corresponding locations.

Further characterization of the glycoproteins and glycosaminoglycans released from TA3 murine adenocarcinoma cells in culture

TA3 murine ascites adenocarcinoma cells were compared for their ability to release radioactive glucosamine and 35SO 4-labeled glycoproteins and glycosaminoglycans into the culture medium. Both TA3-Ha and TA3-St cells contained cell-surface heparan sulfate that was released into culture, but not chondroitin sulfate. Both cells released a membranous aggregate of labeled components from the cell surface and hyaluronic acid from inside the cells that fractionated in the void volume of Sepharose CL-4B. This void-volume fraction from the TA3-Ha cells contained glucosamine-labeled epiglycanin at a higher concentration relative to other glucosamine-labeled components than that found on plasma membranes. Glycoproteins associated with epiglycanin found on the cell surface, as well as released into culture medium, contained sulfate that could not be removed by chondroitinase ABC, heparinase, or keratinase. Kinetic analysis of the glucosamine-labeled material released from TA3-Ha cells indicated that hyaluronic acid was released rapidly with a 45-min half-life, whereas the other membranous components were released much more slowly.

Monoclonal antibody against a cryptic carbohydrate antigen of murine and human lymphocytes. I. Antigen expression in non-cryptic or unsubstituted form on certain murine lymphomas, on a spontaneous murine mammary carcinoma, and on several human adenocarcinomas.

This paper describes an IgM monoclonal antibody (49H.8) which was produced following immunization of BALB/c mice with human neuraminidase-treated erythrocytes (NE-RBC). 49H.8 reacts with NE-RBC, neuraminidase-treated T lymphocytes (NE-T) and NE B lymphocytes of both human and murine origin. Little or no reactivity with untreated T or B cells could be detected. Thus the 49H.8 antigen is "cryptic" in most normal lymphocytes of both humans and mice. In contrast, the 49H.8 antigen was detected in non-cryptic or unsubstituted form on many non-treated murine lymphomas of both B- and T-cell origin, on the spontaneous murine mammary carcinoma, TA3-HA and on several human adenocarcinomas. The 49H.8 antigen appears to be related to the previously described 49H.24 antigen as shown by sugar inhibition experiments. 49H.24 reacts most strongly with the synthetic disaccharide (betaGa1 (I leads to 3)alpha Ga1NAc) but not at all with beta Ga1(I leads to 3)beta Ga1Nac. 49H.24 does not react with any of the murine or human tumors tested. 49H.8 reacts with both the alpha and beta forms of the disaccharide but reacts most strongly with phenyl-beta-galactoside-containing compounds. In contrast, phenyl-alpha-galactoside-containing compounds produced no reaction. The natural determinant detected by this antibody was not determined but various possibilities are considered. 49H.8 was used to detect antigen apparently shed from growing TA3-Ha cells into the serum and ascites of tumor-bearing mice. These observations suggest that the 49H.8 monoclonal antibody will be valuable as a specific reagent for a common tumor-associated antigen shared by certain murine and human tumors, and as a means of assaying shed tumor antigen in circulation as in the TA3-Ha mammary adenocarcinoma model.