The lysosome periphery: biochemical and electrokinetic properties of the tritosome surface.

Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO(3), pH 7.2 at 25 degrees C the tritosomes had an electrophoretic mobility of -1.77 +/- 0.02 microm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm(2), and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10(-7) m. Treatment of the tritosomes with 50 microg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 +/- 0.02 microm/s/V/cm under the same conditions and caused the release of 2.01 microg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven, however, since the tritosomes lysed below a pH of 4 in the present system. Total tritosome carbohydrate (anthrone-positive material as glucose equivalents) was 0.19 mg/mg tritosome protein while total sialic acid was 3.8 microg (11.4 nmol)/mg tritosome protein. A tritosome "membrane" fraction was prepared by osmotic shock, homogenization, and sedimentation. Approximately 25% of the total tritosome protein was present in this fraction. Analysis by gas-liquid chromatography and amino acid analyzer showed the following carbohydrate composition of the tritosome membrane fraction (in microgram per milligram tritosome membrane protein): N-acetylneuraminic acid, 14.8 +/- 3; glucosamine, 24 +/- 3; galactosamine, 10 +/- 2; glucose, 21 +/- 2; galactose, 26 +/- 2; mannose, 31 +/- 5; fucose, 7 +/- 1; xylose, 0; and arabinose, 0. The results indicate that the tritosome periphery is characterized by external terminal sialic acid residues and an extensive complement of glycoconjugates. Essentially all the tritosome N-acetylneuraminic acid is located in the membrane and about 53% of it is neuraminidase susceptible.

Studies on the relationship of the B700 and B50 murine melanoma antigens.

The B50 and B700 proteins of B16 murine melanoma were studied; they were determined to be distinct, unrelated molecules. This was determined by V8 peptide mapping, N-terminal amino acid sequencing, absence of cross-reactivity with specific polyclonal antibodies, and monoclonal antibodies recognizing different epitopes.

Influence of immune status on the metastasis of three murine fibrosarcomas of different immunogenicities.

Three fibrosarcomas of different immunogenicities were tested for their ability to form spontaneous and experimentally induced metastases in normal, sham-suppressed, immunosuppressed, and immunologically restored syngeneic mice. Immunosuppression, achieved by adult thymectomy and sublethal X-irradiation (450 R), affected experimental metastasis of the three tumors in different ways. Upon i.v. injection, the highly immunogenic fibrosarcoma formed more pulmonary tumor colonies in immunosuppressed mice than in normal, sham-suppressed (sham thymectomy and 450 R), or immunologically reconstituted animals (thymectomy, X-irradiation, plus 10(7) normal syngeneic lymphocytes given i.v.). A fibrosarcoma of intermediate immunogenicity also formed more pulmonary metastases in immunosuppressed recipients, but this increase could not be reversed by reconstitution with 10(7) lymphocytes. In contrast, the least immunogenic tumor formed fewer pulmonary tumor colonies in immunosuppressed mice than in normal, sham-suppressed, or immunologically reconstituted mice. We conclude that the role of the immune system in experimental cancer metastasis varies for different tumors and that tumor immunogenicity is an important factor in the relationship between host immunity and tumor dissemination.

Characterization in vivo and in vitro of tumor cells selected for resistance to syngeneic lymphocyte-mediated cytotoxicity.

This report describes the selection and behavior of tumor cells resistant to cytolysis by syngeneic lymphocytes. Two B16 melanoma lines, F1 (low metastasis) and F10 (high metastasis), were cultured with lymphocytes from C57BL/6 mice immunized against B16. The selection procedure involved repeated exposure of the tumor cells to lymphocytes in vitro. After each interaction, the viable tumor cells were trpsinized, replated, and designated lines F1Lr-1 and F10Lr-1. The procedure was repeated five times, yielding lines F1Lr-6 and F10Lr-6, which resisted cytolysis by syngeneic lymphocytes. Mice were given s.c. or i.v. injections of cells from lines F1, F1Lr-6, F10, or F10Lr-6. Tumor growth patterns were the same for all four lines when the cells were injected s.c., however, the incidence of pulmonary metastases differed significantly after i.v. injection. Line F10 cells yielded more pulmonary metastases than an equal number of line F1 cells (p less than 0.01). F1Lr-6 cells yielded significantly fewer metastases than an equal number of lines F1 cells (p less than 0.01). A similar difference between F10Lr-6 and F10 cells was observed. The incidence of artificial metastases after i.v. injection of F10Lr-6 cells was similar to that for F1 cells. The quantitative organ distribution, arrest, and survival of i.v.-injected tumor cells were studied by using [125]-5-iodo-2'-deoxyuridinelabeled cells. There was a significantly greater number of cells from line F10, arrested and able to survive for 14 days in lungs, than cells from line F1. In contrast, cells from either line F1Lr-6 or F10Lr-6 had a lower incidence of arrest and survival than their lymphocyte-sensitive counterparts.

Homology of the B50 murine melanoma antigen to the Ro/SS-A antigen of human systemic lupus erythematosus and to calcium-binding proteins.

B50 is a murine melanoma-associated antigen found in tight association with B700, a melanoma-specific antigen. B700-like molecules are produced by all melanomas tested to date, including those of murine, human, swine and hamster origin. We have used rabbit antibodies to B50 to determine whether B50 expression is also restricted to melanomas. The results demonstrate that B50 is a commonly occurring protein, or is immunologically cross-reactive to a commonly occurring protein; 29 of 29 cell lines tested bound anti-B50 antibodies. N-terminal amino acid sequence analysis indicates that B50 has significant homology to the Ro/SS-A antigen of human systemic lupus erythematosus and to calcium binding proteins; hence B50 is likely to be an RNA and/or calcium-binding protein.

Macromolecules mediate prostacyclin release from human umbilical artery.

Previous reports regarding the modulation of prostaglandin release from tissues by serum components did not identify these components. We have found that inhibition of prostacyclin release from human umbilical artery by human serum is attributable to serum macromolecules. We demonstrate that such inhibitory activity depends on macromolecular size and may result from macromolecule/cell surface interactions.

Further studies of the therapeutic effects of murine melanoma-specific monoclonal antibodies.

The results presented here further characterize four murine monoclonal antibodies (mAb) that recognize melanoma-specific antigens (9B6, T97, 2-3-1 and 2-3-3). These melanoma-specific mAbs are of the IgG2b isotype and are significantly therapeutic when administered systemically against established pulmonary melanoma metastases. Here we show a consistent and significant inhibition of the growth of melanoma lung metastases by all four mAbs and the existence of a time 'window' at days 5-8 after tumor inoculation for optimal therapy. Since these mAbs were found not to be cytotoxic or cytolytic in vitro, we looked for host immune response regulation as being responsible for the therapeutic effects. Natural killer (NK) cells were implicated as one arm of the host immune system involved in this response since depletion of NK cells in vivo by alpha asialoGM1 or alpha NK1.1 antibodies partially abrogated the inhibitory effect of the mAbs. The observed antimetastatic effects could also be partially abrogated using antibodies directed against the T-cell subset surface markers, CD4+ and CD8+. Intramuscular melanoma tumor growth was also found to be suppressed by mAb 2-3-1, but only if administered in the area of tumor growth and only if multiple inoculations are administered over a 13-day period. The beneficial effect of mAb antimetastatic therapy was found to be useful against several syngeneic melanomas, including JB/MS, B16 and several sublines of the B16 F10 melanoma.

Production of monoclonal antibodies against the B700 murine melanoma antigen and their antimetastatic properties.

Two unique murine melanoma antigens, termed B700 and B50, have been identified and isolated from several different murine melanoma cell lines. Both antigens can be detected on the cell surface, are actively shed in culture, and are often found in close association intracellularly. In previous studies, the antigen B700, which is related to serum albumin by biochemical and immunological criteria, was shown to function as a melanoma-specific tumor rejection antigen. We have also shown that animals sensitized to irradiated JB/RH melanoma cells produce antibodies which recognize B700 and/or B50, with B700 evoking the stronger humoral response. Animals testing positive by ELISA for antibody production to B700 or B50 were used for preparation of hybridomas and four different murine monoclonal antibodies have been produced whose specificities should facilitate epitope mapping. Clones have been used to generate ascites fluid in nude mice; the antibodies specifically recognize B700 and intact murine melanoma cells, but not B50. Two of these monoclonal antibodies have been administered systemically to C57Bl/6 mice bearing 5 day pulmonary metastases of the JB/MS melanoma, and significant inhibition of metastatic growth was observed for both antibodies.

Cross-reactivity between murine melanoma antigen B700 and a human melanoma-associated antigen (M-66) recognized by autologous antibody: evidence suggesting shared epitopes.

We have previously reported the purification and partial characterization of a human melanoma-associated antigen (M-66) recognized by autologous antibody. This antigen was found to be an unusually acidic 66 kDa glycoprotein. In studies of murine melanoma, a 67-kDa albumin-like melanoma-associated antigen (MAA) isolated from B16 melanoma cells has also been reported by our laboratories. Because the murine MAA, B700, has a molecular weight that is nearly the same as M-66, we sought to determine what similarities and differences existed between these two antigens. Human sera S150, which is known to recognize M-66, was found to bind to murine melanoma cell line B16. The addition of purified M-66 inhibited binding of S150 to B16 cells. Binding by S150 was not noted against murine melanoma cell line S91, which is known not to express cell surface B700. Conversely, reactivity of S150 against Y-Mel 84:420, known to express M-66, could be inhibited by preincubation with B16 cells. Four monoclonal antibodies known to recognize B700 were evaluated for-binding against murine B16 and human melanoma cell line Y-Mel 84:420. Binding was noted against both B16 and Y-Mel 84:420 which could be inhibited by the addition of M-66. Binding of S150 was also noted against purified B700 as tested by ELISA. While a comparison of the amino acid composition of the two antigens revealed similarities, M-66 contained 2.8 times as much serine and 0.4 times as much proline as B700. B700 has been reported to be related to serum albumin, which is not the case for M-66.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte surface membrane alterations.

Electrophoretic mobility measurements were made of red blood cells obtained from patients with Duchenne and myotonic muscular dystrophy, from dystrophic mice and chickens, and from corresponding controls. Alterations in the erythrocyte surface electrokinetic properties were found in dystrophic mice and chickens and in many, but not all, patients with muscular dystrophy. The results are consistent with the concept of muscular dystrophy as a systemic membrane disease not limited to muscle.

Immunostaining of human melanomas by a monoclonal antibody to B700 mouse melanoma antigen.

Previous studies have shown that B700, an albumin-like murine melanoma antigen, has a human homologue termed H700. Polyclonal antibodies to B700 also bind to all cultured human, swine and hamster melanoma cells, suggesting that B700 is a "pan-melanoma" antigen. The objects of this investigation were: (a) to determine if 2-3-3, a monoclonal antibody to B700, can be used to identify human melanomas in formalin-fixed, paraffin-embedded tissues, and (b) to determine the specificity and potential diagnostic value of 2-3-3. Forty-eight of the 49 human melanomas, including spindle melanoma cells, stained positively, as did five of the eight pigmented naevi including cellular spindle naevi. Twenty-six of the 32 human non-melanomatous lesions were negative for 2-3-3 staining (weakly positive on one breast carcinoma and positive on five neural tumours). These results indicate that 2-3-3, a monoclonal antibody to the mouse melanoma antigen B700, can be used to identify H700 in archival specimens. 2-3-3 may have an advantage over HMB45, which is the most commonly used antibody for melanoma diagnosis, because of its immunoreactivity with spindle melanocytic lesions. Antibodies to B700 may prove to be a useful adjunct in the diagnosis of human melanoma and related lesions.

B700, an albumin-like melanoma-specific antigen, is a vitamin D binding protein.

B700, a murine melanoma-specific antigen, is a member of the serum albumin protein family. Other members include serum albumin and vitamin D binding protein. The primary structure and biochemical functions of B700, as well as its in vivo metabolic fate, are largely unknown. We compared murine albumin, vitamin D binding protein and B700 for their ability to specifically bind [3H]-1,25-dihydroxy-vitamin D3. Scatchard analysis revealed a single binding site for B700 with a Ka of 51,000 mol/l and a Bmax of 4.51 x 10(-7) mol/l. There was no significant difference in the Ka and Bmax among the albuminoid proteins. However, differences in the binding sites could be distinguished by competition experiments where vitamin D3, vitamin D2 or 7-dehydrocholesterol competed for the specific binding of 1.25-dihydroxyvitamin D3 to a greater extent by B700 than by vitamin D binding protein. The albumin binding site more closely resembles vitamin D binding protein than B700, but the data indicate that the binding function of the albuminoid proteins is conserved in B700.

A rapid, novel method for the solid-phase derivatization of IgG antibodies for immune-affinity chromatography.

We show that a solid-phase immune adsorbent can be prepared from rabbit antiserum by isolating the IgG fraction with the A protein of Staphylococcus aureus associated covalently with a Sepharose matrix. The IgG is then coupled to the matrix using the cross-linking agent dimethylsuberimidate. IgG antibody bound in this fashion is in the proper orientation for combination with antigen because association with protein A occurs via the Fc portion of the IgG molecule, thus leaving the combining site of the molecule free to interact with antigen.

Quantitative analysis of macrophage phagocytosis by uptake of particulate 192iridium.

This study describes a method for the quantitation of macrophage phagocytosis by measuring the uptake of small particles of the radioactive inert metal, iridium (192Ir). It is shown that macrophage labeling with 192Ir was uniform, and the label was retained by the macrophages up to 21 days post-plating (when all cultures were terminated). 192Ir labeled macrophages were able to phagocytose other particles such as sheep red blood cells and were able to be activated to become tumorcidal in vitro 21 days following initial 192Ir uptake. Therefore, 192Ir could be a useful tool for the quantitative analysis and correlation of phagocytosis, tumor cytotoxicity and other macrophage functions.

Malignant melanoma: relationship to parameters of differentiation.

Malignant melanoma is not only one of the most aggressive and lethal types of neoplasm, but its incidence in the general population is currently increasing at an alarming pace. It is interesting that most melanomas retain many of their characteristics of differentiation, including a dendritic nature and the production of melanin. This review discusses the phenotypic properties of melanoma cells, including their state of differentiation, and their tumourigenic and metastatic potentials, and attempts to provide an overview of the state of current research on the interrelationships between those parameters in murine and human systems.

Studies on the expression and immunogenicity of the B50 melanoma antigen and its relationship with calreticulin.

B50 is a 50 kDa protein antigen originally identified and isolated from cultured B16 murine melanoma cells; it is found in close association with a melanoma-specific antigen termed B700. Using a specific rabbit antiserum, B50 (or B50 cross-reactive molecules) has been shown to be expressed by 35 out of 36 cell lines, including melanomas, sarcomas, fibrosarcomas, carcinomas, gliomas, immortalized and primary fibroblasts, melanocyte and keratinocyte cell lines obtained from murine, human, hamster, swine, and canine donors. B50 expression is localized on the cellular membrane and in the cytoplasm in varying amounts in seven of the nine cell lines tested. Mice immunized to B50 demonstrated a significant tumour rejection response when subsequently challenged with B16 F10 melanoma cells. Previous studies had indicated that B50 has significant N-terminal amino acid sequence homology with calreticulin. Calreticulin, a calcium-binding protein, is part of the Ro/SS-A complex. This complex is the primary autoantigenic determinant of the autoimmune diseases systemic lupus erythematosus and primary Sjogren's syndrome. We now show that sera from patients with those diseases contain antibodies which bind B50, although B50 itself does not bind calcium. Thus, B50 and calreticulin are closely related but distinct antigens.

Melanoma antigens as modified normal gene sequences.

Melanomas are highly aggressive tumors with a well-documented antigenic nature. Several melanoma antigens have been reported, four of which, p97, Ia-like antigen, B700, and A have been implicated as having regions in common with normally occurring proteins. P97 has partial sequence homology with transferrin and lactotransferrin, Ia-like antigen is immunologically cross-reactive with alpha and beta chains of Ia-like proteins, A is a variant of alpha actin, and B700 resembles a normal melanosomal membrane protein. In addition, B700 has partial sequence homology to serum albumins. These observations suggest that melanoma tumors can produce antigenic proteins by modification of normally occurring proteins. The possible mechanisms are discussed.

Albuminoid molecules: a novel, variability-generating cell-surface receptor system?

The mechanisms by which lymphoid cells produce infinitely variable molecules of the immunoglobulin protein superfamily have been recently elucidated. These molecules serve, in part, as the mediators of cell:cell recognition and interaction among lymphoid cells. However, the generality of those molecular mechanisms to occur in non-lymphoid cell types has not yet been established. In this paper, we propose that the serum albumin superfamily of proteins has the necessary characteristics to serve analogous functions in epithelioid cells, and we critique recent evidence which leads to this hypothesis.

Novel experimental approaches to melanoma diagnosis and therapy.

We have investigated the potential use of immune therapies on the growth of melanoma metastases in a new animal model that more closely approximates the clinical situation. We have found that significant benefits towards decreased metastatic growth and subsequent animal survival can be achieved by treatment of tumor-bearing mice with melanoma-specific monoclonal antibodies or alternatively, with various types of monovalent or polyvalent vaccines. The beneficial effects of those vaccines can be significantly enhanced by concomitant interleukin-2 therapy.

Computer applications in analysis, mapping and cataloging of proteins separated by two dimensional electrophoresis.

Two dimensional electrophoretic separation of complex mixtures of proteins can only be exploited to its fullest potential using sophisticated computerized spot detection, quantification, pattern recognition, pattern normalization, data reduction and data storage. We present a discussion of some of the technical problems and of the options available which will ultimately lead toward full computerization of the data.