MED and PSACH COMP mutations affect chondrogenesis in chicken limb bud micromass cultures.

Mutations in cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). We studied the effects of over-expression of wild type and mutant COMP on early stages of chondrogenesis in chicken limb bud micromass cultures. Cells were transduced with RCAS virus harboring wild type or mutant (C328R, PSACH; T585R, MED) COMP cDNAs and cultured for 3, 4, and 5 days. The effect of COMP constructs on chondrogenesis was assessed by analyzing mRNA and protein expression of several COMP binding partners. Cell viability was assayed, and evaluation of apoptosis was performed by monitoring caspase 3 processing. Over-expression of COMP, and especially expression of COMP mutants, had a profound affect on the expression of syndecan 3 and tenascin C, early markers of chondrogenesis. Over-expression of COMP did not affect levels of type II collagen or matrilin-3; however, there were increases in type IX collagen expression and sulfated proteoglycan synthesis, particularly at day 5 of harvest. In contrast to cells over-expressing COMP, cells with mutant COMP showed reduction in type IX collagen expression and increased matrilin 3 expression. Finally, reduction in cell viability, and increased activity of caspase 3, at days 4 and 5, were observed in cultures expressing either wild type or mutant COMP. MED, and PSACH mutations, despite displaying phenotypic differences, demonstrated only subtle differences in their cellular viability and mRNA and protein expression of components of the extracellular matrix, including those that interact with COMP. These results suggest that COMP mutations, by disrupting normal interactions between COMP and its binding partners, significantly affect chondrogenesis.

Immunologic studies with mouse mammary tumor viruses: comparative studies with spontaneous mammary tumor cell vaccines from five inbred mouse strains.

Vaccines were prepared from cells of primary spontaneous mammary tumors (MT) of RIII/Imr, GR/Imr, C3H/Imr, A/Imr, and C3HfC57BL/Imr mice. Each vaccine was used to immunize "murine mammary tumor virus (MuMTV)-free" BALB/c/Imr and C57BL/Imr mice before challenge with infectious RIII-MuMTV, GR-MuMTV, C3H-MuMTV, and A-MuMTV. RIII-MT and C3H-MT cells protected mice against tumorigenesis by all four MuMTV's; GR-MT cells protected against all but the RIII-MuMTV challenge; A-MT cells were ineffective against challenge by all four MuMTV's and significantly enhanced development of A-MuMTV-induced tumors. The activity of the C3H-MT cells was not seen when C3HfC57BL cells, free of the standard C3H-MuMTV, were used. The same cell vaccines were inoculated into the five donor strains of mice in attempts to prevent spontaneous MT in these high-incidence strains, which are naturally infected with MuMTV at birth. None of the vaccines prevented tumorigenesis in these mouse strains although delays in the appearance of tumors were observed in RIII and GR mice inoculated with either isologous MT cells or C3Hf-MT cells. The role of viral antigens acting alone or in concert with cellular antigens is discussed.

Comparative studies of purified subviral component vaccines and formalin-treated mammary tumor viruses from four inbred strains of mice.

Formalin-treated virus vaccines were prepared from purified murine mammary tumor viruses (MuMTV) from 4 inbred strains of mice: RIII/Imr, GR/Imr, C3H/Imr, and A/Imr. In addition, subviral components were isolated from these 4 strains and purified to homogeneity. The inactivated viruses, their major envelope glycoproteins (gp50-gp55), and their major internal core protein (p28) were emulsified in complete Freund's adjuvant and used as vaccines for prevention of mammary tumors in mice. All 4 Formalin-treated virus vaccines reduced significantly the incidence of mammary tumors in "virus-free" C57BL and BALB/c mice when inoculated prior to challenge with live MuMTV. The RIII-, GR-, and A-MuMTV strains showed extensive heterologous cross-protection, whereas the C3H-MuMTV strain showed significant protection only against C3H- and A-MuMTV challenge. The major viral glycoproteins gp50-gp55 reduced significantly the tumor incidence when mice were challenged with isologous infectious virus after immunization, although these glycoproteins showed different degrees of cross-protection than did the same virus strains used as "intact" but Formalin-treated preparations. RIII-gp55 and GR-gp55 cross-protected against each other but not against challenge with C3H- and A-MuMTV strains; the A-gp50 protected against challenge with A- and RIII-MuMTV strains; C3H-gp55 demonstrated limited activity against C3H-MuMTV challenge only. The internal viral core proteins (p28) were ineffective in all systems studied. The same vaccines were tested in MuMTV-positive, high-tumor-incidence strains from which they were derived. At best, the appearance of spontaneous tumors was delayed in a few experimental sets; eventually, all mice developed mammary tumors. The foster-nursed C3HfC57BL strain of mice, which is not exposed to exogenous MuMTV during suckling and which develops mammary tumors after activation of the endogenous virus genome later in life, was responsive only when the heterologous GR-MuMTV Formalin-treated vaccine was used. The association between the ability of virus vaccines to protect a mouse strain and the degree of natural virus expression in that strain is discussed.

Serologic responses to murine mammary tumor virus (MuMTV) in MuMTV-exposed laboratory personnel.

A retrospective study was conducted to determine whether exposure to murine mammary tumor virus (MuMTV) induced MuMTV-specific serologic responses among intramural laboratory personnel. Results obtained with a panel of five purified structural proteins of the RIII mouse strain milk-derived MuMTV (gp55, gp34, p28, p18, and p12), by means of the enzyme-linked immunosorbent assay to assess antibody binding, established that MuMTV exposure resulted in highly significant increases in serologic responses to these test antigens as compared to age- and gender-matched controls without overt contact with MuMTV. Furthermore, immunoreactions to gp55 and gp34 were found not to be directed to the carbohydrate moieties of these glycoproteins. Similar results were obtained by assays of human immunoglobulin binding to Western blots of MuMTV proteins. These increased MuMTV-specific immunoreactivities, in general, were found to be related to degree and length of exposure to this virus. These results with MuMTV suggest the possibility of important human immune response differences between exposure to type B (MuMTV) and animal type C (leukemia-sarcoma) RNA tumor viruses, perhaps reflective of sensitivity to antibody dependence of complement-induced virolysis.

Responses of serum from breast cancer patients to murine mammary tumor virus: fact or artifact?

For determination of whether breast cancer patients possessed specific serological responses to murine mammary tumor virus (MuMTV), IgG-binding levels were monitored by antibody binding to electrophoretically separated viral proteins (Western blotting and immunodetection) and by the enzyme-linked immunosorbent assay (ELISA) against a panel of five structural proteins (gp55, gp34, p28, p18, and p12) purified from milk-borne MuMTV of the RIII isogeneic mouse strain. No significant antibody reactions were found for sera from 30 cancer patients by the immunoblotting assay, and comparative ELISA studies of 111 patients with malignant mastopathies and 122 healthy, age-matched women revealed no significantly increased mean antibody responses against gp55, gp34, p28, or p12 in breast cancer patients as compared to the responses in the control group. Only for p18 was there a significant increase in mean IgG-binding levels in cancer patients. Additional assays of antibody binding to viral antigens were performed by the cellular immunofluorescence test on MuMTV-expressing cells. These studies also failed to demonstrate greater immunoreactivity of sera from patients as opposed to the immunoreactivity of sera from healthy controls.

Procedures for radioimmunoassay of the mouse mammary tumor virus.

A procedure for radioimmunoassay of the major glycoprotein antigen derived from murine mammary tumor virus is described. The assay is sensitive to 0.05 ng of antigen and is highly reproducible. The antigen, gp55, has been found to be group specific and will detect viruses in 13 separate mouse strains, as wel .l as from continuous cell lines. Factors affecting the assay have been examined.

Two new monoclonal antibodies, EPB-1 and EPB-2, reactive with human lymphoma.

Using the Raji cell line as the immunogen source, we established stable murine hybridomas that secrete B-lymphocyte-specific monoclonal antibodies (MAbs). Two clones, designated EPB-1 and EPB-2, were selected for further study. Both EPB-1 and EPB-2 were typed by reaction with subclass-specific rabbit anti-mouse antibodies and were found to be IgG1 and IgG2a, with kappa-light chains, respectively. In radioimmunobinding and flow cytometric assays, EPB-1 and EPB-2 did not react with T-cell lines, nor with normal diploid cells. EPB-2, in contrast to EPB-1, did not react by flow cytometry with normal peripheral or bone marrow leukocytes. In normal lymphoid tissues, both antibodies were positive with germinal center and mantle zone B-lymphocytes. Additionally, EPB-1 also labeled interdigitating histiocytes in the T-cell zones. Both MAbs discriminated very well between lymphoid and nonlymphoid tissues; neither MAb cross-reacted with tested normal and solid tumor tissue specimens, or with cell lines of non-B-cell origin. Immunohistology also revealed that both MAbs stained malignant lymphoma tissue sections and cloned B-cell lymphomas, both in tissue culture and as human xenografts in nude mice. Due to the radiosensitivity of human lymphomas and the relative specificity of these two new MAbs, they appear to be potential radioimmunotherapeutic agents for human lymphoma.

Experimental infection of a cat kidney cell line with the mouse mammary tumor virus.

A cat kidney cell line, CRFK-F2, was successfully inoculated in suspension and in monolayer culture with a purified mouse mammary tumor virus derived from RIII milk. The virus produced by the infected cells was identified by immunogluorescence, electron microscopy, and RNA-directed DNA polymerase assays; it was a B-type virion that did not cross-react with mouse or feline leukemia-sarcoma viruses, had spikes on its envelope, and had a RNA-directed DNA polymerase reaction that was typical of mouse mammary tumor virus. The producing cells were identified as cat cells by chromosome number, cytotoxic assays, and isoenzyme migratory patterns. A standardized method for the in vitro inoculation of cat cells is described that presently permits highly reproducible results. For the first time, the mouse mammary tumor virus is seen replicating in cells from another species, thus offering an opportunity to study the kinetics of infection of that virus.

Breast tumor radioimmunodetection with a 111In-labeled monoclonal antibody (MA5) against a mucin-like antigen.

Monoclonal antibody MA5 recognizes a determinant displayed on high molecular weight antigens associated with secretory and malignant breast epithelial cells. MA5 reactivity with greater than 95% of primary and metastatic breast tumors, surface expression of the antigen, as well as its ability to localize within breast tumor xenografts prompted this initial study to determine the efficacy of MA5 to localize breast tumors by radioimmunoscanning. A total of 17 patients was monitored, each receiving 2 mg of purified MA5 labeled with 5 mCi of 111In. Some patients also received 3 or 18 mg of unlabeled carrier antibody (MA5); no serious allergic reactions were noted. Primary tumors, bone lesions, soft tissue recurrences, and lung metastases greater than 3 cm in diameter were detectable, whereas only one lesion (hilar node) less than 3 cm was localized. Significant antibody accumulation was noted in the liver and less significant uptake in the spleen and bone. The extensive fibrosis and poor vascularization of breast tumors may partly explain the limited sensitivity obtained thus far. The imaging results obtained with MA5 are compared with other antibodies which we show recognize the same antigens.

Patterns of antigen distribution in human carcinomas.

Ten epithelial-specific monoclonal antibodies, including monoclonal antibodies to antigens that have been used extensively in immunodiagnosis and immunotherapy experiments, were tested for reactivity with 20 human carcinomas each of the colon, lung, and breast. The antibodies tested included B72.3, OC125, and antibodies to carcinoembryonic antigen, the 17-1A antigen, and the milk fat globule mucin antigen (epithelial membrane antigen). Striking differences in the pattern of antigen distribution were seen, with each antibody having a fairly consistent staining pattern, which was dependent on the tumor type. Two antibodies reacted with most or all tumor specimens and, when positive, reacted homogeneously with apparently every cell in the specimen. Other antibodies consistently produced a variegated staining pattern, typically with areas of positive cells surrounded by areas of negative tumor cells. A third pattern was strong localization to the luminal edge and/or secretions of glandular tumors; this pattern was seen primarily in colon carcinomas which have more well-developed glandular structures than breast or lung carcinomas. A correlation with biochemical properties of the antigens was evident, in that mucins or mucin-related antigens generally produced variegated staining of lung and breast carcinomas and luminal edge/secretion staining of colon carcinomas. Such differences in antigen distribution are likely to be a major factor in developing methods for immunodiagnosis and immunotherapy.

Cellular hypersensitivity of gp55 of RIII-murine mammary tumor virus and gp55-like protein of human breast cancers.

Previous studies suggested that immunogenic breast cancer tissues contained a component(s) that is antigenically similar to some component of murine mammary tumor virus (MuMTV) and resembles the glycoprotein, M.W. 55,000 (gp55), of RIII-MuMTV in molecular weight and charge density. This investigation measured in vitro cellular hypersensitivity responses of breast cancer patients to RIII mouse milk, purified RIII-gp55, C3H-MuMTV, autologous and homologous breast cancer tissues, gp50 of A-MuMTV, and preparations of Rauscher leukemia virus and Mason-Pfizer monkey virus. Particular attention was paid to cross-reactivity between gp55 and the other targets. The data indicate that responsiveness to C3H-MuMTV and RIII milk are linearly correlated with responsiveness to gp55. A preferential relationship was demonstrable between responses to gp55 and to those breast cancer tissues containing a gp55-like protein component (S-p50). The critical role of a gp55-like protein as the antigen responded to by breast cancer patients' in leukocytes was also suggested by the ability of anti-gp55 antiserum to decrease leukocyte responsiveness to RIII-gp55, C3H-MuMTV, and breast cancer tissues. In vitro cellular hypersensitivity against RIII-gp55 was preferentially found in prognostically favorable cases with immunogenic lesions. Further studies are needed to test the possibility that gp55 might be of value in the immunodiagnosis of early breast cancer, the monitoring of prognostically significant cellular hypersensitivity, and the induction of such hypersensitivity (immunoprophylaxis).

Prognostically significant protein components of human breast cancer tissues.

Cryostat sections of clinicopathologically characterized breast cancer tissues were eluted with phosphate-buffered 0.9% sodium chloride solution, pH 7.2. The proteins were then characterized by polyacrylamide gel electrophoresis with and without prior treatment with sodium dodecyl sulfate. Approximately 65% of the brease cancer tissue eluates contained a prominent protein fraction with a molecular weight of 47,000 to 55,000 (p50). No such component was found in 15 of 17 eluates of benign breast tissue. Charge density studies disclosed that the p50 component included three populations of proteins that could be characterized according to the migration relative to gp55 derived from RIII murine mammary tumor virus, namely, fast (F-p50), intermediate (I-p50), and slow (S-p50). Prognostically favorable pathological characteristics, i.e., stage, nuclear grade, and lymphoreticuloendothelial responses, were proportionately most frequently found among S-p50 bbreast cancers and were least frequently found among F-p50 breast cancers. It appears that the S-p50 component acts in vivo as a prognostically significant immunogen. Further knowledge of the relationship between protein characteristics and clinicopathological features of human breast cancers would contribute to our understanding of mammary carcinogenesis and biological behavior.

COOH-terminal propeptides of the major human procollagens. Structural, functional and genetic comparisons.

The sequences of the carboxy-terminal extensions (COOH-propeptides) of at least one chain of all of the major human procollagens have only recently been deduced, and include those of the interstitial (alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III)), basement membrane (alpha 1(IV)) and pericellular (alpha 2(V)) procollagens. Comparisons of DNA and protein sequences, corresponding to these COOH-propeptides domains, established the early divergence of the basement membrane alpha 1(IV) COOH-propeptide from the corresponding sequences of the interstitial and pericellular procollagens. The latter are relatively highly conserved and share 58% primary peptide sequence similarities, whereas sequence similarities relative to alpha 1(IV) are limited. Hydropathy profiles and secondary structure potentials further emphasize the clustering of conserved and variable regions among the interstitial and pericellular COOH-propeptides, and provided further evidence for significant structural differences between these sequences and the alpha 1(IV) COOH-propeptide. The most highly conserved sequences of the alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III) and alpha 2(V) COOH-propeptides include regions surrounding the carbohydrate attachment site, cysteine-containing regions and the COOH-terminal sequences. Cysteinyl, tyrosyl and tryptophanyl residues were found to be highly conserved as were most charged residues. Localization of variable regions, in general, occurs within hydrophilic sequences with high beta-turn potentials that are proximal to intron/exon splice junctions. The most variable sequences are associated with the telopeptides and adjoining NH2-terminal portions of the COOH-propeptides as demonstrated by predictive secondary structure analyses. These results, combined with similar analyses of abnormal alpha 2(I) COOH-propeptide (osteogenesis imperfecta) permitted the identification of subsequences that are likely to be a prerequisite for COOH-propeptide functions, namely procollagen chain recognition and nucleation sites for triple helix formation. These functions are also common to the alpha 1(IV) COOH-propeptide; however, the lack of cleavage of this region and its additional postulated structural role in extracellular matrix interactions likely account for its divergent primary and secondary structure.

Construction and characterization of a humanized, internalizing, B-cell (CD22)-specific, leukemia/lymphoma antibody, LL2.

The murine monoclonal antibody, LL2, is a B-cell (CD22)-specific IgG2a which has been demonstrated to be clinically significant in the radioimmunodetection of non-Hodgkin's B-cell lymphoma. The antibody carries a variable region-appended glycosylation site in the light chain and is rapidly internalized upon binding to Raji target cells. Humanization of LL2 was carried out in order to develop LL2 as a diagnostic and immunotherapeutic suitable for repeated administration. Based on the extent of sequence homology, and with the aid of computer modeling, we selected the EU framework regions (FR) 1, 2 and 3, and the NEWM FR4 as the scaffold for grafting the heavy chain complementarity determining regions (CDRs), and REI FRs for that of light chains. The light chain glycosylation site, however, was not included. Construction of the CDR-grafted variable regions was accomplished by a rapid and simplified method that involved long DNA oligonucleotide synthesis and the polymerase chain reaction (PCR). The humanized LL2 (hLL2), lacking light chain variable region glycosylation, exhibited immunoreactivities that were comparable to that of chimeric LL2 (cLL2), which was shown previously to have antigen-binding properties similar to its murine counterpart, suggesting that the VK-appended oligosaccharides found in mLL2 are not necessary for antigen binding. Moreover, the hLL2 retained its ability to be internalized into Raji cells at a rate similar to its murine and chimeric counterparts.

Human cDNA clones transcribed from an unusually high-molecular-weight RNA encode a new collagen chain.

Human collagen (COL) cDNA clones were isolated from a library representing transcripts synthesized by an established rhabdomyosarcoma (RH) cell line. The 0.6-kb insert of the first isolate encodes a discontinuous collagenous sequence not homologous to type I-XVI COL chains. Sequencing of a second clone with a 4-kb insert revealed an open reading frame (ORF) of 2154 nucleotides. The deduced amino acid (aa) sequence begins with an 186-aa noncollagenous region containing seven cysteines (Cys). Several of the Cys and surrounding aa residues can be aligned with those in type XVI, XII and IX COL. Due to the presence of two long interruptions, the 524-aa collagenous region is separated into three subdomains that each have smaller interruptions of 1-6 aa. The protein terminates with an 8-aa noncollagenous peptide including an unusual single Cys which would be expected to form an interchain disulfide bond. Results of Northern blot hybridization suggest that the new COL chain may be uncommonly large since the clone identified a low-abundance RNA at least 12.4 kb in size. The gene coding for RH COL is located on human chromosome 6. It is now important to elucidate the role of this unusual COL in the infrastructure of extracellular matrix.

An extended primer set for PCR amplification of murine kappa variable regions.

The design and successful usage of an extended primer set for the PCR amplification of murine variable kappa light chain sequences from mouse hybridomas are described. Since some of these primer pairs also amplify the endogenous SP2/0 aberrant light chain sequence, strategies to distinguish the irrelevant SP2/0 from the sequence of interest are also provided.

Expression of a gene coding for breast tumor-associated antigen in thyroid papillary carcinoma.

The expression of the H23 gene, previously shown to be overexpressed in breast cancer tissue, was examined in various thyroid pathologies. Thyroid papillary carcinomas demonstrate significant H23 mRNA levels, whereas benign thyroid pathologies have very low levels of expression. H23 gene expression in thyroid cancer inversely correlated with that of thyroperoxidase and thyroglobulin genes. Immunoblot assays of thyroid cancer tissues revealed overexpression of H23 gene at the protein level as well The data presented indicate that dedifferentiation of thyroid tissue to the malignant state is associated with increased H23 gene expression and suppression of some thyroidal differentiation marker genes.

Recognition of peptidyl epitopes by polymorphic epithelial mucin (PEM)-specific monoclonal antibodies.

Peptidyl epitope recognition by several murine monoclonal antibodies (MAbs E29, H23, HMFG-1, HMFG-2, MA5, MA6 and MA9) which react with the polymorphic epithelial mucins [PEM; epithelial membrane antigen (EMA)] was studied by using ten synthetic peptides representative of the 20 residue tandem repeat as test antigens. Antibody binding to 6-10 residue overlaps and to peptides having a common carboxy-terminus and staggered amino-termini (8-31 residues) was assessed by solid phase and competition ELISA techniques. From these analyses, all MAbs except MA9 were found to react predominantly with the carboxy-terminal half of the repeat motif. Polyclonal antibody responses in mice immunized with intact EMA/PEM-containing preparations also displayed significant reactivities against synthetic repeat peptide antigens and, conversely, synthetic peptides as carrier-conjugated immunogens induced antibodies recognizing intact antigens. These results are discussed vis-à-vis peptide conformation, the potential effects of O-glycosylation on secondary structure, and the possible effects of these parameters on immunogenicity and antigenicity.

Chimerization of LL2, a rapidly internalizing antibody specific for B cell lymphoma.

LL2 is a murine monoclonal antibody (MAb) that has been shown to be effective for the diagnosis and treatment of patients with non-Hodgkin's B cell lymphoma. Studies have also shown that radiolabeled murine LL2 (mLL2) or mLL2 and fragments thereof coupled to Pseudomonas exotoxin (PE) can effectively target human B cell lymphoma in mice. We have obtained the DNA sequences encoding the VK and VH domains of mLL2, an IgG2a MAb, which were combined with their respective human kappa and IgG1 constant region domains and expressed in SP2/0 cells. Like its murine counterpart, the chimeric LL2 (cLL2) antibody is glycosylated in the light chain variable region. Chimerization did not interfere with the immunoreactivity of the antibody, as determined by a competitive binding assay, where either antibody shows equivalent inhibition of the binding of its counterpart to the Raji cell membrane surface antigen, CD22. Both antibodies bind and are rapidly internalized by Raji cells, whereas an irrelevant humanized antibody did not bind and was not internalized under similar conditions. The internalization rates of the bound murine or chimeric antibodies were nearly identical, with Ke values of 0.106 and 0.118 min-1 for mLL2 and cLL2, respectively. The observed close equivalence between the murine and chimeric antibodies suggests potential advantages of the latter as a less immunogenic agent. Studies are currently underway to evaluate the chimeric antibody as a potential therapeutic immunoconjugate.