A monoclonal antibody directed against a human cell membrane antigen prevents cell substrate adhesion and tumor invasion.

It was the aim of this study to design mouse monoclonal antibodies (MAbs) that can inhibit the invasion of breast cancer cells in the host tissue. Therefore, MAbs were raised against epitopes on the extracellular domain of SK-BR-3 human breast cancer cells, and biological assays were performed to test the capability of the MAbs to inhibit cell substrate adhesion. MAb 14C5 bound an extracellular plasma membrane antigen of SK-BR-3 and MCF-7 human breast cancer cells and inhibited the cell substrate adhesion of these cells in vitro. The MAb delayed the adhesion of MCF-7 and SK-BR-3 cells on precultured embryonic heart fragments (PHFS). It inhibited the destruction of the PHF by MCF-7 cells and the invasion of the PHF by SK-BR-3 cells. The MAb reacted with an epitope on the cell membrane of in situ and invasive ductal carcinomas of the breast in immunohistochemistry. Poorly differentiated, highly invasive ductal carcinomas show extensive staining of long plasma membrane extensions. Normal multilayered epithelia, normal connective tissue, and tumors derived from these tissues as well as normal breast tissue were negative. From both cell lines a protein complex consisting of two subunits with molecular weight of 50 and 90 kd, respectively, was immunoprecipitated. It is concluded that the 14C5 antigen plays a role in cell substrate adhesion and subsequently also in invasion of breast cancer cells. The 14C5 MAb was able to inhibit cell substrate adhesion and invasion in vitro of breast cancer cells.

Molecular forms of dopamine beta-hydroxylase in rat superior cervical ganglion and adrenal gland.

Dopamine beta-hydroxylase (DBH) enzyme activity was associated in rat superior cervical ganglion with tetrameric DBH-A (294,000 D) and dimeric DBH-B (147,000 D) and in rat adrenal gland with DBH-A and a novel molecular form of DBH, defined as DBH-C, with a molecular weight of 125,000 D. Pretreatment of the rats with cycloheximide markedly reduced DBH activity without altering the molecular heterogeneity.

A monoclonal antibody-based enzyme-linked immunosorbent assay of alpha 1-acid glycoprotein.

A "sandwich"-type enzyme-linked immunosorbent assay for determining concentrations of human alpha 1-acid glycoprotein (AGP) is described. Microtiter plates coated with a polyclonal rabbit antibody to human AGP were subsequently incubated with the antigen, with a specific murine monoclonal antibody, and with goat anti-mouse immunoglobulins conjugated to alkaline phosphatase. To evaluate the method for assay of AGP in human sera, we compared it with single radial immunodiffusion and "rocket" electroimmunoassay. The respective correlations were r = 0.988 (n = 45) and r = 0.973 (n = 47). Repeated assays of a human serum sample with an average AGP concentration of 859 mg/L yielded within-day and between-day CVs of 1.4% (n = 5) and 6.3% (n = 10), respectively. Because of its low detection limit (4.4 micrograms/L), this assay is also suitable for determination of AGP concentrations in other biological fluids, such as dialysates of patients being treated by continuous ambulatory peritoneal dialysis.

A study of the heterogeneity of human alpha 1-acid glycoprotein with monoclonal antibodies.

Monoclonal antibodies (MABS) were raised against human alpha 1-acid glycoprotein (AGP) and their reaction with the polymorphic forms of this plasma protein were evaluated. Spleen cells of BALB/c mice, immunized with native or desialylated human AGP, were fused with NSO mouse myeloma cells. The hybridoma products were screened with a direct ELISA test, in which the immunoplates were coated with a mixture of native and desialylated AGP. In this test, 14 anti-AGP antibody producing clones were retained. Coating the wells with either native or desialylated AGP showed that eleven clones reacted with both types of AGP ('Type I' MABS), while three MABS reacted specifically with the desialylated form ('Type II' MABS). Precoating the immunoplates with polyclonal anti-human AGP followed by incubation with native or desialylated AGP before the addition of hybridoma supernatant (indirect ELISA), confirmed the specificities observed in the direct ELISA. The molecular heterogeneity of both native AGP and desialylated AGP, based on Concanavalin A reactivity and isoelectric point, was not reflected by any specificity in the antibody reactions. Thus 'Type I' MABS reacted with all molecular forms present in native and desialylated AGP while 'Type II' MABS reacted with all molecular forms present in desialylated AGP.

Release of dopamine, noradrenaline and dopamine B-hydroxylase from mouse neuroblastoma.

The murine C1300 neuroblastoma tumor was found to secrete dopamine, noradrenaline and dopamine B-hydroxylase into the circulation of tumor-bearing A/J mice. The plasma levels of dopamine, noradrenaline and dopamine B-hydroxylase increased with the size of the tumor, and the increase in noradrenaline paralleled the increase in dopamine B-hydroxylase (r = 0.86). The vesicular storage of dopamine and noradrenaline in the tumor was evidenced by a decrease of the tissue content of dopamine and noradrenaline 24 hours after the administration of reserpine (5 micrograms/g) respectively to 17.6% and 7.8% of control values. A similar observation could be made for the levels of dopamine and noradrenaline in the plasma of reserpinized C1300 mice. The total activity of dopamine B-hydroxylase in the tumor and in plasma was unaffected by the reserpine treatment. Chronic administration of 6-hydroxydopamine (100 micrograms/g for 8 days) had no effect on the tissue contents of dopamine, noradrenaline or dopamine B-hydroxylase. The release of catecholamines and dopamine B-hydroxylase from the C1300 neuroblastoma was studied in vitro on superfused tumor slices. Stimulation of these slices with 56 mM KC1 or with 5.10(-5) M tyramine failed to induce the release of endogenous dopamine, noradrenaline or dopamine B-hydroxylase above the basal outflow levels. These results are suggestive for a non-exocytotic release of catecholamines and dopamine B-hydroxylase from the neuroblastoma tumor.

Multiple molecular forms of dopamine beta-hydroxylase in the C1300 mouse neuroblastoma tumor and in the serum of tumor-bearing mice.

The distribution of the enzymatic activity of dopamine beta-hydroxylase (DBH) in linear sucrose gradients was studied for a soluble fraction of the C1300 mouse neuroblastoma tumor, for the serum of tumor-bearing A/J mice, and for adrenal tissue and serum of control mice. In controls (adrenal gland and serum of A/J mice), about 75% of the DBH activity was associated with a high-molecular-weight form, denoted as DBHA, with an apparent sedimentation coefficient of 11.3 S. About 25% of the DBH activity was attributable to a slower-sedimenting species (7.1 S), denoted as DBHB. In tumor supernatants and in the serum of tumor-bearing mice, about 55% of the DBH activity was present as the 7.1 S species (DBHB), while only 35% was recovered as the high-molecular-weight form (DBHA). Approximately 5% of the activity could be attributed to a separate form, with a sedimentation coefficient of about 4.5 S. This form is designated DBHC. The ratio DBHB/DBHA is significantly higher in tumor tissue and in serum of tumor-bearing mice than in controls. The three enzymically active forms of DBH in the C1300 tumor are considered to represent the tetrameric (DBHA), dimeric (DBHB), and monomeric (DBHC) forms of the enzyme.

Biochemical evidence for two types of noradrenaline storage particles in rabbit iris.

Centrifugation techniques were used to determine the subcellular distribution of noradrenaline (NA) and dopamine beta-hydroxylase (D beta H) in the rabbit iris. By application of isopycnic and differential gradient centrifugation methods 2 types of NA vesicles could be demonstrated. Of the total particle bound NA about 70% is associated with 'light' and about 30% with 'heavy' vesicles. For both types of vesicles the distribution of D beta H reflected that of NA.