Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction.

A resistant descendant of the human stomach carcinoma cell line EPG85-257 was selected in the presence of increasing concentrations of daunorubicin (DRC). To avoid the expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), cells were cultured in the presence of verapamil. The resulting cells were used to evaluate an induced carbonyl reduction as a new determinant in DRC resistance. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide) toxicity assay was performed to estimate sensitivity to DRC in both cell lines. IC50 values of DRC increased almost 8-fold in the resistant descendants compared to the parental cell line. P-gp transcripts were detectable in both cell lines at only very low levels, and no significant alterations between sensitive and resistant cells were observed. MRP mRNA expression was markedly higher compared to P-gp mRNA, but, as was the case with P-gp, MRP mRNA levels in sensitive and resistant cells showed no alteration. This was probably due to the effect of the presence of verapamil during cell selection. Another known drug resistance factor, the lung resistance-related protein (LRP), was not at all detectable. Interestingly, resistant cells possessed 6-fold higher levels of DRC carbonyl-reducing activity, leading to the less toxic 13-hydroxy metabolite daunorubicinol (DRCOL). The 6-fold higher DRCOL formation roughly parallels the 8-fold increase in DRC IC50 values during cell selection, and therefore may account for DRC resistance in these cells. The determination of specific carbonyl reducing enzymes, known to be involved in DRC detoxification, revealed that mRNA expression of carbonyl reductase (EC 1.1.1.184), aldose reductase (EC 1.1.1.21), and dihydrodiol dehydrogenase 2 (EC 1.3.1.20) increased in the resistant descendant. In contrast, the phase II-conjugating enzyme activities of glutathione S-transferases were significantly lower in resistant than in sensitive cells, whereas those of glucuronosyl transferase were not detectable in either cell line. Apparently, conjugating enzymes are not involved in DRC resistance in human stomach carcinoma cells. These studies indicate that DRC resistance in human stomach carcinoma cells may appear as a result of an induction of metabolic DRC inactivation via carbonyl reduction to the less active 13-hydroxy metabolite DRCOL.

Hinge-thiol coupling of monoclonal antibody to silanized iron oxide particles and evaluation of magnetic cell depletion.

Iron oxide particles of average size 0.5-1.5 microns, covered by a silane coat carrying amino groups (Bio-Mag, Advanced Magnetics, Boston), were derivatized by reaction with N-[(gamma-maleimidobutyryl)oxy]-succinimide (GMBS), N-hydroxysuccinimidyl iodoacetate (NHIA), 2-iminothiolane (2-It), or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The derivatized particles were suitable for the reaction with sulfhydryl groups and subsequently coated with monoclonal antibodies (MoAbs) of different classes and isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) as well as polyclonal rabbit anti-mouse IgG (RAM). The antibodies were reduced by dithiothreitol (DTT) and covalently conjugated to the BioMag derivatives via liberated sulfhydryls of the hinge region. The observed conjugation ratios, expressed as protein/iron (micrograms/mg), could be reproducibly varied for optimization. These ratios were dependent on the type and amount of antibody offered for coupling to the derivatized particles, decreasing as follows: polyclonal = IgM greater than IgG2b greater than IgG2a = IgG3 greater IgG1. The conjugation ratios were also dependent on the type and amount of the spacer used to derivatize the BioMag particles, decreasing as follows: GMBS greater than NHIA greater than 2-It greater than SPDP. The magnetically responsive magnetite-antibody conjugates ("magneto-beads"), carrying MoAb BMA 081 (anti-CD8; IgG2a), MoAb BB10 (anti-CD10/CALLA; IgG2b), MoAb VIL-A1 (anti-CD10; IgM), and polyclonal RAM, coupled similarly via 3.6 mumol of GMBS spacer per mg of Fe, were further investigated with respect to a depletion effect on specific cell subsets. The rates of cell depletion were found to be strongly dependent on the individual characteristics of the antibody used.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytodiagnosis with monoclonal antibodies.

Monoclonal antibodies against cellular antigens are of extreme specificity, reproducibility and in principle can be made available in unlimited amounts. In cellular diagnosis the application of monoclonal antibodies became already established. Different methods to target the reaction of these antibodies, with the corresponding antigens, are available and adaptable if necessary. Monoclonal antibodies at the first time allow application of a reagent in diagnostic pathology by different laboratories and of objective comparison of their results. Normal and malignant cells of the human hematopoietic system and of almost all other tissues of the human are well characterized to day by monoclonal antibodies, this was absolutely impossible with conventionally made antisera. The unique features of these reagents can be utilized to better diagnosis, prognosis and even systemic treatment of tumors, where the latter remains to be seen.

Monoclonal murine antibodies with specificity for tissue culture lines of human squamous-cell carcinoma of the lung.

Monoclonal antibodies directed against human melanoma and colon carcinoma cells were described essentially by the groups of L.L. Old and K.E. Hellström. Reports dealing with monoclonal antibodies reactive with human lung tumors are still rather rare. Therefore, we used a squamous cell carcinoma line of the lung as immunogen in Balb/c mice and fused the spleen cells of the immunized animals with the X63-AG8.653 mouse myeloma cell line. The clones producing antibodies reactive with squamous cell carcinomas of the lung in an indirect immunofluorescence assay were recloned, and a fine specificity analysis was performed with 44 cultured cell lines of different origin. The data indicated that most of the antigens recognized by these monoclonal antibodies seem to be of embryonic type because of their reactivities with some of the tested human embryonic cell lines. Radioimmunoprecipitation in combination with sodium dodecyl sulfate-polyacrylamide gel electrophoresis is underway to characterize in more detail the antigens recognized by the antisquamous cell lung cancer monoclonal antibodies.

Possible synthesis of pregnancy-'specific' beta-1-glycoprotein (sp1) and placenta-'specific' tissue proteins (PP10,PP12) by human and cynomolgus monkey leukocytes.

Using an enzyme-bridge immunoperoxidase (PAP) technique, it was revealed that pregnancy- and/or placenta-specific proteins, SP1, PP10 and PP12 were localized in human and cynomolgus monkey (Macaca fascicularis) polymorphonuclear neutrophils, and PP10, in mononuclear phagocytes. There was no difference in staining results, irrespective of whether the specimens were from human or monkey males, or pregnant or nonpregnant females. The results suggest that SP1, PP10 and PP12 may be synthesized by polymorphonuclear neutrophils and/or mononuclear phagocytes and could represent markers not only for trophoblasts and some kinds of malignant neoplasms but also for normal neutrophils and/or monocytes.

In-vivo phagocytosis: enhancement of bacterial clearance by native and enzyme-treated immunoglobulins.

The enhancement of bacterial blood clearance in mice by native and enzymatically derived fractions of rabbit anti-E. coli hyperimmune serum was tested. Native immune serum, the corresponding IgG, F(ab')2 and Facb fractions strongly augmented the phagocytosis rate of bacteria, whereas Fab/Fc, Fab, Fc fragments, corresponding preparations from normal serum, and E. coli-absorbed preparations showed no marked enhancing capacity. In one experiment opsonization by IgM was demonstrable. Mice that were injected with fatal doses of bacteria could be protected by subsequent treatment with IgG or F(ab')2 preparations of rabbit anti-E. coli serum. Conclusion is drawn that the Fc region of the IgG molecule is not predominantly responsible for opsonized clearance while the intact divalent antigen cross-linking F(ab')2 fragment - presumably by virtue of complement activation via the alternate pathway - could mediate enhanced bacterial clearance.

Electrophoretic mobility test (EMT): studies on lymphocyte response and mechanism of the test using a rat tumor model.

After incubation with an encephalitogenic factor from human (HEF) or rat (REF) brain, lymphocytes of Fischer 344 rats bearing a spontaneous mammary adenocarcinoma produced a soluble substance which reduced the mobility of tanned sheep red blood cells in the electrophoretic mobility test (EMT). For studying the kinetics of this lymphocyte response, 6 X 10(5) tumor cells were injected into the hind footpad. In correlation with time and tumor size, one was able to influence the appearance of metastases by amputation of the leg. As early as 16 hours after inoculation of tumor cells, sensitivity of lymphocytes against HEF and a KCl-extract of the tumor could be shown in the EMT. It decreased on days 2 and 5, but was still seen until the day of amputation. Rats without metastases showed sensitivity up to four weeks after amputation and then returned to normal levels. Rats with metastases showed sensitivity until death at about seven weeks later. With the use of Amicon membranes, Sephadex G-50, and ion-exchange chromatography, a protein could be isolated from human basic myelin extract with a molecular weight of about 16,000-20,000 daltons. It had no direct influence on the EIC by itself, but after incubation with lymphocytes from tumor-bearing rats it evoked the production of a slowing substance. Using Sephadex G-100, the slowing substance appeared in the region in front of BSA indicating a molecular weight of greater than 80,000 daltons. It was heat-stable for 30 min at 56 degrees C and was sensitive to trypsin.

Incidence of circulating immune complexes in patients with lung cancer and their effect on antibody-dependent cytotoxicity.

Circulating immune complexes were determined in patients with lung cancer by the C1q deviation test and by column chromatography on Sepharose 6 B. Immune complexes could be demonstrated by the two different methods in 50--80% of patients' sera at the time of diagnosis. Patients with extended disease had more immune complexes than patients with limited disease. Serial determinations showed a good correlation between immune complex levels and clinical course of disease. The size of the immune complexes present in patients' sera was determined by sucrose density ultracentrifugation and by column chromatography in the region between 10 S and 30 S. Furthermore in this study no inhibition of antibody-dependent cell-mediated cytotoxicity by circulating immune complexes could be seen.

PAL-test for malignant disease.

Peripheral blood lymphocytes from a total of 300 subjects including normal controls and patients with malignant and non-malignant disorders were investigated to determine their ability to agglutinate with Poly-l-lysine 3400 (PAL-test). A high level of association between clinically evident cancer and a positive reaction was confirmed. Lymphocytes from 72 of 90 patients with malignant diseases as well as 33 of 140 patients with non-malignant processes showed positive reactions. All healthy controls were negative. The positive evidence of the PAL-test amounted to over 80%. The results and possible mechanisms of the PAL-test are discussed.

[Immunologic potency of pregnancy associated alpha 2-glycoprotein (alpha 2-PAG)]. / Immunologische Potenz des schwangerschaftsassoziierten Alpha2-Glykoproteins (Alpha2-PAG).

The suggestion that the pregnancy-associated alpha2-glycoprotein could depress the immune response was investigated using the modified electrophoretic mobility test. The presence of the glycoprotein, at physiological levels, significantly reduced the inhibition of the migration velocity of indicator cells in the electric field caused by the MSF. In the contrary alpha2-PAG-free serum had no effect in the test system. Therefore, it can be supposed that alpha2-PAG may have an immunoregulatory function within the immunological mechanism which protects the conceptus and malignant tumor.

Effect of Vibrio cholerae neuraminidase on the phagocytosis of E. coli by macrophages in vivo and in vitro.

The mechanisms by which Vibrio cholerae neuraminidase VCN acts as an adjuvant is unclear. We have studied the effect of VCN on the phagocyotsis of E. coli 0111 K58 by macrophages in vivo and in vitro. (1) Using and in vivo intraperitoneal clearance test we have shown that prior injection of VCN (50 units/mouse) into the peritoneal cavity of mice enhances the clearance of the bacteria as efficiently as an optimal amount of opsonizing antiserum. We have also shown that the enhancement of intraperitoneal clearance by VCN is due to phagocytosis and intracellular killing of the organisms by macrophages. (2) VCN does not enhance the i.v. clearance of E. coli o111 K58. (3) VCN promotes the attachment of the organisms to and phagocytosis by peritoneal macrophages in vitro. Data are provided which suggest that the promotion of attachment and phagocytosis by VCN cannot be explained by a mere increase of passive absorption of the bacteria to the cell wall.

[Diagnosis of malignant processes of the colon and rectum using a new method for the demonstration of sensitized lymphocytes (PAL-test)]. / Diagnostik maligner Prozesse des Colon und Rectums mittels eines neuen Verfahrens zum Nachweis sensibilisierter Lymphocyten (PAL-Test).

Peripheral blood lymphocytes of 45 patients with colon-rectum disease and 30 healthy controls were tested in a simple rapid microagglutination test (PAL test) for cancer diagnosis. In 30 cancer patients 28 positive reactions were obtained. All healthy controls were negative. The 15 patients with benign or premalignant lesions showed 5 positive and 10 negative reactions. Results of the PAL test were about 90% accurate.