Granulocyte-macrophage colony-stimulating factor and tetradecanoyl phorbol acetate induce a distinct, restricted subset of primary-response TIS genes in both proliferating and terminally differentiated myeloid cells.

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.

Imaging of human melanoma xenografts in nude mice with a radiolabeled monoclonal antibody.

External photoscanning with display of radioactivity data as a color-scaled image detected xenografts of human melanoma in male nude inbred mice of BALB/c background 48 hours after injection of 131I-labeled monoclonal IgG 225.28S that is specific for human melanoma. A 131I-labeled polyclonal goat IgG against human melanoma-associated antigens could also image the tumor, but with this preparation there was considerable localization of radioactivity in normal tissues, resulting in less satisfactory tumor definition. Labeled normal mouse IgG did not image the melanoma grafts. Assay of radioactivity in tissues of melanoma-grafted mice confirmed tumor-specific localization of the antimelanoma antibodies. The tumor:blood ratio of radioactivity was 6.55 with the monoclonal antimelanoma IgG and 0.45 with the polyclonal IgG.

Immunoprecipitation of a Mr 64,000 glial tumor-associated antigen by monoclonal antibody 217c.

Monoclonal antibody 217c binds to a tumor-associated surface antigen of transformed rat glial cells. Treatment of C6 glioma cells with 2.5% 1-butanol yielded an extract which was active in competitive inhibition of antibody 217c to cell monolayers in an 125l-protein A assay as well as in binding antibody 217c in an enzyme-linked immunodot assay. The antigen, however, was not released in soluble form, but in a particulate fraction which could be pelleted by ultracentrifugation for 2 h at 120,000 X g. Antibody binding activity in the immunodot assay could be destroyed by heating the extract to 100 degrees C for 10 min. To determine the molecular weight of the antigenic polypeptide, cell monolayer cultures were surface radioiodinated and extracted with Nonidet P-40. Immobilized antibody 217c bound only a single labeled polypeptide with a molecular weight of 64,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This surface peptide was present in the C6 glioma line as well as in oligodendrocyte and astrocyte cultures transformed either spontaneously or using ethylnitrosourea. It was absent from normal astrocyte and oligodendrocyte cultures of neonatal rat brain. In the glial lines studied the P-64 peptide appears as a surface marker indicating malignant transformation.

Monoclonal antibodies to kidney and tumor-associated surface antigens of human renal cell carcinoma.

Three IgG1 monoclonal antibodies derived from BALB/c mice immunized with the Caki-1 human renal cell carcinoma (RCC) line react with antigens present in most human RCCs but restricted in their expression in normal adult tissues. Antibody DAL-K20 reacted with five of six RCCs and the lining epithelium of normal proximal and distal convoluted tubules. Antibody DAL-K29 reacted with eight of nine RCCs, with glomeruli, where it outlined the capillaries, and more weakly with prostatic glandular epithelium and the basal layer of the epidermis. K29 precipitated molecules with molecular weights of 118,000 and 150,000 from extracts of surface-labeled Caki-1 cells. Antibody DAL-K45 reacted with four of six RCCs but not with any normal adult tissue including kidney. It precipitated Mr 177,000 and 150,000 antigens. The three antibodies showed distinct patterns of reactivity with human tumor cell lines.

Tumor localization of monoclonal antibodies against human renal carcinoma in a xenograft model.

We investigated the localization of intravenously injected DAL K45 and DAL K29, two monoclonal antibodies (MABs) against human renal cell carcinoma (RCC), and their F(ab)2 fragments in nude mice bearing intrarenal transplants of the RCC line Caki-1. More of the MABs or their F(ab)2s specifically localized in the tumor than in any normal tissue with the exception of blood. Compared to parent MABs, F(ab)2s were cleared faster from all tissues. In serum, the MABs and F(ab)2s showed a single radioactive peak retaining partial immunoreactivity. DAL K45-F(ab)2 showed the highest tumor:normal tissue localization ratios and the most distinct gamma-camera image at 24 h.

Monoclonal antibodies against Epstein-Barr virus transformed B lymphocytes from a CLL patient.

Immunization of BALB/c mice with EBV-CLL-1 cells, derived from Epstein-Barr virus transformed B lymphocytes from a chronic lymphocytic leukemia (CLL) patient, yielded 2 monoclonal antibodies (IgG1 Kappa and IgG2a Kappa) against a membrane antigen on a subset of normal B lymphocytes and non-Hodgkin's lymphomas. Immunofluorescence revealed strong reactivity of the antibodies with EBV-CLL-1 cells and with most lymphocytes in tonsil follicles, in the intestinal wall, around splenic arterioles and near Hassall's corpuscles in the neonatal thymus as well as with a small proportion of lymphocytes in some large reactive lymph node follicles, weak reactivity with 1/5 of peripheral blood B lymphocytes (PBL), and no reactivity with platelets, granulocytes and non-lymphoid tissues. PBL from 3 CLL patients showed weak staining of only larger cells. Intense fluorescence was observed in several non-Hodgkin's lymphomas of various histological types and in Burkitt's lymphoma lines but not in the 3 T lymphoblastoid and 12 nonlymphoid tumor lines examined. The antibodies precipitated Mr 22,000 and 33,000 bands from surface labeled RAJI or EBV-CLL-1 cells and cross-competed in a binding inhibition assay. The antibodies had approximately 6 million binding sites per EBV-CLL-1 or RAJI cell but were not cytotoxic. This high antigen-density and limited expression in normal cells may permit their use for immunocytological diagnosis and targeting cytotoxic agents and radionuclides against appropriate lymphoma cells.

A new approach to isoelectric focusing and fractionation of proteins in a pH gradient.

This paper describes a new method of condensation (focusing) of extended volumes of mixtures of proteins (or other ampholytes) into an isoelectric spectrum of discrete zones located at points of a pH gradient corresponding to the pI value of the individual proteins. In contradistinction to the currently practiced isoelectric focusing in "natural" pH gradients which may require as much as 96 hours for completion, the present method yields clear-cut condensations marking the isoelectric pH within about five minutes and complete fractionation within about 15 minutes. No "Ampholines" or other special buffers are required and establishment of the pH gradient requires only about 10 seconds rather than days as is common in natural pH gradient focusing. The pH gradient is generated by utilization of the temperature dependence of pH. By establishing a temperature gradient between 0 degrees and 50 degrees C in an electrophoretic column, a stable pH gradient extending over 1 pH unit can be maintained in the absence as well as the presence of a current. The pH gradient can be easily controlled by variation of the temperature limits so that high resolving power can be achieved in shallow pH gradients. The pI of the focused fractions is determined by measurement (by a thermistor or resistance thermometer) of the local temperature within each of the isoelectric zones that determine the local pH. The individual zones can be pipetted out. The method is illustrated by simultaneous condensation and evacuation of hemoglobin in two pH gradients traversed by opposite currents and by separation of hemoglobins A and S in 16 minutes in a pH gradient where the current passes in the direction of increasing pH in Tris buffer solutions stabilized by a sucrose density gradient.

Comparison of electrophoretic mobility and membrane sialic acid content of erythrocytes from adult and umbilical cord blood.

Determinations of cell electrophoretic mobility at low ionic strength and of ghost sialic acid content show that erythrocytes from umbilical cord blood and from adult donors are identical in these two glycoprotein-related properties. Using a streak deflection electrophoresis in 16.5 mM Tris-acetic acid buffer, pH 7.4, no increased streak width indicating electrophoretic heterogeneity could be detected when mixed suspensions of adult and umbilical cord blood erythrocytes were compared with suspensions of adult cells alone. Sialic acid content of 100 nmol/mg protein were obtained for both populations of cells.

Triazene metabolism. III. In vitro cytotoxicity towards M21 cells and in vivo antitumour activity of the proposed metabolites of the antitumour 1-aryl-3,3-dimethyltriazenes.

In vitro cytotoxicity of a series of antitumour triazenes towards the M21 melanoma cell line has been studied. Dimethyltriazenes are structural analogues of 5-(3,3-dimethyl-1-triazeno-)imidazole-4-carboxamide (Dacarbazine) and are inactive, which is consistent with the requirement for metabolic activation. Monomethyltriazenes and hydroxymethyltriazenes , the proposed metabolites of the dimethyltriazenes, are cytotoxic to the M21 cell line. A new series of 4-hydroxy-1,2,3- benzotriazines has been tested for in vitro cytotoxicity. A series of monoalkyltriazenes (Ar X N = N X NHR ) has been tested for antitumour activity against the P388 lymphoma in vivo. Only monomethyltriazenes had significant antitumour activity, which supports the hypothesis that the monomethyltriazene is the active metabolite of the antitumour dimethyltriazenes. The activity of monomethyltriazenes in vivo is correlated with the chemical stability and t1/2 measurements in pH 7.5 phosphate buffer.

Use of a fluid dispensing apparatus for bronchoalveolar lavage in mice.

The volume of the mouse lung is small, so bronchoalveolar lavage (BAL) in mice is generally performed with 1 ml syringes to infuse smaller volumes of fluid. Multiple infusions are required to obtain enough recovered fluid for multiple analyses. This paper introduces the use of one type of a simple fluid dispensing apparatus as an infusion device. It proved to be a faster and a less tedious method than the syringe infusion method. The results of studies in normal mice using both infusion techniques showed no differences between the two with respect to the recovery of cells and protein and to differential leukocyte counts. Thus, the results obtained with this device can be compared with those previously obtained with syringes.

Effects of proteases and neuraminidase on RBC surface charge and agglutination. A kinetic study.

Electrophoretic mobility, membrane sialic acid content and agglutinability by "incomplete" antisera against Rh-o, hr' and k antigens were determined for red blood cells in the course of treatment with trypsin, ficin and neuraminidase. Neuraminidase gradually produces a slight to moderate agglutinability as it reduced surface charge density in proportion to the amount of sialic acid removed. Proteases acted in two distinct steps. The first stage is characterized by the cells rapidly becoming highly agglutinable and by the unmasking of new negative charge as the first half of the sialic acid is removed. In the second stage the cells show a slight gain in agglutinability as surface charge is removed in proportion to sialic acid removal as in the case of neuraminidase. Neuraminidase-treated cells are considerably less agglutinable than cells reduced to the same zeta-potential by protease treatment. The greater efficacy of proteases compared to neuraminidase in making cells agglutinable could be because they not only reduce surface charge density but also increase antigen-antibody bond strength, render antigens more mobile in the membrane to allow clustering in regions of cell to cell antibody bridging and remove glycopeptide chains which may be causing steric hindrance to antigen-antibody binding or to cell-cell contact.

Cell electrophoretic, membrane sialic acid and quantitative hemagglutination studies on some MN variants.

The group of conditions exhibiting diminished MN antigenicity, increased saline agglutinability, decreased electrophoretic mobility and reduced membrane content of sialic acid includes enzyme-treated cells, the hereditary MNSs variants Mk and Mg, En(a-) and the acquired condition of persistent mixed-field polyagglutinability. Here we report our studies on the above serological, chemical and biophysical properties of Mg, Mk and EnaEn? CELLS AND ON TWO ADDITIONAL HEREDItary variants, Miltenberger III and V, (Mi-III and Mi-V). The latter clearly fits into this group of conditions. On the other hand, Mi-III shows its kinship to the broad group of abnormalities of membrane glycophorin but it deviates from normal in the opposite direction. That is we find evidence of decreased saline agglutinability, increased electrophoretic mobility and of increased sialic acid content. Moreover, in the rare MsMi-III Mk phenotype, the opposing effects evident in the heterozygotes tend to balance out their serologic and physicochemical expressions in the double heterozygote.

Heteroconjugate antibody-directed killing of autologous human renal carcinoma cells by in vitro-activated lymphocytes.

Tumor cell lysis can be enhanced significantly in vitro when heteroconjugate (HC) antibodies (anti-CD3 x anti-tumor mAb) are used to specifically direct lymphocyte effector cells to the tumor cell target. In order to effectively utilize HC antibodies in an immunotherapy protocol, methods must be identified for the optimum expansion, activation, and retargeting of lymphocyte-effector populations from cancer patients. In this study, we have compared the proliferative responses of different normal and renal cell carcinoma (RCC) patient lymphocyte preparations (PBL, tumor-infiltrating lymphocytes) stimulated in vitro for periods up to 12 days with a variety of growth factor combinations (anti-CD3, rIL-2, rIL-4). These activated lymphocyte preparations were then tested in vitro for their ability to kill RCC tumor cells and tumor cell lines in the presence of HC preparations (anti-CD3 mAb covalently linked to mAb reactive to different RCC tumor-associated Ag). RCC patient PBL cultured with anti-CD3 plus rIL-2 for 12 days resulted in a 3- to 160-fold expansion of effector cells. These cells, as well as tumor infiltrating lymphocytes, when retargeted with appropriate HC antibodies were capable of mediating high levels of killing of autologous tumor cells. No constitutive autologous anti-tumor cell response was detected in the absence of added HC antibodies. Of the five anti-RCC mAb tested (A6H, K29, K20, UR07, and URO 3), HC containing URO 3 x anti-CD3 and K20 x anti-CD3 elicited the highest level of tumor cell lysis by the activated lymphocyte effector cells. Together these results demonstrate that HC antibodies may be a useful imunotherapeutic reagent for directing the killing of RCC tumor cells by autologous lymphocytes.