Anchorage independent growth and plasminogen activator production by bovine endothelial cells.

Endothelial cells obtained from the aortae of 1- to 2-d-old calves were cloned at high efficiency using fibrin-coated dishes. Primary cultures as well as clones derived from them produced high fibrinolytic activity when grown on 125I-fibrin-coated dishes which was 90% dependent upon the presence of plasminogen. High plasminogen-dependent proteolytic activity was also demonstrated in endothelial cell lysates and in the culture medium of the cells. The production and secretion of the plasminogen activator(s) were found to increase during the log phase of cell growth and to reach a maximum level at confluence. These endothelial cells exhibited morphological phenotypes comparable to those of transformed cells when grown in the presence of acid-treated fetal calf, dog, or human serum. Furthermore, they demonstrated anchorage independent growth, and large colonies were formed in semisolid media. Spontaneous neoplastic transformation of these cells was excluded by karyotypic analysis, lack of tumorigenicity in athymic nude mice, and limited lifespan in culture. Cell clones isolated from colonies grown in agarose demonstrated the same growth characteristics and proteolytic activity as before plating in agarose. High fibrinolytic activity, morphological changes in the appropriate serum, and growth in semisolid media may therefore be indicative of the migratory and/or invasive capacity of both nontransformed endothelial cells as well as tumor cells.

Liposome-encapsulated methotrexate interactions with human chronic lymphocytic leukemia cells.

The uptake of free and liposome-entrapped methotrexate (MTX) by human chronic lymphocytic leukemia cells was compared under incubation conditions resembling those encountered in vivo. Liposome encapsulation enhanced up to 200 times the association of MTX with these cells. Autoradiographic studies established an association of [3H]MTX liposomes with the cells. Such a mode of delivery did not significantly increase the amount of drug available to inhibit dihydrotolate dehydrogenase, which suggested that the liposome content was not readily available to the cytoplasmic sites of action. Increased doses of encapsulated MTX resulted in enzyme inhibitions that were similar to those obtained with the free drug. This finding demonstrates that higher doses of MTX delivered by liposomes could be potentially useful in overcoming drug-transport resistance.

Evaluation of a radioimmunoassay for alpha 1-acid glycoprotein to monitor therapy of cancer patients.

A new radioimmunoassay for alpha 1-acid glycoprotein (AGP) for monitoring the therapy of cancer patients was evaluated. Plasma levels of this glycoprotein were measured in 49 normal healthy volunteers, 71 patients with illnesses other than cancer, 190 patients with solid tumors, and 58 patients with hematologic neoplasms. Plasma levels of AGP were elevated in 89% of the solid tumor patients and 87% of the patients with hematologic neoplasms who had newly diagnosed, locally recurrent, or metastatic cancer. Only 18% of patients with illnesses other than cancer and normal renal function had elevations of plasma AGP. Serial measurements of AGP may be useful for monitoring therapy in several tumor types, including small-cell lung cancer, colon cancer, lymphoma, and non-small-cell lung cancer.

Improved therapeutic benefits of doxorubicin by entrapment in anionic liposomes.

When used as drug carriers, anionic liposomes can reduce the chronic cardiac toxicity and increase the antileukemic activity of doxorubicin (DXN; Adriamycin). Continuing investigations, reported here, have now established the therapeutic benefits of this mode of drug delivery. Liposome encapsulation caused a prolonged elevation in DXN plasma levels and a 2-fold reduction in the exposure of cardiac tissue to the drug. This reduction, however, was not proportional to the substantial decrease in chronic heart toxicity observed in the earlier study. In vivo studies have demonstrated that the entrapped drug retains its full activity against Sarcoma 180 and significantly increases its action against Lewis lung carcinoma, as measured by reduced tumor volume. The increased antineoplastic activity was again not proportional to the increased association of drug with tumor tissue. The effect of liposome entrapment on the immune-suppressive activity of DXN was also examined to determine if factors other than the direct delivery of drug to tumor tissue might improve the therapeutic response. The suppression of the humoral immune response and peripheral leukocyte counts by free DXN was nearly abolished when the drug was administered in the liposome form. These experiments suggest that the improved therapeutic effect of encapsulation may be the outcome of three different mechanisms: (a) altered disposition into subcellular compartments, which reduces cardiotoxicity; (b) increased plasma drug exposure to tumor cells; and (c) significant reduction in the immune suppressive activity of DXN.

Immunohistochemical study of the expression of human milk fat globule membrane glycoprotein 70.

Human milk fat globule membrane, which is said to derive from apical plasma membrane of secretory epithelial cells in breast, was analyzed by sodium dodecyl sulfate-two:dimensional gel electrophoresis. More than 35 components were detected in the gels. One of the major glycoproteins with an apparent molecular weight of 70,000, human milk fat globule membrane glycoprotein, was purified to homogeneity. The pattern of distribution of this glycoprotein in tissues was studied using polyclonal rabbit antibodies to the purified component. The localization of the antigen was accomplished by an indirect immunoperoxidase staining method. Normal mammary epithelial cells display this antigen mostly on the apical plasma membrane, whereas poorly differentiated breast carcinoma cells retained it predominantly in the cytoplasm. These observations suggest that the proper insertion of this glycoprotein into an apical membrane domain may be impaired in malignant tumor cells. In addition, a small population of tumor cells in each case examined failed to express detectable amounts of this component, indicating the presence of antigenic heterogeneity among the tumor cell population.

Cell surface-mediated cytotoxicity of polymer-bound Adriamycin against drug-resistant hepatocytes.

Growth inhibition properties of Adriamycin-coupled polyglutaraldehyde microspheres have been assessed using a highly resistant rat liver cell line, RLC. Covalent attachment of Adriamycin to microspheres increased the cytostatic activity of the drug 1000-fold for this cell line as measured by 50% inhibitory concentration determinations. The effects of Adriamycin-polymer complexes were investigated and compared to free drug, using trypan blue dye exclusion and 51Cr release as indicators of cell viability. When carcinogen-altered drug-resistant rat hepatocytes were tested at an Adriamycin concentration of 10(-6) M, the polymer-bound drug killed 32% of the cells, while the free drug had no detectable cytotoxic effect. However, with normal rat hepatocytes at the same drug concentration, the Adriamycin-coupled microspheres were shown to be less toxic than free drug at 24 hr. It was demonstrated that greater than 99.5% of the drug remains covalently coupled to the microspheres throughout the experiments. Scanning electron micrographs are presented for both cell types, which demonstrate the effects of free and bound Adriamycin on the ultrastructure of the cell surface. The cells lose their microvilli, exhibit numerous blebs, and develop holes and pits in the surface. Transmission electron microscopy demonstrates that less than 1% of the microspheres is internalized by either cell type. Multiple interactions of the drug-polymer complexes with the cell surface are presented as the most probable explanation for the results.

Generation and immunohistological characterization of human monoclonal antibodies to mammary carcinoma cells.

The purpose of this study was to identify human antibodies generated against autologous breast tumor cells by the host's immune response. Accordingly, lymphocytes from lymph nodes of seven different patients with metastatic breast carcinomas were immortalized by fusing them with a nonsecreting variant of murine myeloma cells. The screening for binding of antibodies to tumor cells was performed by indirect immunoperoxidase staining of paraffin-embedded tissue sections of the autologous tumor. The selected hybrid cells, after being cloned three times, were stable for the secretion of immunoglobulins for over 2 years. A total of 81 human immunoglobulin-producing clones was obtained from an initial 595 wells with hybrid growth. Nine of these clones produced immunoglobulin M, none of which showed detectable binding to tissue antigens in breast. Seventy-two clones produced immunoglobulin G monoclonal antibodies, and 15 of these showed preferential binding to breast carcinoma cells. Three of these immunoglobulin G monoclonal antibodies were subjected to detailed immunohistological evaluations. Using these antibodies at concentrations ranging from 10 to 100 ng/tissue section, the morphologically normal mammary epithelial cells could be discriminated from their malignant counterparts. The antibodies showed diffuse staining of cytoplasmic components in the malignant counterparts. Under these conditions, lymphocytes, erythrocytes, and stromal cells in breast tissues were unstained. The antibodies showed variable reactivity with malignant epithelial cells of colon and stomach, and with normal epithelial cells lining the renal tubules and sebaceous glands in skin. Antigenic heterogeneity of malignant mammary epithelial cells was revealed. The antibodies may have value in the characterization of tumor-associated antigens responsible for inducing autologous immune responses.

Differential release of active proteinase inhibitors by two rat mammary adenocarcinoma variants possessing different metastatic potentials.

The ability of tumor cells to express elevated levels of proteinases capable of degrading tissue matrix and basement membrane components in vitro has been correlated to their invasive and metastatic potential. Many in vitro invasion assays have been performed either in the presence of serum or with tumor cells that had been previously grown in serum. Since serum contains large amounts of active proteinase inhibitors, their presence could complicate interpretations. We have, therefore, attempted to measure the amounts of serine proteinase inhibitors released into culture medium by two rat mammary adenocarcinoma metastatic variants selected in vitro for serum-independent growth and differing in their in vivo metastatic behavior. Concentrated spent media (CSM) derived from cultures of poorly metastatic MTLn2(T42D) and highly metastatic MTLn3(T17D) tumor cells, grown in the presence and absence of fetal bovine serum (FBS) for 20-24 h, were compared for the presence of serine proteinase inhibitors capable of inactivating alpha-chymotrypsin. Our results show that when MTLn2(T42D) and MTLn3(T17D) tumor cells were exposed to FBS, the CSM of MTLn2(T42D) exhibited nearly 5-fold greater amounts of active proteinase inhibitors than that of MTLn3(T17D). The amount of proteinase inhibitory activity detected in the CSM of tumor cells not exposed to FBS was not eliminated but declined by 82% and 37% for MTLn2(T42D) and MTLn3(T17D), respectively. Analysis for enzyme-inhibitor (E-I) complex formation by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography confirmed the kinetic results and revealed that the major inhibitor present in CSM/FBS of both variants forms a heat- and sodium dodecyl sulfate-stable E-I complex with an apparent molecular weight of approximately 79,000, identical to that formed when FBS or purified alpha 1-proteinase inhibitor is incubated with [alpha-125I]chymotrypsin. E-I complexes with apparent molecular weights of 44,000 and 50,000 were formed from CSM/bovine serum albumin of MTLn3(T17D) and MTLn2(T42D), respectively, that were not detected when [alpha-125I]chymotrypsin was incubated with bovine serum albumin. We infer from these observations that, in culture, poorly metastatic MTLn2(T42D) tumor cells, as compared to their highly metastatic MTLn3(T17D) counterparts, exhibit an increased capacity to retain and subsequently release significantly greater amounts of serum-derived active proteinase inhibitors. Moreover, the detection of proteinase activity by kinetic analysis and E-I complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography in CSM prepared from cultures not exposed to FBS indicates that both variants have the capacity to produce their own inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)

Preferential expression of a Mr 155,000 milk-fat-globule membrane glycoprotein on luminal epithelium of lobules in human breast.

An integral membrane glycoprotein with an apparent molecular weight of 155,000 and isoelectric points ranging from 7.2 to 7.6 has been found to be predominantly expressed on the apical plasma membrane of luminal epithelial cell lining the lobules and terminal ducts in breast. The glycoprotein was purified to homogeneity from human milk-fat-globule membrane and was termed MFGM-gp 155. Polyclonal antibodies were raised which specifically reacted to a single component that electrophoretically comigrated with this glycoprotein in immunoblotting experiments. These antibodies appear to recognize epitopes which are expressed on the protein segment of the glycoprotein. Using an indirect immunohistological method, the glycoprotein was localized predominantly on the apical plasma membrane of luminal epithelial cells lining the alveoli of normal breasts. The expression of the antigen was maintained in both morphologically well- and poorly differentiated lobular carcinoma cells. The antigen was weakly detectable on normal epithelial cells lining the terminal ducts and malignant cells of infiltrating ductal and medullary carcinomas. Expression of the glycoprotein is not organ specific as it is detectable in normal epithelial cells of kidney, pancreas, salivary gland, and stomach and in malignant cells of colon and stomach. The antibodies did not react with large ductal, myoepithelial, stromal, endothelial, epidermal squamous epithelial cells, melanocytes, eccrine sweat glands and ducts, sebaceous glands, erythrocytes, and lymphocytes in breast and skin tissues. Thus, antibodies to this glycoprotein appear to be useful phenotype markers to study differentiation of mammary epithelial cells and the pathogenesis of different histological types of mammary carcinomas, including Paget's disease and signet-ring cell carcinoma of mammary gland.

Synthesis of alpha 1-antichymotrypsin and alpha 1-acid glycoprotein by human breast epithelial cells.

Malignant and uninvolved human breast tissues were maintained in organ culture for 3 to 6 days. Under these conditions, the three-dimensional glandular architecture is maintained with the least disruption of tissue integrity. The biosynthesis and release of glycoproteins were studied by using the incorporation of [14C]glucosamine and [14C]leucine by the breast surgical specimens. Five major families of labeled glycoproteins were identified from culture supernatants using two-dimensional gel electrophoresis. Quantitative immunoprecipitation established that 16 to 30% of the total of labeled glycoproteins were recognized as normal serum components. Two of these glycoproteins were antigenically related to normal human serum components as demonstrated with crossed immunoelectrophoresis. Evidence was obtained for the active synthesis of alpha 1-antichymotrypsin and alpha 1-acid glycoprotein by human breast epithelial cells. alpha 1-Antichymotrypsin accounted for 0.9 to 7.8% of the biosynthetically labeled glycoproteins from organ culture supernatants. This component was 11.9% of the glycoproteins released by a monolayer culture of the established breast carcinoma cell line, MCF-7. alpha 1-Acid glycoprotein made up 0.7 to 3.1% of the labeled glycoproteins. alpha 1-Antichymotrypsin is a known neutral serine proteinase inhibitor with a particularly strong affinity for cathepsin G. alpha 1-Acid glycoprotein may function primarily as a potent immunomodulator by suppressing lymphoblastogenesis. These glycoproteins may thus have regulatory roles in the proteolytic modification of breast tissue and represent the tissue's own protecting shield against invading leukocytes.

Induction of erythroid differentiation in human leukemic K-562 cells by membrane-directed action of adriamycin covalently bound to microspheres.

Covalent coupling of Adriamycin (ADR) to polyglutaraldehyde microspheres focuses the drug action to the plasma membrane which leads to growth inhibition and also to induction of erythroid differentiation in human leukemic K-562 cells without any evidence of cellular internalization of the drug-microsphere complexes. As observed with the free drug, a reduction in cell growth by the coupled drug correlated with a recruitment of differentiating cells. Treatment of sensitive cells with ADR-microspheres results in 40% of cells containing hemoglobins as determined by benzidine staining at 87% growth inhibition. Similar treatment of ADR-resistant cells produces 24% of benzidine-positive cells at 72% growth inhibition. Furthermore, free and coupled forms of ADR stimulate heme synthesis 12- and 20-fold. Hemoglobin analysis of ADR-polymer induced cells demonstrates additional embryonic (Gower-2, X, Portland) and fetal (F) types of hemoglobin in comparison to uninduced cells which synthesize only small amounts of Gower-1 in sensitive cells and Gower-1 plus hemoglobin X in resistant cells. In addition, free and bound forms of Adriamycin differ markedly in the relative proportion of hemoglobin types that they induce. Free and coupled forms of ADR produce an increase in the gamma-globin mRNA synthesis in sensitive K-562 cells. These results demonstrate that both ADR-sensitive and -resistant K-562 cells can be induced to differentiate at the cell surface by ADR-microspheres and that this induction differs qualitatively from that of free ADR.

Expression of an active proteinase inhibitor, alpha 1-antichymotrypsin, by human breast epithelial cells.

The synthesis of an active proteinase inhibitor, gp 66, by human breast epithelial cells is reported. This glycoprotein is identical to serum alpha 1-antichymotrypsin, which inhibits proteinases that cleave at hydrophobic residues. Immunohistological studies show the in vivo expression on normal secretory and ductal epithelial cells and on primary and metastatic adenocarcinomas. Immunoaffinity-purified gp 66 from MCF-7 culture supernatants is an active inhibitor of chymotrypsin as determined in a fluorogenic enzyme assay and can form stable 88 kDa enzyme-inhibitor complexes. The synthesis of a functional inhibitor may represent the epithelial cell's attempt to stabilize its extracellular milieu.

Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes.

At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest 125-I-labelled casein, covalently linked to latex beads, have been examined. Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8-9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade 125-I-labelled casein when their surfaces are brought into contact with substrate-coated beads.

Synthesis, biological evaluation, and quantitative structure-activity relationship analysis of 2-hydroxy-1H-isoindolediones as new cytostatic agents.

A series of 16 derivatives of 2-hydroxy-1H-isoindole-1,3-diones was designed and synthesized as potential antitumor agents. The cytostatic activity against L1210 cell growth of these compounds was studied, and their IC50 values were found to be in the range of 10(-4) to 10(-8) M. Quantitative structure-activity relationship analysis of these compounds showed that the inhibitory effect was well correlated with the electronic and the lipophilic parameters. Derivatives having a substituent with strongly electron-donating properties at the 6-position showed enhanced inhibitory activity while compounds having an electron-withdrawing group at the same position showed lower activity.

Immunoperoxidase localization of a glycoprotein on plasma membrane of secretory epithelium from human breast.

A glycoprotein component of human milk-fat globule membrane, which is said to derive form apical plasma membrane of mammary secretory epithelium, has been purified. The purified glycoprotein yields a single band under reduced condition and has an estimated molecular weight of 70,000 daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The glycoprotein was termed "secretory epithelial membrane antigen" (SEMA-70) to indicate its origin and molecular size. Specific antisera to SEMA-70 were raised. Using the peroxidase-antiperoxidase method of staining, the presence of SEMA-70 was demonstrated on the apical plasma membrane of the mammary epithelial cells lining the ducts of both normal and lactating breast. No significant staining of cytoplasm was observed. these observations add further evidence as to the origin of milk-fat globule membrane. Although the antigen continues to be expressed on breast adenocarcinoma cells, its cellular distribution appears to be different. The potential usefulness of specific antisera to SEMA-70 is suggested to identify rearrangements in cellular architecture due to pathological changes.

Novel mode of cytotoxicity obtained by coupling inactive anthracycline to a polymer.

The inactive anthracycline analog 4-demethoxy-7,9-di-epi-daunorubicin was covalently coupled to polyglutaraldehyde microspheres. The polymer-bound analog acquired significant cytostatic activity as evaluated with doxorubicin resistant and sensitive murine L1210 leukemia cells. A suggested multiple membrane interaction at the cell surface may represent a novel mechanism of cytotoxicity.

Liposome encapsulation enhancement of methotrexate sensitivity in a transport resistant human leukemic cell line.

A stable mutant of human leukemia CCRF/CEM cells has recently been isolated which is transport resistant for methotrexate (MTX). Encapsulation of MTX in cationic unilamellar liposomes increased the association of the drug 5-fold with the sensitive, and 50-fold with the resistant, cells as compared to the uptake of free drug. The liposome-mediated associations of MTX with sensitive and transport deficient cell lines were similar. Cytostatic studies demonstrated that liposome encapsulation increased MTX activity 4-fold towards the transport resistant cell line. The addition of cholesterol to the vesicles decreased their effectiveness. A 4-fold increase in drug sensitivity due to encapsulation may allow such transport resistant tumor cells to become responsive to chemotherapeutic doses of MTX which are currently feasible in human clinical protocols.

Phenotypic variations dictate the intracellular compartmentalization of doxorubicin in normal human bone marrow cells.

In vitro accumulation of doxorubicin in intracellular compartments of normal bone marrow cells was studied with the use of fluorescent microscopy. Both the cytoplasmic and nuclear compartments had distinguishable drug accessibility in the diverse hemopoietic series and in different stages of maturation of each lineage. Nuclei appeared to be more sheltered in the myelogranulocytic series than in the nucleated erythroid cells. Nuclei of activated phagocytic cells of the myelogranulocytic series and macrophages appeared to be the least accessible to doxorubicin uptake. These observations establish that phenotypic variations dictate the patterns of anthracyclines' subcellular compartmentalization. They also suggest that the molecular mechanism contributing to the intracellular trafficking of doxorubicin deserves more substantial investigation that may contribute to our understanding of drug resistance and sensitivity.

Neurotoxicity and dermatotoxicity of cyanomorpholinyl adriamycin.

The highly lipophilic cyanomorpholinyl adriamycin (CMA) is the most potent antineoplastic anthracycline yet described. CNS distribution and toxicity were examined after i.v. administration of CMA to mice. At doses greater than or equal to 0.1 mg/kg, a neurotoxic syndrome including ataxia, hypokinesia, and tremors appeared. At doses of less than or equal to 0.05 mg/kg, which have been reported to be antineoplastic, no neurotoxicity was observed. On histopathologic examination, no changes were observed in the brain, spinal cord, or dorsal root ganglia. Unlike adriamycin (ADR), which rapidly appears in the nuclei of several tissues, CMA showed no fluorescence, suggesting a different cellular microcompartmentalization. The i.d. injection of CMA disclosed a 200-fold increase in toxicity compared with that of adriamycin. In comparisons of CMA and ADR, neurotoxicity and cardiotoxicity occurred equally only at higher doses; however, the dermatotoxicity and antineoplastic activity of CMA were increased several hundred-fold.

Inhibition of tumor cell growth by Schiff bases of hydroxysemicarbazide.

The inhibitory activities of Schiff bases of hydroxysemicarbazide (HSC) against eight human and murine tumor cell lines and one non-cancer cell line were studied using MTS/PES microculture tetrazolium and methylene blue assays. Compounds 1 (1-[9-(10-methylanthryl)methylene]-4-HSC), 2 (1-[2-hydroxy-3,5-dibromobenzylidene]-4-HSC) and 3 (1-[2,3,4-trihydroxybenzylidene]-4-HSC), which have been shown to be active against murine leukemia L1210 cells in our laboratories, inhibited human leukemia CCRF-CEM cells with similar IC50s ranging from 2.7 to 7.0 microM. Of the compounds tested against attached tumor cell lines (B16, CHO, HT29, ZR75) at 50 microM concentration, compound 1 showed the strongest inhibition, followed by 4 (1-[2-(5-nitrothienyl)methylene]-4-HSC), 2 and 5 (1-[2-hydroxy-3,5-diiodobenzylidene]-4-HSC) with more than 50% inhibition. The IC50s of compound 1 were found to range from 2.7 to 12 microM against the attached tumor cell lines examined. As compared with hydroxyurea, compound 1 had more favorable selectivity against tumor cells. Further more, compound 1 was found to have IC50s of 2.8 and 6.5 microM against hydroxyurea-resistant and gemcitabine-resistant KB cells, respectively, but had no cross-resistance with hydroxyurea and gemcitabine (two known ribonucleotide reductase inhibitors acting at different sites of the same enzyme). In conclusion, several Schiff bases of HSC showed inhibition of tumor cell growth at micromolar concentration and had no cross-resistance with hydroxyurea-resistant KB cells.