Search

Murine fibrosarcoma UV-2237MM cells were implanted into different organs of syngeneic C3H/HeN mice. The resultant tumors were treated by i.v. administration of Adriamycin (ADR). Despite the high sensitivity of the fibrosarcoma cells to ADR in vitro, the established tumors growing in vivo exhibited marked differences in their responses to ADR. Tumors growing in the subcutis and the spleen were ADR-sensitive, whereas lung metastases were not. The resistance of lung metastases to ADR was not due to selection of a drug-resistant population since tumor cells isolated from lung metastases were highly sensitive to ADR under in vitro conditions. The responsiveness of skin and spleen tumors to ADR was due neither to increased blood supply nor to preferential accumulation of ADR, since both parameters were higher in lung metastases. Protein kinase C activity levels correlated with ADR resistance in the closely related murine fibrosarcoma cell line UV-2237 and its ADR-selected multidrug-resistant variants. However, nearly identical levels of protein kinase C activity were found in UV-2237MM tumors growing in the lung, spleen, and subcutis, indicating that protein kinase C activity levels did not account for the different responses to ADR. The present studies suggest that the organ environment influences the response of UV-2237MM to ADR administered systemically. This finding may have implications for the design of animal models for therapy of disseminated cancer.

Retention of the vital dyes rhodamine 123 (R-123) and hydroethidine (HET) correlates inversely with the multidrug resistant phenotypes of the adriamycin (ADM)-selected variants of a uv-induced murine fibrosarcoma cell line (UV-2237M). The differential affinity of these dyes for specific cellular organelles makes them unique compounds for studies of cellular transport. HET enters viable cells freely, is dehydrogenated to ethidium bromide (EtBr), and is subsequently accumulated in the nucleus. Viable cells are impermeable to extracellular EtBr, facilitating kinetic analysis of the efflux of intracellular EtBr. We found that the metabolite EtBr was rapidly cleared by ADM-resistant but not by ADM-sensitive cells. R-123 has a high affinity to mitochondria. Our results show that ADM-sensitive cells retain R-123 whereas the ADM-resistant cells do not. The clearance of both R-123 and EtBr from these cells was inhibited by verapamil. Therefore, R-123 and HET may be considered MDR-associated compounds useful in studying the MDR phenotype of cancer cells. Previously we reported a direct correlation between the level of activity of the calcium- and phospholipid-dependent protein kinase (protein kinases C) and ADM resistance in UV-2237M variant lines. In this report, we demonstrate a direct correlation between cellular calcium and MDR in these cells. Although chelation of extracellular calcium by EDTA did not alter the fluorescence profile of R-123 of the various cell lines, treating the ADM-resistant variants with verapamil restored cellular calcium to the same level as that of the parental cells and, at the same time, retarded the facilitated efflux of R-123 and EtBr and partially reversed cancer cell resistance to ADM.

The purpose of this study was to characterize the antiproliferative activity of a recombinant interferon-alpha (IFN-alpha) against a newly established human adenocarcinoma cell line (DMS-4C) and to determine whether IFN-alpha entrapped in multilamellar liposomes had superior antitumor effects compared with free IFN-alpha. Treatment of DMS-4C cells with 100 U/ml of free IFN-alpha resulted in 34% cytostasis. IFN-alpha encapsulated in phosphatidylcholine/phosphatidylserine multilamellar vesicles produced growth inhibition of 67%, which was significantly greater than that produced by free IFN-alpha or by control liposomes containing only medium combined with free IFN-alpha. Moreover, kinetic analysis revealed that to produce significant cytolysis, free IFN-alpha had to be incubated with target cells for at least 24 h, whereas IFN-alpha encapsulated into liposomes required only 30 min of exposure.

In vitro incubation of mouse UV-2237M fibrosarcoma cells with liposomes containing Adriamycin (ADR) produced significant cytotoxicity in drug-sensitive cells and in multidrug-resistant variants of this tumor. ADR was encapsulated in the aqueous space of multilamellar liposomes composed of phosphatidylcholine and phosphatidylserine. The preparation was stable in medium at 37 degrees C for up to 7 days. Free unencapsulated ADR produced cytostasis in parental ADR-sensitive cells but not in variant lines selected for resistance to the drug. In contrast, ADR encapsulated in multilamellar liposomes (MLV) produced high levels of cytostasis in both ADR-sensitive and ADR-resistant cells. The phospholipid composition of the MLV influenced the outcome of ADR-mediated cytostasis. ADR encapsulated in MLV consisting of only phosphatidylcholine did not produce cytostasis. Increasing the proportion of phosphatidylserine in the MLV increased the level of ADR-mediated cytotoxicity in cells resistant to free ADR. This effect was not due to simple modification of tumor cell surface by liposomes since ADR added to resistant cells together with liposomes containing buffer produced less cytostasis. The cytostasis of resistant cells by ADR in liposomes was not due to appreciable changes in the intracellular ADR concentration or localization within the cells because ADR-induced DNA cleavage was not found in ADR-resistant cells treated with cytostatic amounts of liposomal ADR. Whether the enhanced sensitivity of tumor cells to ADR was due to localized damage to the plasma membrane through a phosphatidylserine-mediated release of the drug to the cell surface is now under active investigation.

Cells from eight different human melanomas and two murine melanomas were injected into the internal carotid artery of anesthetized nude mice. Although all were injected by the same route, particular melanomas produced lesions in different regions of the brain. Two melanoma cell lines isolated originally from brain metastases in patients produced metastases predominantly in the brain parenchyma. In contrast, melanoma cells from subcutaneous or lymph node metastases produced more lesions in the meninges, choroid plexus, and ventricles than in the brain parenchyma. All of the melanomas grew in the brain after a direct intracerebral injection. The pattern of brain metastasis did not correlate with tumorigenicity per se or with the ability of the melanomas to grow in the lungs of nude mice. Two mouse melanomas showed different patterns of experimental metastasis after internal carotid artery injection, with one growing predominantly in the parenchyma and the other more frequently in the meninges and choroid plexus. The growth pattern of human melanoma metastasis in the brain of T cell-deficient nude mice suggests that it is determined by properties unique to each tumor interacting with the host's organ microenvironment.

A new technique is described for the detection of intracellular antigens by immunofluorescence and flow cytometry. The technique utilizes lysolecithin (lysophosphatidylcholine), a naturally occurring phospholipid, to permeabilize cell membranes and allow antibodies to reach intracellular antigens. The technique is rapid and sensitive, and retains sufficient integrity of the cells being treated to enable differentiation of cell types on the basis of light scatter (e.g., lymphocytes from monocytes). Permeabilization of cells following lysolecithin was assessed using standard techniques including trypan blue exclusion, propidium iodide staining, and hydrolysis of fluorescein diacetate. Lysolecithin treatment was accompanied by only minimal increases in non-specific background fluorescence, and no increase in autofluorescence. Our studies have demonstrated that lysolecithin treatment of human mononuclear cell populations permits flow cytometric analysis of cytoskeletal structures, including intermediate filaments, as well as cytoplasmic immunoglobulin. Studies currently in progress in our laboratory have demonstrated broad intracellular reactivity with some monoclonal antibodies identifying leukocyte differentiation and tumor-associated antigens. These findings contrast with the more restricted expression of these antigens on the cell surface and demonstrate not only the value of the lysolecithin technique but also the importance of the study of intracellular antigens in our overall understanding of the specificity, distribution, synthesis, and function of cellular antigens.

Interleukin 2 induced alterations in the morphology of bovine aortic endothelial cells in vitro. The changes observed in confluent cultures of bovine aortic endothelial cells included retraction and elongation of cells leading to enlarged gaps between cells quantified by image analysis. Purified IL-2 (1 U/ml medium) increased the gaps between endothelial cells 3-4-fold compared with control cultures. The effect was transient, since the cells reverted to their original morphology 6-12 hours after the removal of IL-2. Correlative scanning electron microscopy (SEM) studies using fresh bovine aorta showed a dose-dependent alteration of the endothelial surface by IL-2 characterized by rounding and elongation of endothelial cells and prominent perinuclear areas. Gaps between the endothelial cells were observed when aorta samples were incubated with 2 U of IL-2/ml of medium. This was confirmed by SEM, transmission electron microscopy and Evans blue dye staining. These results suggest that IL-2 caused morphological alterations in endothelial cells that enhanced the permeability of the vascular endothelium.

Murine Kupffer cells (RC) were isolated in sufficient number and purity to allow in vitro investigations of their tumoricidal capabilities. The identity of the adherent cells as KCs was established by morphologic, histochemical, and functional criteria. The yield of KCs varied from young (high) to old (low) mice but was not affected by the mouse strain. KCs activated in vitro by either endotoxins (lipopolysaccharide) or lymphokines were rendered highly cytotoxic against syngeneic melanoma or fibrosarcoma target cells. These studies indicate that KCs may indeed play a role in destruction of tumor cells in vivo and thus be important in host defense against developing hepatic cancer metastases.

A new transplantable leukemia (KSL) of unknown etiology that arose in a female Sewall-Wright strain 2 guinea pig is described. KSL cells morphologically resembled medium to large lymphocytes, displayed surface Ia antigen and receptors for complement and for the Fc portion of immunoglobulin, and synthesized surface immunoglobulin (IgM). These characteristics suggest a leukemia of B-lymphocyte origin. KSL cells were shown to be sensitive in vivo to both cyclophosphamide and 1,3-bis(2-chloroethyl)-1-nitrosourea; however, neither drug effectively prevented eventual recurrence of the disease. KSL leukemia was also shown to be distinct from another guinea pig lymphatic leukemia (L2C) with respect to cell morphology, antigenicity, and in vivo growth rate. In this last respect, KSL appeared more closely related to the chronic lymphocytic leukemias. Thus KSL is the first chronic lymphocytic leukemia in the guinea pig to be characterized; it is also the only guinea pig model of lymphatic leukemia that is distinct from L2C leukemia currently available for study.

The purpose of this study was to determine whether the degree of anchorage-independent growth of human tumor cells in increasing concentrations of agarose correlated with the capacity of the cells to produce experimental metastases in nude mice. Human melanoma, breast carcinoma, and colon carcinoma cells from parental lines and variants selected in vivo for metastasis and in vitro cloned lines were plated into medium containing 0.3%, 0.6%, 0.9%, or 1.2% of agarose. These cells were also injected into nude mice: intravenously for melanoma, into the mammary fat pad for breast carcinoma, and into the spleen for colon carcinoma. Production of tumor cell colonies in dense agarose (greater than 0.6%) correlated with production of experimental metastases in the lung (melanoma, breast carcinoma) or liver (colon carcinoma). We conclude that the degree of anchorage-independent growth of tumor cells can predict their biological behavior and metastatic potential in vivo. Thus, this technique may be useful for the isolation of metastatic cells from heterogeneous human neoplasms.

Present therapy for human bladder cancer includes the intravesical administration of antiproliferative agents, such as recombinant human interferon alfa (IFN-alpha). The administration of cytotoxic molecules encapsulated in liposomes could provide a more efficient method for such therapy. Therefore, we determined whether encapsulation of the recombinant human IFN-alpha hybrid BBDD within liposomes will produce antitumor effects against the human bladder cancer cell line 253J superior to those observed with free IFN-alpha. Adherent cells were cultured in medium alone, in medium containing different concentrations of IFN-alpha, or in medium containing multilamellar liposomes (phosphatidylcholine-phosphatidylserine at a molar ratio of 7:3) that encapsulated saline or IFN-alpha. Cell growth was determined 96-120 hours later. Additional control groups consisted of target cells cultured with free IFN-alpha or with IFN-alpha plus liposomes containing saline. Cytostasis mediated by free IFN-alpha alone or IFN-alpha in the presence of liposome-saline was identical and ranged from 0%-30% (10 IU/mL) to 45%-70% (1,000 IU/mL). Liposomes containing saline produced no effects. Liposome-encapsulated IFN-alpha produced significantly greater growth inhibition than free IFN-alpha: 40%-70% (10 IU/mL) and 80%-90% (1,000 IU/mL), respectively. Moreover, a 253J variant subline selected for resistance to free IFN-alpha was sensitive to IFN-alpha presented in liposomes. These data suggest that the encapsulation of antiproliferative agents such as IFN-alpha in liposomes can improve therapeutic results.

A reproducible and convenient microassay was developed by us to measure the antiproliferative activity of biological materials and anticancer drugs. The assay involved the labeling of target cells after their incubation with anticancer agents with the fluorescent vital dye hydroethidine at a dose of 28 micrograms/ml for 30 minutes. Once internalized into live cells, hydroethidine is dehydrogenated to ethidium, which then intercalates into DNA. Hydroethidine labeled the cells uniformly. The cells were lysed with detergent, and the relative fluorescence of this dye was monitored in a microfluorimeter at the speed of 30 seconds per 96-well plate. Because this assay is accurate and reproducible, does not present problems of disposal of labeling agents, and is extremely rapid, it should prove very useful for the screening of many potential anticancer agents with antiproliferative activity.

Variant cell lines of the murine fibrosarcoma UV-2237, selected for doxorubicin [adriamycin (ADM)] resistance, were used to study the tumoricidal activity of macrophages that had been exposed to ADM. Free ADM (0.01-1 microgram/ml) was cytotoxic to the sensitive UV-2237M parent and to the partially sensitive UV-2237M-revertant cell lines, whereas the ADM-resistant UV-2237M ADMR line was unaffected by these levels of ADM. Macrophages harvested from the peritoneal cavities of mice given ip injections of 10 mg ADM/kg body weight 1 day or 4 days previously inhibited proliferation of the 3 cell lines, an effect that was directly correlated to the degree of ADM sensitivity of each cell line. Macrophages exposed in vitro to ADM (from 0.01 to 1 microgram/ml) inhibited the growth of, and eventually were cytolytic to, the parent and the revertant cell lines; these ADM-exposed macrophages did not affect the UV-2237M-ADMR cell line. The correlation between the antitumor effects of macrophages exposed to ADM and the ADM susceptibility of tumor cells suggests that ADM-exposed macrophages exert their effect by the release or transfer of the stored drug.

The characteristics of the binding of human monocytes to tumor cells were studied by a newly developed microassay. First, we determined the kinetics and optimal conditions of the binding. Monocytes recognized and bound to tumor cells very rapidly within 10-20 min of cellular interaction. Binding was also more efficient at 37 degrees C suggesting that active metabolism of monocytes is required. Second, we determined that selective binding of monocytes to cells with tumorigenic phenotypes occurs. For this purpose, lymphocytic leukemia cell lines versus normal lymphocytes, and tumorigenic versus nontumorigenic hybrids from the same parental lines were compared as the targets of the binding assay. In both cases, neoplastic cells were selectively bound by monocytes. Although tumor cells were bound rapidly and selectively by monocytes, initial recognition and binding did not necessarily lead to subsequent tumor cell lysis. This is based on the observation that some tumorigenic parental and hybrid lines were avidly bound by monocytes yet not subsequently killed in a cytotoxicity assay.

Human blood monocytes were activated to become tumoricidal by incubation with liposomes containing muramyl tripeptide-phosphatidylethanolamine, a lipophilic derivative of muramyl dipeptide. The interaction of both tumoricidal and control monocytes with target melanoma cells was analyzed by means of light microscopy and scanning and transmission electron microscopy. The authors found increased clustering around the melanoma cells by tumoricidal monocytes as compared with the control monocytes. The initial clustering of the tumoricidal monocytes around the tumor cells was followed by the establishment of numerous focal points of contact (binding), some of which actually exhibited areas of discontinuous membrane, a finding confirmed by stereophotography. By 24-48 hours of cocultivation, many of the target cells exhibited zones of vacuolation in the immediate vicinity of the tumoricidal monocytes, suggesting target cell damage. (This finding was confirmed by time-course cytotoxicity assays.) The authors conclude that tumor cell lysis mediated by activated human blood monocytes occurs as the final step in a process that includes the establishment of a direct cell-cell contact, damage to the target cell membrane, and the development of areas of vacuolation in the target cells.

Although methapyrilene (MP) produces hepatocellular carcinomas in rats, it does not elicit many of the cellular responses induced by other hepatocarcinogens. We have investigated the early changes induced in rat liver epithelial cell cultures by MP using morphological, cytochemical, and cytofluorometric techniques. Within 2 h of MP treatment, inclusion bodies which were stainable with lipid stains were observed in the cytoplasm. Ultrastructurally, they resembled lamellar bodies with alternating light and dark lamellae. These bodies were transient in nature, since they disappeared within 24 h of removal of MP. They were, however, retained in the cytoplasm as long as MP was present in the medium. Lamellar bodies appear to be induced in the presence of histamine H1 receptor-blocking agents, since methaphenilene and diphenhydramine produced this reaction, but cimetidine, an H2 antagonist, did not. Morphologically, mitochondria of control cells were long and rod-like, whereas they were short or bizarrely shaped in the MP-treated cells. Moreover, a quantitative increase was observed in the mitochondrial content of the treated cells, when intact liver cells vitally stained with Rhodamine 123 were analyzed with a fluorescence-activated cell sorter. A significant increase in binucleated cells was observed when liver cells were exposed for 10 to 12 days with MP. Collectively, these results suggest that MP might perturb the cytoskeletal elements leading to an alteration in the nuclear and mitochondrial makeup of rat liver cells.

Pineal glands from young adult hamsters (Mesocricetus auratus Water-house) kept on a 14-10 light-dark photoperiod contain annulate lamellae. These annulate lamellae are apparently limited to the light pinealocytes. They are found in close proximity to nuclei, the Golgi apparatus, and agranular endoplasmic reticulum. There is also an interesting association or microtubules with the annulate lamellae. It seems reasonable that the presence of annulate lainellae in the pinealocytes may have some correlation with the physiological function or functions of these cells.

Harderian glands of male and female hamsters have nerve endings associated with the secretory cells, myoepithelial cells, and the blood vessels. The nerve endings adjacent to the myoepithelial cells also have myoneural junctions.