Anticarcinoma activity in vivo of rhodamine 123, a mitochondrial-specific dye.

Carcinoma cells and normal epithelial cells differ in the mitochondrial retention of a permeant cationic compound, rhodamine 123. The possibility of utilizing this difference in carcinoma chemotherapy was investigated. Rhodamine 123 exhibited anticarcinoma activity in mice, and this activity was potentiated by 2-deoxyglucose.

Rhodamine-123 selectively reduces clonal growth of carcinoma cells in vitro.

Rhodamine-123, a cationic laser dye, markedly reduced the clonal growth of carcinoma cells but had little effect on nontumorigenic epithelial cells in vitro. This selective inhibitory effect of Rhodamine-123 on some carcinomas is unusual since known anticancer drugs, such as arabinosyl cytosine and methotrexate, have not been shown to exhibit such selectivity in vitro.

Use of SM-1 monoclonal antibody and human complement in selective killing of small cell carcinoma of the lung.

SM-1 is a murine monoclonal antibody strongly reactive with a cell membrane antigen of small cell carcinoma (SCC) of the lung but unreactive with the membrane of most other carcinomas and normal tissues including normal bone marrow. We have found that in the presence of human complement, SM-1 antibody is highly cytotoxic to SCC cells. Using three treatments with antibody and complement, more than 99% of SCC cells in culture were lysed, as determined by the chromium release and clonogenic assays. Similar efficiency of SCC cell lysis was observed when one SM-1 antibody treatment was followed by three treatments with human complement. In contrast, there was little antibody-dependent lysis of non-small cell lung cancer cells, other carcinomas, and leukemia cell lines. The amount of chromium released from normal bone marrow cells treated with SM-1 antibody and complement was minimal and was mainly due to the effect of complement alone. Clonogenic assays, including colony-forming unit-granulocytic/monocytic, erythroid burst-forming unit, and colony-forming unit-granulocytic/erythroid/monocytic/megakaryocytic, also showed no significant SM-1 antibody-dependent cytotoxicity on normal bone marrow precursors. Since SM-1 antibody is selectively cytotoxic to SCC cells in the presence of human complement, it is a potentially useful agent for the selective eradication of tumor cell contamination in marrows of patients with metastatic small cell lung cancer and possibly for in vivo serotherapy.

Membrane antigen in small cell carcinoma of the lung defined by monoclonal antibody SM1.

Mouse myeloma cells were fused with spleen cells from BALB/c mice immunized with a cell line derived from human small cell carcinoma (SCC) of the lung. The cloned hybridoma SM1 produced antibody that was reactive with the surface membrane of SCC cell lines and SCC tumors but not with the membrane of several non-SCC cell lines and tumors. SM1 ascites fluid was used to screen for reactivity of the antibody with other human cancer cell lines, tumor tissues, and normal tissues. SM1 antibody was found to be unreactive with neuroblastoma, adrenal carcinoma, melanoma, and bronchial carcinoid. Reactivity was detected with some breast carcinoma cell lines but not with breast cancer tissue specimens. In the same individual, the antibody was reactive with SCC lung tumor and SCC metastatic to the liver but not with normal tissues, including bronchus, lung parenchyma, liver, kidney, and brain. Human erythrocytes and marrow cells were also unreactive. Since SM1 detects an antigen that is present in greatest amounts on the surface membrane of SCC of the lung, this antibody may be useful in tracing the lineage patterns of human lung cancers.

Induction of cytoskeleton-associated proteins during differentiation of human myeloid leukemic cell lines.

Alterations in the expression of proteins associated with the cytoskeletal framework during differentiation of two human myeloid leukemia cell lines were analyzed by two-dimensional gel electrophoresis of Triton-insoluble cellular framework fractions. During in vitro differentiation of HL60 (human promyelocytic leukemia line) and U937 (human monocytoid leukemia line), several new cytoskeleton-associated (CSK) proteins are induced. All of these CSK proteins are also present in freshly isolated normal granulocytes and macrophages. One of these differentiation-induced proteins comigrates with vimentin. There are several differentiation-sensitive proteins, i.e., those that are no longer synthesized upon differentiation. The changes in CSK protein synthesis during differentiation of HL60 and U937 cells do not seem to be related to drug treatment per se since exposure to conditioned medium from phytohemagglutinin-stimulated lymphocytes as well as to dimethyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate results in the production of many similar proteins. In vitro conditions that do not result in differentiation of HL60 and U937, such as cultivation in serum-free medium, do not induce the CSK proteins that we describe. A notable finding in this study is that all of the qualitative changes in the proteins synthesized during differentiation are detected in the cytoskeletal (Triton-insoluble) fraction, whereas only minor quantitative alterations are observed in the Triton-insoluble extract. The changes in CSK protein components occur in an orderly fashion. Vimentin, an intermediate-filament protein, is synthesized in large amounts prior to changes in cellular morphology and the induction or loss of other CSK proteins. Vimentin may play an important role in the reorganization of the cytoskeleton to support the process of differentiation. The other CSK proteins are synthesized sequentially along with the morphological and functional changes during differentiation. These model systems, therefore, present an opportunity to investigate the role of specific cytoskeletal components in cellular differentiation.

Keratin subtypes of small cell lung cancer.

The molecular forms of keratin in small cell lung cancer (SCLC) cell lines and tumors were examined with antikeratin monoclonal antibodies. Immunostaining of SCLC by antikeratin antibody and examination by fluorescence microscopy indicates population heterogeneity in keratin content. Intensity of immunostaining is often weak. However, polyacrylamide gel electrophoresis and immunoblotting reproducibly demonstrate the presence of keratin and allow analysis of the keratin subtypes. The finding of keratin subtypes closely associated with the development of keratinizing epithelium (the 68 kDa basic keratin) in SCLC was unexpected in a tumor that is regarded as poorly differentiated. The cytoskeletal composition of SCLC suggests the presence of a heterogeneous population with a significant proportion of cells expressing highly differentiated epithelial properties.

Selective toxicity of rhodamine 123 in carcinoma cells in vitro.

The study of mitochondria in situ has recently been facilitated through the use of rhodamine 123, a mitochondrial-specific fluorescent dye. It has been found to be nontoxic when applied for short periods to a variety of cell types and has thus become an invaluable tool for examining mitochondrial morphology and function in the intact living cell. In this report, however, we demonstrate that with continuous exposure, rhodamine 123 selectively kills carcinoma as compared to normal epithelial cells grown in vitro. At doses of rhodamine 123 which were toxic to carcinoma cells, the conversion of mitochondrial-specific to cytoplasmic-nonspecific localization of the drug was observed prior to cell death. At 10 microgram/ml, greater than 50% cell death occurred within 7 days in all nine of the carcinoma cell types and lines of different origin studied, while six of six normal epithelial cell types and lines remained unaffected. Cotreating carcinoma cells with 2-deoxyglucose and rhodamine 123 enhanced the inhibition of growth by rhodamine 123 alone in clonogenic survival assays. The observation of the selective toxicity of rhodamine 123 appears to be unique in view of the absence of selective toxicity reported in vitro for the various antitumor agents currently in clinical use. Preliminary results with rhodamine 123 in animal tumor systems indicate antitumor activity for carcinomas.

Selective cytotoxicity of the SM1 monoclonal antibody towards small cell carcinoma of the lung.

A murine monoclonal antibody, SM1, is strongly reactive with the surface membrane of small cell carcinoma of the lung. SM1 antibody is unreactive with most other cancers and various normal tissues including bone marrow cells. We now find that SM1 antibody is selectively cytotoxic to small cell carcinoma (SCC) in vitro. The antibody is present in high titers in supernatant fluids or ascites obtained by i.p. injection of SM1 hybridoma cells into pristaned BALB/c mice. The cytotoxic effect of the antibody is reduced to one-half maximal activity only at dilutions greater than 1:40,000. The efficiency of tumor cell lysis is greatly enhanced by repeated treatments with antibody and complement. Using three treatments with antibody and complement, 99.9% of SCC cells are lysed, as determined by the chromium release. Similar efficiency of SCC cell kill was observed by clonogenic assays. SM1 antibody produces no significant antibody-dependent lysis of cell lines derived from non-SCC lung carcinomas and leukemia cells. The results from chromium release assay and clonogenic assays also indicate that the effect of SM1 antibody and complement on bone marrow cells is minimal and could be accounted for by the effect of complement alone.

Effects of dibutyryl cyclic adenosine 3':5'-monophosphate on the growth of cultured human small-cell lung carcinoma and the specific cellular activity of L-dopa decarboxylase.

High activity of L-dopa decarboxylase separates small (oat)-cell from non-small-cell lung cancer in cell culture. The present study investigates relationships between the specific cellular activity of this enzyme and: (a) cell growth kinetics of an established line (O-H-1) of human small cell lung carcinoma, and (b) responses of these cells to treatment with cyclic adenosine 3':5'-monophosphate and sodium butyrate. The O-H-1 cells, as for most other established small-cell lines, grow as suspended cell aggregates. During growth, the specific cellular activity of L-dopa decarboxylase parallels levels for [3H]thymidine labeling index and the ratio of cells in G2-M to those in G1-G0 phases of the cell cycle. Each of these parameters is 2- to 3-fold higher during exponential versus stationary growth. Continuous treatment with dibutyryl cyclic adenosine 3':5'-monophosphate (dcAMP; 0.1 or 1 mM) and 1 mM theophylline produces simultaneous cessation of cell growth and an increase in cellular L-dopa decarboxylase activity. During this period, analyses of DNA histograms reveal an increase in the number of cells in the G2-M phase; the rate of increase in the ratio of G2-M to G1-Go cells paralleled the rate of increase in specific activity of the enzyme. The effects of the dcAMP were promptly reversible; release of the apparent G2-M block preceded regrowth of the cells and was accompanied by a return of L-dopa decarboxylase activity to base-line levels. The changes in enzyme activity were specific for cyclic adenosine 3':5'-monophosphate; another cyclic adenosine 3':5'-monophosphate analogue, 8-bromo adenosine cyclic 3':5'-monophosphate yielded similar increases in L-dopa decarboxylase to those seen with dcAMP, while 0.01 to 1 mM butyrate alone produced the inhibition of cell growth but no changes in specific activity of L-dopa decarboxylase or percentage of cells in the different phases of the cell cycle. We conclude that the specific activity of L-dopa decarboxylase, a key neuroendocrine marker for cultured small-cell lung carcinoma, is highest during proliferative growth and/or when these cells are in the G2M phase of the cell cycle. The differential effects of dcAMP and sodium butyrate offer potential for exploring neuroendocrine differentiation in this important lung cancer and related endocrine neoplasms.

Cytotoxic murine monoclonal antibody LAM8 with specificity for human small cell carcinoma of the lung.

The reactivity of the murine immunoglobulin monoclonal antibody LAM8 directed against a membrane antigen of human small cell carcinoma (SCC) of the lung was investigated on human cell lines and tissues. Indirect immunofluorescence staining, radioimmunoassays, and cytotoxicity assays showed LAM8 antibody to selectively react with SCC but not with non-SCC lung cancer cell lines and extrapulmonary tumor cell lines. Unlike other SCC antibodies, including those we have previously described, highly preferential reactivity with SCC tissues was also demonstrated by immunoperoxidase staining of deparaffinized formalin-fixed tissue sections. Membrane and cytoplasmic staining was seen in of 9 of 12 SCC tissues. No significant staining was seen in non-SCC lung cancer and a wide range of other tumors, including mesothelioma and bronchial carcinoids. Significant LAM8 reactivity was also absent in normal tissues of all major organs. Few tumors and epithelial tissues, including bronchial epithelium had rare LAM8 positive cells which were always less than 2% of the entire cell population. In vitro treatment with antibody and human complement was highly cytotoxic to SCC cells, but had not effect on bone marrow progenitor cells. Immunoblotting of membrane extracts separated on sodium dodecyl sulfate-polyacrylamide gels showed the LAM8 antigen to have a band of an approximate molecular weight of 135,000 and a cluster of bands with approximate molecular weights of 90,000. This reactivity was lost after incubation of the extracts with periodate. LAM8 antibody shows a highly preferential reactivity with SCC cell lines and formalin-fixed paraffin-embedded SCC tissues and is selectively cytotoxic to cells expressing LAM8 antigen.

In vitro and in vivo killing of acute lymphoblastic leukemia cells by L-asparaginase.

L-Asparaginase (ASNase) is a potent antileukemic enzyme routinely used in the treatment of children with acute lymphoblastic leukemia. As part of investigations of the biological activity of ASNase, we have developed techniques which measure the in vitro and in vivo cell killing ability of ASNase. To study the effect of ASNase on in vitro survival of primary lymphoblasts, bone marrow mononuclear cells obtained from untreated patients with acute lymphoblastic leukemia were cultured with and without ASNase. After 5 days, viable cells were counted using trypan blue exclusion to calculate total cell kill due to ASNase. Propidium iodide exclusion, leukemia cell surface antigens, and flow cytometry were used to determine leukemia cell kill due to ASNase. Comparison of leukemia cell kill and total cell kill showed a direct linear relationship (n = 24, r = 0.7), preferential killing of leukemia cells by ASNase (slope = 0.66), and that use of leukemia cell surface markers yielded a more accurate measurement of leukemia cell killing. ASNase at concentrations from 0.0001 to 0.1 IU/ml had equal effects on extent of leukemia cell killing (P = 0.3 to 0.7), suggesting the absence of a dose response at the ASNase concentrations tested. As a measure of the in vivo response to ASNase treatment, the number of viable bone marrow leukemia cells in the patient prior to and 5 days after treatment with ASNase was measured as the product of (% of rhodamine 123 fluorescent [viable] cells) x (absolute leukemic infiltrate). The change which occurred in the viable leukemic infiltrate was the same for patients whether they received 25,000 or 2,500 IU/m2 of ASNase as a single drug. There was a linear correlation (n = 8, r = 0.9) between in vivo and in vitro leukemia cell killing by ASNase. Thus, the in vitro assay described here can be used to predict in vivo sensitivity to ASNase in acute lymphoblastic leukemia.

Detection of bone marrow metastasis in small-cell lung cancer by monoclonal antibody.

A murine monoclonal antibody against a surface antigen of small-cell carcinoma of the lung (SM1 antibody) was investigated for its use in detecting bone marrow metastasis. Bone marrow cells of healthy volunteers and of patients with small-cell carcinoma of the lung (SCCL) were examined for reactivity with SM1 antibody and indirect immunofluorescence and the results compared to conventional histochemical staining (Wright-Giemsa stain of bone marrow aspirates and hematoxylin-eosin stains of bone marrow biopsies). No SM1 reactivity was found in marrow cells of eight healthy volunteers. Thirty-six samples from 33 patients with SCCL were examined; tumor involvement was found in 69% by SM1 antibody and in 16% by histochemical stains. All bone marrow samples from patients with SCCL that were unreactive with SM1 antibody also showed no evidence of tumor involvement by histochemical stains. Samples of 29 patients were investigated at initial staging; SM1 reactive cells were found in 50% of 16 patients with limited disease and in 77% of 13 patients with extensive disease. Overall, the proportion of patients recognized to have disseminated disease at diagnosis was increased from 45% to 72% by monoclonal antibody staining. Indirect immunofluorescence with SM1 antibody allows detection of bone marrow metastasis of SCCL that cannot be seen by conventional morphology and can identify disseminated disease in patients otherwise staged limited disease.

Cytoskeleton-associated proteins: their role as cellular integrators in the neoplastic process.

The cytoskeleton (CSK) of eukaryotic cells is composed of a complex interconnected network of filaments which is important in a wide variety of cellular functions including changes in cell shape, cell motility, mitosis, anchorage-dependent growth, and the localization of cellular organelles such as mitochondria, polyribosomes, and secretory granules. The various proteins comprising the cytoskeleton include actin in microfilaments, tubulin in microtubules, and the heterogeneous group of intermediate filament proteins that are associated with different cell types (keratin in epithelial cells, vimentin in fibroblasts, desmin in muscle cells, glial filament protein in glial cells, and the neurofilament protein subunits in neural tissue). Many other proteins in glial cells, and the neurofilament protein subunits in neural tissue). Many other proteins are closely associated with the cytoskeleton and influence its organization. In neoplastic cells, the expression of these different CSK proteins, especially the intermediate filament proteins, reflects their morphologic and functional differentiation. The carcinomas contain keratin; identification of individual keratin components may allow further sub-classification of carcinomas which is consistent with their tissue of origin. The sarcomas of muscle origin contain desmin. Vimentin is found primarily with cells of mesenchymal origin, but may coexist with other intermediate filament proteins in other tumors. One example is the coexistence of keratin and vimentin in tumors, such as mesotheliomas, which are derived from epithelial cells of embryonic origin. Glial fibrillary acidic protein is the most specific marker for glial tumors. Tumors of neural origin are characterized by the presence of neurofilament subunits. Therefore, analysis of CSK composition would be useful in diagnosis of clinical specimens and aid in studies of lineage relationships of neoplasms. Although no consistent differences in cytoskeletal structure between neoplastic and normal cells have been identified so far, the presence of more subtle biochemical alterations in the cytoskeletal structure of neoplastic cells that contributes to malignant behavior has not been ruled out. Since the cytoskeletal network plays an important role in cell shape and cell locomotion, which in turn are thought to be involved in growth control, invasion, and metastasis, further work is directed at identifying the various alterations in cytoskeletal architecture that may influence the malignant behavior of neoplastic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of fluosol-DA/carbogen on etoposide/alkylating agent antitumor activity.

The tumor growth delay produced by the combination of etoposide with the alkylating agent CDDP or BCNU and Fluosol-DA with carbogen breathing in three model tumor systems was examined. The addition of Fluosol-DA to etoposide treatment increased tumor growth delay 2.8-fold, 3.3-fold and 2.2-fold in the FSaIIC fibrosarcoma, the Lewis lung carcinoma and the SW2 small-cell xenograft, respectively. In both the FSaIIC fibrosarcoma and the Lewis lung carcinoma the combination of etoposide treatment with CDDP produced an additive effect. When Fluosol-DA was added to this combination the tumor growth delay increased 1.9-fold and 1.4-fold in the FSaIIC fibrosarcoma and the Lewis lung carcinoma, respectively. Adding Fluosol-DA to a treatment regimen with etoposide and BCNU produced a 2.2-fold, 2.0-fold and 1.6-fold increase in the tumor growth delay of the FSaIIC fibrosarcoma, the Lewis lung carcinoma and the SW2 small-cell xenograft, respectively. The effect of these various treatment combinations on tumor cell survival was assessed in the FSaIIC fibrosarcoma. When the alkylating agents CDDP or BCNU were prepared in Fluosol-DA, there was an additional increase in tumor cell kill, so that with CDDP there was 2.1-fold and 4.7-fold increase in tumor cell kill and with BCNU there was 1.5-fold and 1.2-fold increase in tumor cell kill compared to the drug plus Fluosol-DA and the drug plus Fluosol-DA/carbogen breathing, respectively. The combination of etoposide and CDDP led to less than additive cell killing, and the combination of etoposide and BCNU appeared to be additive, as predicted by simple product summation, in all of the treatment conditions examined. Both etoposide + CDDP and etoposide + BCNU produced additive or less than additive toxicity to bone marrow as measured by CFU-GM.

Reversal of methotrexate resistance in human squamous carcinoma cells by SQM1-liposome.

SQM1, a membrane protein associated with methotrexate (MTX) transport was incorporated into unilamellar vesicles, forming. SQM1-Liposome. Human squamous carcinoma of the head and neck (SqCHN) cells resistant to MTX with defective MTX transport and low SQM1 expression were treated with SQM1-Liposome. SQM1 content was found to be increased in these treated SqCHN cells by radioimmunoassay. Concurrent increases in MTX uptake and MTX cytotoxicity in these treated SqCHN cells were found. The ability to reverse MTX-resistance in vitro opens further possibilities for in vivo applications.

Multiple NF-I-site binding factors associated with different cell-types of human lung carcinomas.

Nuclear extracts from human lung adenocarcinoma and squamous carcinoma cells were found to form two types of DNA-protein complexes with a consensus nuclear factor I (NF-I)-binding site. Three NF-I-site-binding cofactors (NCF90, NCFIIO and NCF120) were found to be differentially expressed in these two cell-types of human lung carcinomas. These data suggest that the NF-I-binding enhancer element with its differentially expressed NF-I cofactors may play an important role in the differentiation of neoplastic human bronchial epithelium.

Expression of a new surface membrane antigen (SQM1) in tumor cells cultured in media with different calcium ion levels.

Terminal differentiation is usually achieved in normal as well as transformed squamous epithelial cells when cultured. On the other hand, tumor cells at various differentiation stages and with different biological characteristics comprise the heterogeneous properties of tumors which have been one of the barriers to effective treatments. Recently, a surface membrane protein has been reported in squamous cell carcinomas of the head and neck, which is recognized by a murine monoclonal antibody, SQM1. This glycoprotein was further suggested to be related to squamous cell differentiation and intercellular adhesion. In a recent study, the esophageal carcinoma cells of EC/CUHK2 cell line were induced to various differentiation stages as evidenced by the increasing amount of intracellular desmosomes and tonofilaments and greater binding ratios of cytokeratin and involucrin antibodies than in those cells that maintained lower calcium ion concentrations. The expression of SQM1 antigen was found to increase in intensity when the tumor cells were cultured in moderate to high calcium ion levels for 10 to 15 hours when the differentiation patterns were beginning to appear. The intensity declined gradually thereafter. Thus SQM1 protein might be related to the stage when the cells started committing with squamous differentiation.

A new membrane marker for commitment of human bronchial epithelial cells to terminal squamous differentiation.

Squamous differentiation in human bronchial epithelial (HBE) cells is a metaplastic change that occurs primarily after chronic injury and often precedes squamous cell carcinoma. After treating HBE cells with inducing agents for various periods followed by removal of these agents and assay for terminal squamous differentiation, HBE cells were found to become committed sqamous differentiation after 12-36 hours of treatment. Expression of a membrane marker, SQM1 protein, increased during the commitment stage, peaked shortly after commitment and gradually decreased in the later stages of squamous differentiation of HBE cells. The lack of marked morphological and biochemical changes associated with the commitment and early stages of squamous differentiation makes SQM1 protein a useful marker for the study of squamous differentiation and metaplasia of HBE cells.

Ultrastructural localization of a new surface membrane antigen (SQM1) related to squamous differentiation.

Normal squamous epithelial cells readily undergo terminal differentiation in culture and are commonly used in differentiation studies. Several intracellular markers of squamous differentiation such as keratin, involucrin, transglutaminase and cholesterol sulfate have been well-studied and described by other workers. We have recently reported a surface membrane antigen in squamous carcinoma of the head and neck antigen in squamous carcinoma of the head and neck which is recognized by a murine monoclonal antibody SQMI. In this paper, we present our studies on the ultrastructural localization of SQMI antigen in cultured squamous epithelial cells using gold-labelled antibody. The cells studied included both normal and cancer cells at different degrees of differentiation. Under both transmission and scanning electron microscopy examination, the SQMI antigen was localized at the membrane surface of cultured cells, particularly at sites of cell-cell interdigitation. No association with desmosomal structure was observed in any of the specimens examined. There was however an association of SQMI antigen with microvilli of cell membrane. No non-specific cytoplasmic localization of SQMI antigen was observed. The intensity of SQM1 antigen revealed by gold-labelling appeared to have a positive correlation with the degree of differentiation of the cells in culture.

Immunohistochemical identification of squamous carcinoma of the head and neck with monoclonal antibody SQM1.

Frozen tissue sections of biopsies from head and neck squamous cancer lesions were examined for immunohistochemical staining with a recently developed monoclonal antibody, designated as SQM1 antibody and directed against the surface membrane of squamous carcinoma cells. SQM1 antibody stained selectively squamous carcinoma, while normal mucosa and cells of the stroma were non-reactive. Positive staining of tumor was found in 33/35 specimens obtained from several major sites of the head and neck area and was observed in primary manifestations and lymph node metastases as well as in recurrences. The most consistent reactivity was seen with carcinomas of the tongue. Well differentiated squamous carcinomas contained a higher proportion of SQM1 positive tumor cells than poorly differentiated carcinomas. We suggest that the SQM1 antibody may aid in the immunohistochemical identification of squamous carcinoma of the head and neck area.